JP3767597B2 - Novel polypeptide and method for measuring biological components using the same - Google Patents

Description

  The present invention provides a novel polypeptide having an acid residue derived from a strong acid and a biological fluid such as serum, blood, plasma, urine, lymphocytes, blood cells, various cells, etc. The present invention relates to a method for measuring a substance to be measured in a sample.

  Specific substances such as antigens and antibodies, proteases and proteinaceous protease inhibitors, sugar chains and lectins, enzymes and substrates and coenzymes for them, physiologically active substances such as hormones and receptors and transport proteins for them, double chains It is known that a pair of polynucleotide strands of DNA and the like have a strong interaction with each other (that is, show affinity for each other) to form a strong complex.

  As a method for measuring a substance to be measured in a sample using such an interaction, a complex formed by a reaction between the above-described combinations of substances on the solid phase is formed, and then the solid phase is used. Performing so-called B / F separation, for example, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA) (for example, JP-A-1-27061, JP-A-3-44399, etc.) And the like, and a method of performing so-called B / F separation using a high-performance liquid chromatography (HPLC) developed by some of the present inventors (so-called B / F separation, for example). JP-A-2-28557, JP-A-3-206964, JP-A-3-221865, JP-A-6-66800, etc.).

  In such a measurement method, since the accuracy of measurement and the time required for analysis are affected to some extent depending on how efficiently the B / F separation is performed, various studies have been made on the B / F separation method. .

  For example, a method for performing B / F separation using a solid phase such as JP-A-1-27061 and JP-A-3-44399, or HPLC disclosed in JP-A-6-66800, etc. In the method of performing B / F separation, for example, an anionic substance is bound in advance to an antibody against a measurement target substance in a biological sample, and so-called B / F separation is performed using the measurement target substance and an antibody against the substance. This is performed by utilizing the property (anionic property) of the anionic substance in the composite.

  Although these methods are methods that can perform B / F separation more efficiently than in the past, the anionic substances used are all derived from polyamino acids and their carboxyl groups Since the B / F separation is performed using the anionic nature derived from the method, the method of performing the B / F separation using HPLC developed by the inventors of the present application is a slightly problematic method.

  That is, in the method using HPLC, it is necessary to separate the complex from a substance or free antibody that may affect the coexistence in the sample, together with B / F separation. In order to sufficiently perform separation using the anionic nature of the carboxyl group derived from polyamino acid, about 200 carboxyl groups (about 200 amino acid residues) must be introduced into the anionic substance. There wasn't. For this reason, firstly, it takes time to prepare the anionic substance, and furthermore, it is difficult to prepare a large amount of anionic substance having a uniform molecular weight. When B / F separation is performed, there is a problem in that the peak may tail or read during the separation, and the measurement accuracy may decrease. In addition, when a polyamino acid having a carboxyl group, such as polyglutamic acid, is used for separation, a non-specific reaction may occur, and a phenomenon such as a blind value (blank value) or an increase in measured value may occur. In order to prevent this, there is a problem that an appropriate polymer anion such as γ-polyglutamic acid needs to be added. For this reason, there is a need for further improvements.

Japanese Patent Publication No. 227061 published in 1991 1991 Patent Publication No. 44399 Heisei 2 published patent publication 28557 1991 Published Patent Publication 206964 1991 Patent Publication No. 221865 1994 Published Patent Publication No. 66800

  The present invention has been made in view of the above situation, and is abbreviated as a substance to be measured in a biological sample and a substance having binding ability (affinity) to the substance to be measured (hereinafter referred to as a binding ability substance). When the complex resulting from the interaction with) is separated from the free binding substance and the coexisting substance that may affect the detection of the complex using a method using a negative charge, It is intended to provide a novel polypeptide that can be used to more effectively separate a complex from a free binding substance and the like, and a method for measuring a substance to be measured in a biological sample using the polypeptide. Objective.

The present invention relates to a polypeptide having 3 to 30 acid residues derived from a strong acid selected from sulfuric acid and phosphoric acid and having a total number of amino acid residues of 3 to 30, and a substance to be measured in a biological sample. It is an invention of a combination with a substance having a binding ability to. "
The present invention also relates to “a polypeptide having 3 to 30 acid residues derived from a strong acid selected from sulfuric acid and phosphoric acid , and a total number of amino acid residues of 3 to 30 via a spacer at the N-terminus. A compound having a maleimide group bonded thereto. "
Furthermore, the present invention provides a “polypeptide having 3 to 30 acid residues derived from a strong acid selected from sulfuric acid and phosphoric acid and having a total number of amino acid residues of 3 to 30, and a measurement target in a biological sample. It is an invention of a reagent for measuring a substance to be measured in a biological sample, comprising a conjugate with a substance capable of binding to the substance.
Furthermore, the present invention provides a biological sample, a polypeptide having 3 to 30 acid residues derived from a strong acid selected from sulfuric acid and phosphoric acid , and a total of 3 to 30 amino acid residues, Reaction with a reagent comprising a conjugate with a substance capable of binding to the substance to be measured in the sample, and then separating the resulting complex by a method utilizing a negative charge, based on the amount of the complex The biological component measuring method characterized in that the amount of the substance to be measured in the biological sample is determined. "

  That is, the present inventors have found that a complex resulting from the interaction between a measurement target substance and a binding ability substance in a biological sample, and a free binding ability substance [or a free measurement target substance. The substance to be measured may be labeled with a substance that can be detected by any method (hereinafter abbreviated as a detection substance). When the separation from the coexisting substance that may affect the detection of the complex such as] is performed using a method using a negative charge, the complex and the free binding substance are more clearly separated. In the course of earnest research in search of a possible method, a substance having an appropriate property capable of further changing the properties of the complex (hereinafter abbreviated as a separation enhancing substance) is bound to the complex, and the separation is improved. Based on the nature of the substance, when the complex is separated from a coexisting substance that may affect the detection of the complex, such as a free binding ability substance (or a free measurement target substance), By appropriately selecting and using a separation enhancing substance, it is possible to freely adjust the elution position of the complex. In other words, by binding an appropriate separation enhancing substance to the complex, Clearly identify free binding substances, etc. It found that it is possible to have filed a patent application previously (JP-A-6-66800). However, in this method, when an anionic separation-enhancing substance is bound to the complex to try to separate the complex, the problems described above may occur. As a result of further diligent research by the present inventors to find an anionic separation-enhancing substance that does not cause such problems, an anionic separation-enhancing substance comprising a polypeptide having at least three acid residues derived from a strong acid. As a result, it has been found that the problems described above can be solved, and the present invention has been completed.

  The polypeptide of the present invention may be any polypeptide having 3 to 30 acid residues derived from a strong acid, and the type of amino acid residues constituting the polypeptide is not particularly limited. The total number of groups is usually selected appropriately from the range of about 3 to 30, preferably about 5 to 15. Examples of strong acids include acids having a pKa of 3 or less, such as inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid.

  The acid residue derived from a strong acid in the present invention means an acid residue derived from the strong acid. Further, the acid residue in the present invention means a residue obtained by removing a hydrogen atom from the strong acid. An amino acid residue refers to an amino acid having an N-terminal hydrogen atom and a C-terminal hydroxyl group removed.

Specific examples of the polypeptide having 3 to 30 acid residues derived from a strong acid and having a total number of amino acid residues of 3 to 30 according to the present invention include, for example, the following general formula [1]:
^{A- (R 4) j-B} [1]
(In the formula, j represents an integer of 4 to 20. 4-20 of j R ⁴ are sulfated tyrosine residues, and the remaining R ⁴ may be the same or different, and is derived from a strong acid. Represents an amino acid residue in which no acid residue is introduced, and a protective group may be introduced into the reactive group of the side chain of each amino acid residue, A is derived from a hydrogen atom, a sulfated tyrosine residue or a strong acid Represents a protecting group for the N-terminal amino group of an amino acid in which no acid residue is introduced, and B represents a protecting group for a hydroxyl group or a C-terminal carboxyl group of an amino acid).
The following general formula [2]
^{A- (R 5) k-B} [2]
(In the formula, R ⁵ represents an amino acid residue into which an acid residue derived from a strong acid is introduced, and may be the same or different. K represents an integer of 4 to 20, A represents a hydrogen atom, an acid derived from a strong acid. Represents a protecting group for the N-terminal amino group of the residue or amino acid, and B represents a protecting group for the hydroxyl group or the C-terminal carboxyl group of the amino acid, provided that 4 to 20 of k R ⁵ are sulfated tyrosine residues. Group),
The following general formula [3]
^{A- (R 5) j- (R} 6) l-B [3]
(In the formula, R ⁵ represents a sulfated tyrosine residue, j represents an integer of 4 to 20. R ⁶ represents an amino acid residue into which an acid residue derived from a strong acid has not been introduced. Also, (j + 1) Represents a protecting group for the N-terminal amino group of an amino acid into which a hydrogen atom, a sulfated tyrosine residue or an acid residue derived from a strong acid has not been introduced, and B represents a hydroxyl group or a C-terminal carboxyl of an amino acid. Represents a protecting group.)
The thing etc. which are shown by are mentioned.

  In the polypeptide of the present invention, the acid residue as described above is usually introduced into a reactive group of the polypeptide. Examples of the reactive group include a free hydroxyl group, an amino group, and an imino group in the polypeptide. And thiol groups. The amino group of the main chain of the polypeptide (the amino group at the N-terminal of the polypeptide) can also be used as the reactive group. However, when the polypeptide is used as a separation enhancing substance, It is preferable not to introduce the acid residue. That is, if the amino group at the N-terminal of the polypeptide is used for binding the polypeptide and the binding substance, the binding mode between the polypeptide and the binding substance can be made constant to some extent. This is because the target measurement can be carried out with higher accuracy if the binding ability substance to which the obtained polypeptide (that is, the separation enhancing substance) is bound is used for the measurement of the measurement target substance in the biological sample.

Examples of the polypeptide of the present invention include the following general formula [I]:
A- (R) m-B [I]
(In the formula, m represents an integer of 3 or more, preferably 3 to 30, more preferably 3 to 20, still more preferably 4 to 20, still more preferably 5 to 15, and at least of m Rs. 3 are amino acid residues into which an acid residue derived from a strong acid is introduced, and the remaining R is an amino acid residue into which an acid residue derived from a strong acid is introduced or an amino acid residue into which the acid residue derived from the strong acid is not introduced The amino acid residue in which a strong acid-derived acid residue represented as R is introduced and the type of amino acid residue in which the strong acid-derived acid residue is not introduced are the same. They may be different, a protective group or the like may be introduced into the reactive group of the side chain, and the binding order thereof is arbitrary, A is a hydrogen atom, an acid residue derived from a strong acid or the N-terminal of an amino acid Represents a protective group for an amino group, and B represents a hydroxyl group or a C-terminal carboxyl group of an amino acid. It represents a protecting group of the Le group.)
The following general formula [II]
A- (R ¹ ) m′-B [II]
[Wherein, R ¹ represents an amino acid residue into which an acid residue derived from a strong acid is introduced, and m ′ represents an integer of 3 or more, preferably 3 to 30, more preferably 3 to 20, still more preferably 4 -20, even more preferably 5-15, and A and B are the same as above. The types of m R1 (amino acid residues into which a strong acid-derived acid residue is introduced) may be the same or different, and their binding order is arbitrary. ],
The following general formula [III]
^{A- (R 1) m '-} (R 2) n-B [III]
[Wherein R ² represents an amino acid residue having no acid residue derived from a strong acid, n represents a natural number, and R ¹ , m ′, A and B are the same as above. The types of n R ² (amino acid residues) may be the same or different, and the binding order is arbitrary. Further, the bonding order of m R ¹ and n R ² is also arbitrary. Further, (m ′ + n) is usually 4 to 31, preferably 4 to 21, more preferably 5 to 21, and still more preferably 6 to 16. ],
The thing etc. which are shown by are mentioned.

  When the polypeptide of the present invention (including those represented by the above general formula) is used as a separation-enhancing substance when measuring a substance to be measured in a biological sample, it is derived from a strong acid in the polypeptide. The acid residue is preferably an acid residue derived from a polyvalent strong acid such as sulfuric acid or phosphoric acid. In addition, since there is a possibility that phosphatase coexists in a biological sample, a sulfate residue is desirable particularly when a polypeptide is used as a separation enhancing substance. In addition, it is sufficient that at least three acid residues are present in the polypeptide of the present invention. However, when the number of acid residues is too large, it is difficult to use as a separation improving substance (for example, anion exchange method). In this case, the number of acid residues when used as a separation-enhancing substance is usually 3 to 30, preferably 3 to 20, and more preferably Is appropriately selected from the range of 4 to 20, more preferably 5 to 15, and the total number of amino acid residues is usually about 3 to 30, preferably about 3 to 20, considering the ease of synthesis and the like. More preferably, it is appropriately selected from the range of 4 to 20, more preferably about 5 to 15. If the number of acid residues is less than 3, the separation peak based on the separation-enhancing substance overlaps with the elution peak derived from the serum component, resulting in a problem that the measurement (analysis) accuracy is lowered. In addition, in order to eliminate the overlap between the separation peak based on the separation-enhancing substance and the elution peak derived from a component in a biological sample such as serum, and to increase the measurement accuracy, the number of acid residues is 4 or more, Preferably it is 5 or more. Furthermore, when introducing a sulfuric acid residue as an acid residue derived from a strong acid, considering the stability in an aqueous solution after the introduction of the sulfuric acid residue, it should be introduced using the phenolic hydroxyl group of the tyrosine residue. Is desirable.

  In the polypeptide of the present invention, the amino acid residue into which an acid residue derived from a strong acid is introduced is an amino acid residue having a free reactive group, and an acidic residue derived from a strong acid is present in the reactive group. There is no particular limitation as long as it is introduced. More specifically, amino acid residues having a free hydroxyl group such as tyrosine, serine, threonine, for example, amino acid residues having a free amino group such as lysine, arginine, such as histidine, tryptophan, proline, oxyproline, etc. Amino acid residue having a free imino group, for example, an amino acid residue having a free thiol group such as cysteine, which is derived from a strong acid derived from a free reactive group such as a hydroxyl group, amino group, imino group or thiol group And those having an acid residue introduced therein. In view of ease of introduction of acid residues derived from strong acids, amino acid residues such as serine, threonine, tyrosine and the like are preferable. The amino acid residue having no acid residue derived from a strong acid in the polypeptide of the present invention is not particularly limited as long as it is generally recognized as an amino acid residue in the field of peptide chemistry. , Amino acid residues such as glycine and β-alanine are preferred.

  The polypeptide of the present invention comprises (a) a method for introducing an acid residue derived from a strong acid into at least three free active reactive groups of a polypeptide having an appropriate number of free active reactive groups (J. Chem. Soc Perkin Trans I, (1990), 1739-1744, etc.), (b) A polypeptide having at least three acid residues derived from a strong acid is synthesized using an amino acid into which an acid residue derived from a strong acid has been introduced as a synthetic raw material. And the like (Chem. Pharm. Bull., 41 (2), (1993), 376-380, etc.), etc. In view of the yield of the target polypeptide, the method (a) is preferred among the above methods (especially when the number of amino acid residues increases). However, since the method (b) does not require a sulfation reaction, a functional group capable of binding to a binding ability substance such as an antibody, such as a maleimidocaproic acid residue, at the N-terminus of the obtained polypeptide. Since the group can be directly introduced, there is an advantage that the obtained polypeptide can be immediately bound to the binding substance.

  As a method for introducing an acid residue derived from a strong acid into a free active reactive group of a polypeptide or amino acid, for example, an acid residue derived from a strong acid in a free reactive active group such as a hydroxyl group, an amino group, an imino group, or a thiol group Any method can be used as long as it can be introduced.

More specifically, for example, an amino acid having a free hydroxyl group such as tyrosine, serine, threonine (or a polypeptide having a residue of these amino acids) and 1 to 20 with respect to the free hydroxyl group of the amino acid (residue) A double equivalent of a sulfating agent such as HSO ₃ · Cl, HSO ₃ · dimethylformamide, HSO ₃ · pyridine, or a phosphorylating agent such as phosphoryl halide such as phosphoryl chloride or phosphorus trichloride, for example, dimethylformamide (DMF ), In a solvent (anhydrous) such as dimethyl sulfoxide (DMSO), for example, by reacting an alkaline substance such as pyridine, triethylamine, NaH or the like as a catalyst at 0 to 40 ° C. for 1 to 24 hours, the hydroxyl group of the amino acid residue is derived from a strong acid For example, amino acids having a free amino group such as lysine and arginine (or polypeptides having residues of these amino acids) Is, for example, an amino acid having a free imino group (or a polypeptide having residues of these amino acids) such as histidine, tryptophan, proline, oxyproline, and the free amino group (or / and imino group) of the amino acid (residue). 1) to 20 times equivalent of a sulfating agent such as HSO ₃ · Cl, HSO ₃ · dimethylformamide, HSO ₃ · pyridine or a phosphorylating agent such as phosphoryl chloride or phosphorus trichloride, for example, dimethylformamide (DMF), in a solvent (anhydrous) such as dimethyl sulfoxide (DMSO), for example, by reacting at 0 to 40 ° C. for 1 to 24 hours using an alkaline substance such as pyridine, triethylamine or NaH as a catalyst, the amino group of the amino acid residue Examples thereof include a method of introducing an acid residue derived from a strong acid into (or / and an imino group). In view of the ease of synthesis and the stability of the introduced acid residue in an aqueous solution and the stability of the enzyme such as phosphatase present in a biological sample, the acid residue derived from the strong acid according to the present invention Is more preferably a sulfuric acid residue.

  In addition, as a raw material for the polypeptide of the present invention, for example, a polypeptide having a free reactive group such as a hydroxyl group, an amino group, an imino group, or a thiol group, a commercially available product may be used, or in the field of peptide chemistry. Commonly used conventional methods such as the active ester method, mixed acid anhydride method, azide method, etc. ("Peptide Synthesis Fundamentals and Experiments", pages 89-142, written by Nobuo Izumiya et al., January 20, 1985, Maruzen ( May be synthesized by the following method. In synthesizing such a polypeptide, the protecting group for the amino group at the N-terminus is preferably one that is stable in the subsequent sulfation step (a protecting group that is removed under acidic conditions is inappropriate), for example, under alkaline conditions. Preferable examples include urethane-type protecting groups such as a detached 9-fluorenylmethoxycarbonyl (Fmoc) group. The protecting group for the reactive group on the side chain of the amino acid (residue) may be appropriately selected in consideration of, for example, polypeptide synthesis conditions, conditions for introducing acid residues derived from strong acids, and the like. Group, benzyl group and the like are preferred.

  The binding ability substance according to the present invention is not particularly limited as long as it is a substance capable of binding to the measurement target substance in the biological sample to be measured. Specifically, for example, antibodies having an ability to bind to a substance to be measured in a biological sample, antigens such as concanavalin A, lentil lectin, kidney bean lectin, duck lectin, wheat germ lectin and other lectins such as amylase and creatine Preferred examples include inhibitors for enzymes such as kinase (CK) and glutamic acid oxaloacetate transaminase (GOT), and polynucleotides complementary to nucleic acid chains as substances to be measured, such as receptors for thyroid stimulating hormone, acetylcholine, glutamic acid and the like.

  The method for preparing a conjugate of the binding ability substance according to the present invention and the polypeptide of the present invention (hereinafter abbreviated as the conjugate of the present invention) comprises a specific reaction group of the binding ability substance and a specific reaction of the polypeptide. A method of binding to a group, a method of substituting a specific reactive group of the binding ability substance with the polypeptide, a substance capable of binding to the binding ability substance (eg, antibody, lectin, antigen, inhibitor, DNA, etc.) And a method of binding the binding substance to the polypeptide. Specifically, for example, 1) a per se known labeling substance and an antibody generally used in a known enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), etc. Binding method (for example, Medical Laboratory, Vol. 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawaio, 1st edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, School of Medicine, 1982, etc.) 2) Modification and binding methods of substances known per se (for example, chemical modification of proteins <top> <bottom> , Shinzo Sugaya, Kensuke Shimura, Michinori Nakamura, Katsunori Funatsu, 1st edition, Society Publishing Center, 1981; Polyethylene glycol modified protein, Yuji Inada et al., Biochemistry, Vol. 62, No. 11, P1351- 1362, Japan Biochemical Society, 1990; DNA PROBES, George HK and Mark MM STOC KTON PRESS, 1989, etc.) are all listed without exception, and may be carried out according to these methods.

  Specific examples of the method for binding the specific reactive group of the binding substance to the specific reactive group of the polypeptide include, for example, first, a maleimide group bound to the N-terminus of the polypeptide of the present invention via a spacer. Compound obtained by reacting compound (hereinafter abbreviated as maleimide compound of the present invention) with SH group of Fab ′ of antibody prepared by a conventional method and binding ability substance (antibody) of the present invention The method of preparing is mentioned.

The maleimide compound of the present invention used here is, for example, a compound represented by the following general formulas [IV] to [VI].
Formula [IV]
D-E- (R) m-B [IV]
(In the formula, D represents a maleimide group, E represents a spacer, and R, m and B are the same as above.)
General formula [V]
^{D-E- (R 1) m'} -B [V]
(In the formula, D, E, R ¹ , m ′ and B are the same as above.)
General formula [VI]
^{D-E- (R 1) m} '- (R 2) n-B [VI]
(In the formula, D, E, R ¹ , R ² , m ′, n and B are the same as above.)
Here, as the spacer represented by E in these general formulas, the property that the N-terminus and the maleimide group of the polypeptide of the present invention can be bonded to both ends, and the maleimide compound of the present invention are included in the present invention. It is not particularly limited as long as it has a property that does not prevent binding with such a binding substance (antibody). For example, a group represented by —R ⁴ —CO— (wherein R ⁴ is a divalent carbon atom) Represents a hydrogen group.) More specifically, for example, the following general formulas [VII] to [IX]
_{- (CH 2) p-CO-} [VII]
(In the formula, p represents an integer of 1 to 10.)

（式中、ｑ及びｒは０又は１〜５の整数を表す。）

(In the formula, q and r represent 0 or an integer of 1 to 5.)

（式中、ｑ及びｒは前記に同じ。）
で示される基が挙げられる。

(Wherein q and r are the same as above)
The group shown by these is mentioned.

  In the general formulas [VII] to [IX], a substituent may be introduced into the alkylene group, phenylene group and cyclohexylene group. Examples of such a substituent include a methyl group, an ethyl group, and a propyl group. A lower alkyl group having 1 to 4 carbon atoms such as a group or a butyl group (which may be linear or branched), for example, a hydrophilic group such as a hydroxyl group, a carboxyl group, a sulfo group or an amino group Etc.

  The maleimide compound of the present invention can be easily obtained, for example, by the following method.

  That is, the polypeptide of the present invention and 1 to 100 times mol, preferably 1 to 50 times mol, more preferably 1.2 to 10 times mol of a bivalent cross-linking agent with respect to the N-terminal amino group of the polypeptide ( A compound having a maleimide group at one end and a group capable of reacting with an amino group on the other end), and if necessary, 2 to 5 moles of a reaction accelerator with respect to the amino group, pH 6-9 After reacting at 4 to 37 ° C. for 0.2 to 10 hours in an appropriate buffer or an appropriate organic solvent, the maleimide compound of the present invention can be obtained by purifying the reaction solution by appropriate column chromatography.

  The divalent crosslinking agent used in the above reaction is not particularly limited as long as it can finally introduce a maleimide group into the N-terminus of the polypeptide of the present invention. For example, a maleimide group and a succinimide group are used. More specifically, succinimidyl-N-4-maleimidobutyrate, succinimidyl-N-6-maleimidocaproylate, succinimidyl-N-8-maleimidocaprylate, succinimidyl-N-11- Maleimidoundecanoate, succinimidyl-N-4- (2-maleimidoethoxy) succinilate, sulfosuccinimidyl-N-4-maleimidobutyrate, sulfosuccinimidyl-N-6-maleimidocaproylate, sulfosk Cyimidyl-N-8-maleimidocaprylate, succinimidyl-N-11-maleimide undecanoate, succinimidyl-4- (N-maleimidomethy ) Cyclohexane-1-carboxylate, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, succimidyl-m-maleimidobenzoate, sulfosuccinimidyl-m-maleimidobenzoate, succinimidyl-4 Preferred examples include-(p-maleimidophenyl) butyrate and sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate.

  Examples of the reaction accelerator include bases such as triethylamine and pyridine.

  Examples of the pH 6-9 buffer used as a solvent during the reaction include a phosphate buffer having such a pH and a tris (hydroxymethyl) aminomethane buffer such as N, N-bis (2-hydroxyethyl). Good buffers such as glycine (Bicine) buffer, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, etc. Examples of the organic solvent include dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

  Examples of the chromatography used for purifying the maleimide compound of the present invention after completion of the reaction include gel column chromatography, silica gel column chromatography, octadecyl silica gel (ODS) column liquid chromatography and the like.

  In addition, as a solvent at the time of reaction, the organic solvent is more preferable. That is, the decomposition of the maleimide group is less in the organic solvent, and as a result, the maleimide compound of the present invention can be obtained in a high yield.

  Further, purification by ODS column liquid chromatography is preferable because an excess of the divalent crosslinking agent and the maleimide compound of the present invention can be more clearly separated.

  The maleimide compound of the present invention obtained as described above can be stably stored, for example, by treatment by concentration, drying, lyophilization, etc., so that the polypeptide of the present invention can be appropriately bound according to the present invention. It is extremely useful as an intermediate for binding to an active substance (antibody).

  In addition, as a method for obtaining a conjugate of the polypeptide of the present invention and the binding ability substance by binding the maleimide compound of the present invention and the binding ability substance of the present invention, for example, the following may be performed.

  That is, a solution (for example, a buffer solution usually used in the field of immunological assay) in which the maleimide compound of the present invention and the SH group of Fab ′ of an antibody prepared by a conventional method are not lost. Etc.), the conjugate of the polypeptide of the present invention and the binding ability substance (antibody) can be prepared by reacting at 4 to 37 ° C. for 1 to 16 hours.

  Examples of the method for binding the amino group of the polypeptide of the present invention and the amino group of the binding ability substance include the per se known glutaraldehyde method, and the like. Examples of the method for binding the sugar chain include periodic acid known per se, in which the sugar chain is treated with periodic acid to form an aldehyde group and the aldehyde group is bound to the amino group of the polypeptide of the present invention. Law.

  In addition, when the polypeptide of the present invention is bound to the binding ability substance, it may be performed using the reactive group on the side chain of the polypeptide, but it is preferable to use the N-terminal amino group. .

  That is, since there is usually only one amino group at the N-terminus, when this is used to bind to the binding ability substance, the binding molar ratio of the polypeptide of the present invention to the binding ability substance is 1: 1. For this reason, when the substance to be measured is separated using the combined substance, it is separated as one peak, so that the accuracy of the target measurement can be further improved. The maleimide compound of the present invention is a useful substance for preparing such a conjugate.

  Further, when the conjugate of the present invention is prepared by utilizing the N-terminal amino group, it is preferable not to introduce a strong acid-derived acid residue into the N-terminal amino acid residue of the polypeptide of the present invention. That is, when the polypeptide of the present invention is bound to the binding ability substance, the yield of the target conjugate is increased.

  As a method for measuring a biological component using the conjugate of the present invention (hereinafter abbreviated as “measurement method of the present invention”), any method can be used as long as the target measurement can be performed using the conjugate of the present invention. Although not particularly limited, specific examples include the following methods.

(1) Measurement method for the amount of target substance in biological samples using non-competitive reactions (Measurement method (1))
First, a sample derived from a living body containing a measurement target substance, a binding ability substance labeled with a detection substance (hereinafter abbreviated as “label binding ability substance”), and the conjugate of the present invention (in the measurement target substance, The binding site of the label-binding substance is different from the binding site of the conjugate of the present invention), if necessary, added to an appropriate buffer, mixed and reacted, and the substance to be measured in the biological sample After forming a complex in which the binding substance of the present invention and the conjugate of the present invention are bound, the complex and the free label binding substance are subjected to a method using a negative charge, for example, anion exchange chromatography. Separation using an anion-exchange mechanical device (anion exchange method) such as an HPLC apparatus equipped with a column packed with packing material, an anion exchange membrane, an anion exchange reaction tube, etc. . Next, the amount of the detection substance contained in the separated complex is determined by a measurement method according to the property of the detection substance. Separately, a sample having a known concentration of the substance to be measured is measured by the same method, and a calibration curve representing the relationship between the amount of the substance to be measured in the biological sample and the amount of the detection substance in the complex is prepared. When the amount of the measurement target substance in the biological sample corresponding to the amount of the detection substance in the complex is obtained, the amount of the measurement target substance in the biological sample is obtained.

  In the above reaction, the concentration of the label-binding ability substance and the conjugate of the present invention used when forming the complex varies depending on how much the calibration limit of the measurement target substance in the biological sample is set. Usually, in the reaction solution, the concentration is higher than the concentration capable of binding to all the substances to be measured in the biological sample corresponding to the set calibration limit concentration, preferably twice the concentration, more preferably 5 times the concentration. As described above, it is desirable that 10 times or more is present in the reaction solution.

  If the substance to be measured itself is a substance that can be measured (detected) by some method, the above reaction is performed without using a label binding substance, and the properties of the substance to be measured in the resulting complex are determined. Similarly, the amount of the substance to be measured in the biological sample in the sample can also be obtained by obtaining with a corresponding measurement method.

(2) Measurement method for the amount of target substance in biological samples using competitive reaction (Measurement method (2))
First, a sample derived from a living body containing a measurement target substance, a measurement target substance labeled with a detection substance (hereinafter abbreviated as a labeled measurement substance), and the conjugate of the present invention are added to an appropriate buffer solution if necessary. , Mixed and reacted to form a complex of the measurement target substance in the biological sample and the conjugate of the present invention, and a complex of the label measurement substance and the conjugate of the present invention (hereinafter abbreviated as a labeled complex). After forming the label complex, the label complex and the free label measurement substance are subjected to a method using a negative charge, for example, an HPLC apparatus equipped with a column packed with a packing material for anion exchange chromatography, an anion exchange membrane, an anion. Separation is performed using a mechanical instrument having an anion exchange action such as a reaction tube having an ion exchange action. Next, the amount of the detection substance contained in the separated label complex is determined by a measurement method according to the property of the detection substance. Separately, using a sample with a known concentration of the substance to be measured, the same method is used to create a calibration curve indicating the relationship between the amount of the substance to be measured in the biological sample and the amount of the detection substance in the labeled complex. If the amount of the measurement target substance in the biological sample corresponding to the amount of the detection substance in the labeled complex is determined using, the amount of the measurement target substance in the biological sample in the sample can be determined.

  In the above reaction, the concentration of the conjugate of the present invention and the concentration of the labeled measurement substance used to form the labeled complex are set to the calibration limit and measurement sensitivity of the measurement target substance in the biological sample. It may be set as appropriate depending on whether or not it is performed, and is not particularly limited. However, it goes without saying that the use concentration of the labeling measurement substance must be set to be higher than the concentration at which it can bind to all of the conjugates of the present invention present in the reaction solution.

(3) Measurement method (measurement method (3)) in which the substance to be measured in a biological sample is the two or more substances having the same action and the same detectable chemical properties
First, a biological sample containing a substance to be measured and a polypeptide that specifically binds to at least one of the substances to be measured in the biological sample but does not bind to at least one of them, and the polypeptide of the present invention A substance bound to (hereinafter abbreviated as “the conjugate A of the present invention”) is added to an appropriate buffer, mixed, and reacted if necessary, and the substance to be measured in a specific biological sample is mixed with After the complex with the conjugate A of the present invention is formed, the complex and the substance to be measured in the free biological sample are filled with a method using a negative charge, for example, a filler for anion exchange chromatography. Separation is performed using an HPLC apparatus equipped with a column, an anion exchange membrane, a mechanical instrument having an anion exchange action such as a reaction tube having an anion exchange action. Next, the amount of the measurement target substance in the biological sample contained in the separated complex and / or the amount of the measurement target substance in the free biological sample is measured by a measurement method according to the property of the measurement target substance in the biological sample. Ask. Separately, for example, measurement is performed by a similar method using a sample whose measurement target substance concentration is known, and the relationship between the amount of the measurement target substance in the biological sample and the measurement value based on the detectable property of the measurement target substance in the complex is determined. If a calibration curve to be expressed is created and the amount of the measurement target substance in the biological sample corresponding to the measurement value is obtained, the amount of any of the measurement target substances in the biological sample in the sample can be obtained.

  In the above reaction, the concentration used of the conjugate A of the present invention when forming a complex may be set as appropriate depending on how much the calibration limit and measurement sensitivity of the substance to be measured are set, and is particularly limited. Not.

(4) Measuring method (measurement method (4)) in which the substance to be measured in a biological sample is two or more substances having the same action or two or more substances having similar structures but different actions
First, a sample derived from a living body containing a substance to be measured and a substance capable of binding to all of the substances to be measured in the biological sample (hereinafter abbreviated as “binding substance A”) labeled with a detection substance (hereinafter, referred to as “binding substance A”). Abbreviated as “label binding substance A”) and a substance capable of binding to a specific substance to be measured in a biological sample and bound with the polypeptide of the present invention (hereinafter referred to as “binding substance B of the present invention”). (Abbreviated) is added to an appropriate buffer, mixed, and reacted if necessary, and a complex of the substance to be measured and the labeled binding substance A in the biological sample (hereinafter abbreviated as complex A). ) And a specific measurement target substance in a biological sample, a labeled binding ability substance A, and a conjugate B of the present invention (hereinafter abbreviated as complex B), and then complex A, complex A method using a negative charge between the body B and the free labeled binding substance A, for example, HPLC system equipped with a column filled with an anion-exchange chromatographic packing material, an anion exchange membrane, is separated using mechanical instruments or the like having an anion-exchange action of such reaction tube having an anion-exchange effect. Next, the amount of the detection substance contained in the separated complex A and / or the amount of the detection substance contained in the separated complex B is determined by a measuring method according to the property of the detection substance. Separately, for example, a sample having a known concentration of the substance to be measured is used in the same manner, and the amount of the substance to be measured in the biological sample and the amount of the detection substance contained in the complex A or / and contained in the complex B Create a calibration curve that represents the relationship with the amount of the detected substance, and use this to determine the amount of the substance to be measured in the biological sample corresponding to the amount of the detected substance in the complex. The amount of any of the substances to be measured is determined.

  In the case where the binding ability substance A itself is a substance that can be measured (detected) by any method, the above reaction is carried out using the binding ability substance A that is not labeled with the detection substance, The amount of the substance to be measured in the biological sample in the sample can also be obtained in the same manner by obtaining the amount of the binding ability substance A by the measuring method according to the property of the binding ability substance A.

(5) Measuring method (measurement method (5)) in which the measurement target substance in the biological sample is two substances having the same action or two substances having a similar structure but different actions
First, a biological sample containing a measurement target substance, a label binding substance A, a substance having binding ability to the measurement target substance 1 in the biological sample and bound to the polypeptide 1 of the present invention (hereinafter referred to as “the present invention”). And a substance having a binding ability to the substance to be measured 2 in the biological sample and bound with the polypeptide 2 of the present invention (hereinafter abbreviated as “the combined substance D of the present invention”). ) Is added to a suitable buffer, mixed, and reacted if necessary, and a complex of the measurement target substance 1 in the biological sample, the conjugate C of the present invention, and the label binding substance A (hereinafter, complex) And a complex of the substance to be measured 2 in the biological sample, the conjugate D of the present invention, and the label binding substance A (hereinafter abbreviated as complex D), Complex C, Complex D, and free labeled binding substance A are combined with polypeptides 1 and 2. Methods using negative charges based on differences in properties, for example, an anion exchange such as an HPLC apparatus equipped with a column packed with a packing material for anion exchange chromatography, an anion exchange membrane, a reaction tube having an anion exchange action, etc. Separation is performed using a machine tool or the like having an action. Next, the amount of the detection substance contained in the separated complex C and / or the amount of the detection substance contained in the complex D is determined by a measurement method according to the property of the detection substance. Separately, for example, a sample having a known concentration of the substance to be measured is used and measured in the same manner, and the amount of the substance to be measured 1 (or 2) in the biological sample and the amount or / and the amount of the detection substance contained in the complex C are measured. A calibration curve representing the relationship with the amount of the detection substance contained in the body D is created, and using this, the amount of the measurement target substance in the biological sample corresponding to the amount of the detection substance in the complex is obtained. The amount of any substance to be measured in the biological sample is determined.

  In the case where the binding ability substance A itself is a substance that can be measured (detected) by any method, the above reaction is carried out using the binding ability substance A that is not labeled with the detection substance, The amount of the substance to be measured in the biological sample in the sample can also be obtained in the same manner by obtaining the amount of the binding ability substance A by the measuring method according to the property of the binding ability substance A.

(6) A measuring method in which a substance to be measured in a biological sample is two or more glycoproteins having different sugar chain structures but substantially the same protein structure (measuring method (6))
First, a biological sample containing a glycoprotein to be measured, a lectin that recognizes at least one specific sugar chain structure of the glycoprotein to be measured, and a property that binds to all the glycoproteins to be measured, but the lectin binds A conjugate of the antibody of the present invention and the polypeptide of the present invention (hereinafter abbreviated as “polypeptide-bound antibody”), a saccharide to be assayed to which the lectin is bound. Reaction with an antibody (hereinafter abbreviated as “labeled anti-glycoprotein antibody”) having a property capable of binding to all glycoproteins to be measured, including proteins, and a detection substance bound thereto, After forming a complex with a labeled anti-glycoprotein antibody and a complex of a glycoprotein, a polypeptide-bound antibody and a labeled anti-glycoprotein antibody, these complex and the free labeled anti-glycoprotein antibody are negative. For example, an HPLC apparatus equipped with a column packed with a packing material for anion exchange chromatography, an anion exchange membrane, a mechanical instrument having an anion exchange action such as a reaction tube having an anion exchange action, etc. To separate. Next, the amount of the detection substance contained in the separated complex is determined by a measurement method according to the property of the detection substance. Separately, for example, a sample having a known glycoprotein concentration to be measured is measured by the same method, and a calibration indicating the relationship between the amount of the glycoprotein to be measured in the biological sample and the amount of the detection substance contained in these complexes. By creating a line and using this to determine the amount of glycoprotein to be measured in the biological sample corresponding to the amount of the detection substance in these complexes, any of the glycoproteins to be measured in the biological sample in the sample can be obtained. Is required.

  Needless to say, when the glycoprotein to be measured is a substance that can be measured (detected) by any method, the above measurement can be carried out without using a labeled anti-glycoprotein antibody.

The measurement target substance in the biological sample that can be measured by the measurement methods (1) and (2) using the present invention includes i) a strong complex that interacts strongly with the measurement target substance in the biological sample. There is a substance that can form a substance, and the substance itself can be measured (detected) by some method, or can be labeled with some detectable substance, or ii) the substance to be measured itself is detected in any way Any substance that can be labeled with a substance and has a substance that can interact strongly with a measurement target substance in a biological sample and can form a strong labeled complex is not particularly limited. Examples include proteins, peptides, nucleic acids, sugar chains, lipids, hormones, drugs, etc. contained in biological samples such as serum, blood, plasma, urine, and other biological samples such as lymphocytes, blood cells, and various cells. But Table specific be mentioned as. More specifically, for example, cancer markers such as α-fetoprotein (AFP), CA19-9, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), a substance having a special sugar chain produced by cancer cells, For example, serum proteins such as immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), β ₂ -microglobulin, albumin, ferritin, such as C-peptide, peptides such as angiotensin I, such as amylase, Enzymes such as alkaline phosphatase and γ-glutamyltransferase (γ-GTP), for example, antiviral antibodies against viruses of clinical interest such as Rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, and pathogens such as viruses Deoxyribonucleic acid (DNA) and ribo Acids (RNA) or single-stranded polynucleotides constituting these nucleic acids, antigenic substances derived from pathogens such as viruses, such as antibodies that react with allergens such as Japanese cedar and other pollen and indoor dust, such as lipoproteins Lipids such as trypsin, plasmin, and serine proteases such as insulin, human chorionic gonadotropin (hCG), thyroxine (T ₄ ), triiodothyronine (T ₃ ), prolactin, thyroid stimulating hormone (TSH), etc. Examples include hormones such as receptors for drugs such as digoxin, phenytoin, morphine, and nicotine, such as thyroid stimulating hormone, acetylcholine, and glutamic acid.

According to the measurement method (1) using the present invention, the binding ability substance to the measurement target substance in the biological sample used for preparing the label binding ability substance is a strong mutual interaction with the measurement target substance in these biological samples. A substance that interacts and forms a strong complex, which can be measured (detected) by some method, if necessary, or can be labeled with any detectable substance (detectable) ( This is not particularly limited as long as the substance to be measured in the biological sample itself can be labeled with any detection substance.), For example, a substance having antigenicity (including a hapten) For example, concanavalin A, lentil lectin, kidney bean lectin, and datsu. Lectins, lectin such as wheat germ lectin, for example alpha ₁ to trypsin - antitrypsin, ₂ alpha for plasmin - macroglobulin, alpha for serine protease ₂ - macroglobulin and alpha ₁ - inhibitors, for specific enzymes antichymotrypsin like, Examples include a polynucleotide chain complementary to a single-stranded polynucleotide that is a substance to be measured in a biological sample, a receptor for a hormone, and the like.

According to the measuring methods (1) and (2) using the present invention, the substance capable of binding to the measurement target substance in the biological sample used for preparing the conjugate of the present invention is the measurement target in these biological samples. Any substance that has a strong interaction with a substance (or a substance to be measured, or a complex of a substance to be measured and a label binding substance) and forms a strong complex can be used without particular limitation. For example, antibodies against antigenic substances (including haptens), antigens against antibodies, concanavalin A, lentil lectin, kidney bean lectin, duck lectin, wheat germ lectin, etc. Lectins such as α ₁ -antitrypsin against trypsin, α ₂ -macroglobulin against plasmin, serine protease Inhibitors for specific enzymes such as α ₂ -macroglobulin, polynucleotide strands complementary to the single-stranded polynucleotides to be measured in biological samples, etc. (however, they are used to prepare labeled binding substances) The binding site is different from that of the binding substance.

  The measurement target substance in the biological sample that can be measured by the measurement method (3) using the present invention can be measured (detected) by any method, and at least one of the measurement target substances in the biological sample. As long as there is a substance having a property of exerting a strong interaction with each other and forming a strong complex but not binding to at least one of them, there is no particular limitation. Typical examples include biological fluids such as blood, plasma, and urine, enzymes included in biological samples such as lymphocytes, blood cells, and various cells. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate Examples thereof include enzymes such as pyruvate transaminase (GPT), renin, protein kinase (PK), and tyrosine kinase.

  The binding substance for a specific measurement target substance in a biological sample used for preparing the conjugate A of the present invention according to the measurement method (3) using the present invention includes the measurement target substance in the biological sample. Although it does not specifically limit as long as it is a substance which exerts a strong interaction with each other and forms a strong complex, but does not bind to at least one substance to be measured in a biological sample. Antibodies having specific properties (including haptens) or antibodies to specific antigenic determinant sites, and those having binding ability to sugar chains of specific structures, such as concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, yellow chawantake Lectins such as lectin, castor lectin, peanut lectin, wheat germ lectin, such as amylase, clear Nkinaze (CK), inhibitors, and the like for enzymes such as glutamic oxaloacetic transaminase (GOT).

As a measurement target substance in a biological sample that can be measured by the measurement method (4) using the present invention, (a) it binds to all of the measurement target substance and has a property that itself can be detected by some method. Or a substance that can be labeled with a detection substance, and (b) at least one substance to be measured in a biological sample exerts a strong interaction with each other to form a strong complex. Examples include substances that form, but do not bind to at least one of them, and are not particularly limited. Examples include biological fluids such as serum, blood, plasma, and urine, lymphocytes, blood cells, Typical examples include enzymes, physiologically active substances, cancer-related antigens, substances having sugar chains, and the like contained in biological samples such as cells. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate Physiological activities of enzymes such as pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase, such as steroid hormone, human chorionic gonadotropin (hCG), prolactin, thyroid stimulating hormone (TSH), luteinizing hormone (LH) Substances such as cancer-related antigens such as prostate-specific antigen (PSA), α ₂ -macroglobulin, carcinoembryonic antigen (CEA), α-fetoprotein and the like are preferable.

  According to the measurement method (4) using the present invention, the binding ability substance for the measurement target substance in the biological sample used for preparing the label binding ability substance A is bound to all the measurement target substances in the biological sample. The substance is not particularly limited as long as it is a substance that itself can be detected by some method or can be labeled with a detection substance. Specific examples include, for example, concanavalin A, lentil lectin, and kidney bean having binding ability to specific partial structures of antigenic substances (including haptens) or antibodies to antigen-determining sites and sugar chains of specific structures. Inhibitors for lectins such as lectin, datsura lectin, yellow tea lectin lectin, castor lectin, peanut lectin, wheat germ lectin, or enzymes such as amylase, creatine kinase (CK), glutamic acid oxaloacetate transaminase (GOT), etc. .

  The binding ability substance used for preparing the conjugate B of the present invention according to the measurement method (4) utilizing the present invention and having the ability to bind to a specific measurement target substance in a biological sample includes: A biological sample that does not inhibit a complex formation reaction between a specific substance to be measured in a sample and a free label-binding substance A and a reaction for detecting a detection substance (or binding substance A) in the complex, In particular, a substance having a binding ability to a specific substance to be measured or a substance having a binding ability to the detection substance when the detection substance is bound to the label binding substance A is particularly limited. Be mentioned without being. Specifically, for example, antibodies having binding ability to a specific substance to be measured in a biological sample, for example, lectins such as concanavalin A, lentil lectin, kidney bean lectin, duck lectin, wheat germ lectin (however, binding ability A substance having a binding site different from that of substance A.) or an antibody having binding ability to binding substance A or the detection substance, such as concanavalin A, lentil lectin, kidney bean lectin, duck lectin, wheat germ lectin, etc. Lectins and the like are preferred.

As a measurement target substance in a biological sample that can be measured by the measurement method (5) using the present invention, (a) it binds to all of the measurement target substance and has a property that itself can be detected by some method. Or a substance that can be labeled with a detection substance and (b) a substance to be measured 1 in a biological sample exert a strong interaction with each other to form a strong complex. However, a substance that does not bind to the measurement target substance 2 and (c) a strong interaction with the measurement target substance 2 in the biological sample and form a strong complex. Any substance that does not bind to the substance to be measured 1 can be used without particular limitation. Examples thereof include biological fluids such as serum, blood, plasma, and urine, lymphocytes, blood cells, and various cells. An enzyme contained in a biological sample, Representative examples include physiologically active substances, cancer-related antigens, substances having sugar chains, and the like. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate Physiological activities of enzymes such as pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase, such as steroid hormone, human chorionic gonadotropin (hCG), prolactin, thyroid stimulating hormone (TSH), luteinizing hormone (LH) Substances such as cancer-related antigens such as prostate-specific antigen (PSA), α ₂ -macroglobulin, carcinoembryonic antigen (CEA), α-fetoprotein and the like are preferable.

  According to the measurement method (5) using the present invention, the binding ability substance for the measurement target substance in the biological sample used for preparing the label binding ability substance A is bound to all the measurement target substances in the biological sample. The substance is not particularly limited as long as it is a substance that itself can be detected by any method or can be labeled with a detection substance. Specific examples include, for example, concanavalin A, lentil lectin, and kidney bean having binding ability to specific partial structures of antigenic substances (including haptens) or antibodies to antigen-determining sites and sugar chains of specific structures. Inhibitors for lectins such as lectin, datsura lectin, yellow tea lectin lectin, castor lectin, peanut lectin, wheat germ lectin, or enzymes such as amylase, creatine kinase (CK), glutamic acid oxaloacetate transaminase (GOT), etc. .

  Binding ability having binding ability to a specific measurement target substance 1 or 2 in a biological sample used for preparing the conjugate C or D of the present invention according to the measurement method (5) utilizing the present invention Substances that do not inhibit the complex formation reaction between the measurement target substance 1 or 2 in the biological sample and the free label binding substance A and the reaction for detecting the detection substance (or binding substance A) in the complex And if it is a substance which has binding ability with respect to the specific measuring object substance 1 or 2 in a biological sample, it will be mentioned without being specifically limited. Specifically, for example, antibodies having binding ability to the measurement target substance 1 or 2 in a biological sample, for example, lectins such as concanavalin A, lentil lectin, kidney bean lectin, duck lectin, wheat germ lectin (however, binding Having a binding site different from that of the active substance A.), or antibodies having binding ability to the binding substance A or the detection substance, such as concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, wheat germ lectin Preferred are lectins such as

  The lectin used in the measurement method (6) using the present invention recognizes the target sugar chain structure from various lectins such as concanavalin A, lentil lectin, kidney bean lectin, duck lectin, and wheat germ lectin. What is necessary is just to select and use what has the property which can do, and it does not specifically limit.

  The antibody used for preparing the polypeptide-binding antibody or the labeled anti-glycoprotein antibody used in the measurement method (6) using the present invention is an ordinary method as long as it is an antibody having the above-mentioned properties. According to the method described in, for example, “Introduction to Immunological Experiments, Second Printing, Nao Matsuhashi, Society Publishing Center, 1981”, etc., animals such as horses, cows, sheep, rabbits, goats, rats, mice, etc. In the case of polyclonal antibodies prepared by immunizing the measurement target, or according to the cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975) Monoclonal antibodies produced by hybridomas obtained by fusing cells from spleen cells and spleen cells of mice previously immunized with the substance to be measured may be used, either alone or in combination Etc. I mean.

Specific examples of glycoproteins that can be separately measured by the measurement method (6) using the present invention include biological body fluids such as serum, blood, plasma, and urine, biological samples such as lymphocytes, blood cells, and various cells. A lectin recognizing at least one specific sugar chain structure of the glycoprotein to be measured, a glycoprotein having a sugar structure that is different but having substantially the same protein structure, As long as there is an antibody capable of preparing a peptide-binding antibody, examples thereof include, but are not limited to, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate tiger Enzymes such as suminase (GPT), renin, protein kinase, tyrosine kinase, physiologically active substances such as human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), luteinizing hormone (LH), such as prostate specific antigen ( PSA), α ₂ -macroglobulin, carcinoembryonic antigen (CEA), cancer-related antigens such as α-fetoprotein, and sugar chain antigens such as CA19-9 and CA125 are preferred.

  The concentration of reagents used in the measurement method (6) of the present invention will be described below.

  That is, first, the concentration of lectin used varies depending on the type of lectin used, the nature of the glycoprotein to be measured, etc., but is usually 10 times or more, preferably 100 times or more of the set detection limit concentration of the glycoprotein to be measured. More preferably, it is desirable to coexist at the concentration of 1000 times or more during the reaction.

  The concentration of the polypeptide-bound antibody used varies depending on the detection limit of the glycoprotein to be measured, but considering the difference in binding constants between the lectin and the polypeptide-bound antibody to the glycoprotein to be measured. It is desirable to set above. For example, although it varies depending on the glycoprotein to be measured, the type and physical properties of the lectin, the physical properties of the polypeptide-bound antibody, etc., it is desirable to set using the general formula shown below.

Polypeptide-bound antibody concentration ≦ (lectin binding constant) ÷ (polypeptide-bound antibody binding)
Constant) x (lectin concentration)
The binding constant related to the above general formula indicates a binding constant in an equilibrium reaction such as the following formula (1), and is a constant determined by the following formula (2).

[A] + [B] ← → [A ・ B] (1)
Coupling constant = [A · B] / ([A] × [B]) (2)
[A]: Concentration (M) of lectin or polypeptide-bound antibody at equilibrium.
[B]: Free glycoprotein concentration (M) to be measured in an equilibrium state.
[A / B]: Complex of lectin (or polypeptide-binding antibody) and glycoprotein to be measured
Body concentration (M).

More specifically, for example, the binding constant of the lectin to the measurement target glycoprotein is 1 × 10 ⁶ M ⁻¹ , and the binding constant of the polypeptide-bound antibody to the measurement target glycoprotein is 1 × 10 ⁸ M ⁻¹ . In some cases, the polypeptide-bound antibody concentration will be 1/100 or less, preferably 1/1000 or less of the lectin concentration. It should be noted that the concentration of the polypeptide-bound antibody used is preferably not less than the concentration at which it can bind to all the glycoproteins to be measured having a set detection limit concentration, but may be less (for example, about 1/10). .

  The concentration of the labeled anti-glycoprotein antibody varies depending on how much the detection limit of the measurement target glycoprotein is set, but all the measurement target glycoproteins with the detection limit concentration set as the concentration in the reaction solution. It is desirable that the concentration be higher than the concentration capable of binding to, preferably 2 times or more, more preferably 5 times or more, still more preferably 10 times or more.

  In carrying out the measurement methods (1) to (6) using the present invention described above, the complex is divided into a free binding substance (including a labeled substance) and a measurement target substance (labeled). In other words, the method uses a negative charge instead of HPLC as described above, and electrically separates them based on the difference in the negative charge of substances usually used in this field. Separation methods, for example, electrokinetic separation methods that do not use a separation carrier (Japanese Patent Publication No. 7-111398, etc.), electrophoresis methods that use a separation carrier, capillary electrophoresis methods, such as nucleic acid hybridization In addition, after promoting the antigen-antibody reaction using a difference in charge, a method of electrically separating free nucleic acid chains, antibodies, etc. (International Publication No. 95/12808 etc.) may be used. . Of these, carriers used in electrophoresis using a separation carrier are also carriers usually used in this field (for example, filter paper such as agar gel, agar gel, agarose gel, polyacrylamide gel, starch gel, etc. , Cellulose acetate film, etc.), and can be used without any particular limitation.

Examples of the detection substance used in the measurement method of the present invention include alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphorus used in enzyme immunoassay (EIA). Enzymes such as acid dehydrogenase, acetylcholinesterase, malate dehydrogenase, luciferase, for example, radioisotopes such as ^99m Tc, ¹³¹ I, ¹²⁵ I, ¹⁴ C, ³ H used in radioimmunoassay (RIA), for example Fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or their derivatives used in fluorescence immunoassay (FIA), such as luciferin, isoluminol, luminol, bis (2,4,6-trifluoro) Luminescent substances such as (phenyl) oxalate, eg For example, substances having absorption in the ultraviolet region such as phenol, naphthol, anthracene or derivatives thereof, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2, 5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α- (3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene) Needless to say, the substance having properties as a spin labeling agent typified by a compound having an oxyl group such as -p-tolyloxyl is not limited thereto.

  As a method of labeling a detection substance as described above on a binding substance, a labeling method known per se generally used in EIA, RIA, FIA, etc. Volume, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawaio, 1st edition, Soft Science Inc., 1983; Enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai Ed., 2nd edition, medical school, 1982, etc.) are all listed without exception. Needless to say, a conventional method using a reaction between avidin (or streptavidin) and biotin may be used as a labeling method.

  Examples of binding substances that can be measured (detected) by any method according to the present invention include, for example, enzymes, fluorescent substances, luminescent substances, or substances having absorption in the ultraviolet region. Examples thereof include those having the above-mentioned properties as detection substances.

  In the measurement method of the present invention, the elution time of the complex can be freely adjusted according to the type of the polypeptide of the present invention (type and number of acid residues derived from strong acid to be introduced, types of constituent amino acid residues, etc.). Therefore, fractional measurement is possible by using this property.

  That is, by using two or more kinds of polypeptides of the present invention having different acid residues derived from strong acids and appropriately selecting the type of binding ability substance that binds each polypeptide, simultaneous measurement of a plurality of measurement target substances ( (Separate measurement) can be performed.

  Moreover, even when there are a plurality of serum components that affect the measurement, if the ionicity of the complex is made larger than the ionicity of these serum components using the polypeptide of the present invention, the serum components will affect the measurement. The effect that an influence can be avoided arises. In this case, the time required for the analysis can be shortened by using the stepwise gradient.

  Furthermore, chromatographic carriers (gel carriers, membrane carriers, glass carriers, etc.) used in the anion exchange method generally have a high exchange capacity (absolute adsorption capacity of ionic substances), so that biological bodies such as serum can be used. Even when analyzing a sample having a large absolute amount of an ionic coexisting substance such as a derived sample, the complex to which the conjugate of the present invention is bound can be adsorbed. It is possible to elute at a position where the influence of can hardly be avoided. In addition, since the polypeptide of the present invention used in the measurement method of the present invention has high solubility in water, the water solubility of the complex to which the polypeptide is bound is higher than that before binding. In this case, there is almost no possibility that the substance to be measured is denatured or deactivated during the formation reaction of the complex to which the separation enhancing substance (polypeptide) is bound.

  In addition, the carrier used in the anion exchange method for separating the target substance and other coexisting substances by utilizing the anionic property of the polypeptide of the present invention may be a carrier having anion exchange ability. For example, a carrier having an anion exchange group such as a diethylaminoethyl (DEAE) group or a quaternary ammonium group (for example, Q group, QAE group, etc.), more specifically, for example, DEAE- MCI gel (Mitsubishi Kasei Co., Ltd. trade name), QAE MCI gel (Mitsubishi Kasei Co., Ltd. trade name), Wakobead DEAE gel (Wako Pure Chemical Industries, Ltd. trade name), etc. Examples include Memsep DEAE (trade name of Nihon Millipo Ltd.) using membrane. Needless to say, a commercially available carrier for chromatography or an anion exchange group as described above bound to the surface of a container made of resin or glass may be used in-house. .

  The method for measuring a biological component using the conjugate of the present invention is an excellent measurement method because it uses a method using a negative charge.

  That is, for example, it is necessary to use a column of an appropriate length in order to perform a method for measuring a biological component using the gel filtration method. There is a drawback in that the separation time becomes longer than in the case of using the method to be used. Therefore, when shortening of the separation time is required, it is a method using a negative charge, for example, an HPLC apparatus equipped with a column packed with a packing material for anion exchange chromatography, an anion exchange membrane, an anion exchange action. It is preferable to use an anion exchange method such as a mechanical instrument having an anion exchange action such as a reaction tube having an anion. Furthermore, the gel filtration method has a drawback that it is not suitable for separating a substance to be measured in a biological sample having a very high molecular weight (a molecular size of about 1000 angstroms or more).

  In the case of the hydrophobic separation method, when the measurement target substance in the biological sample is a physiologically active substance having a higher order structure such as a protein, the higher order structure is destroyed by the organic solvent used in the separation operation and combined. There may be a problem that the activity of the substance to be measured in the biological sample in the body is lost.

  On the other hand, the anion exchange method can more effectively separate a measurement target substance in a biological sample based on a subtle difference in ionicity. Furthermore, according to the anion exchange method, the polypeptide of the present invention having various ionic properties can be arbitrarily selected, so that the substance to be measured in the biological sample can be separated under the optimum pH condition. . Furthermore, since the polypeptide of the present invention itself has a high water solubility, even when it is bound to a measurement target substance in a biological sample, there is almost no risk of precipitation of the measurement target substance in the biological sample. The separation operation can be performed in a stable state.

  When a measurement target substance in a biological sample is measured using the measurement method of the present invention, the peak of the measurement target substance in the target biological sample is moved to a position that is not affected by components such as serum and urine. Can be made. In addition, by selecting and using the conjugate of the present invention having appropriate properties according to the substance to be measured in the biological sample, the elution position of the complex containing the substance to be measured in various biological samples can be determined. Since they can be matched, there is also an effect that the measurement target substance in various biological samples can be measured using an anion exchange method (for example, HPLC) under the same analysis conditions.

  In the measurement method of the present invention, a detection substance (binding substance, binding substance A or substance to be measured in a biological sample) contained in a complex (labeled complex) separated by a method using a negative charge Is measured according to a predetermined method depending on the property of the detection substance (binding substance, binding substance A or substance to be measured in the biological sample) that can be detected by any method. . For example, when the property is an enzyme activity, a conventional EIA method, for example, “Enzyme immunoassay, protein nucleic acid enzyme separate volume No. 31, Kitagawa Tsuneo, Minamihara Toshio, Tsujiakio, Ishikawa Yuji, 51-63, Measurement may be carried out according to the method described in Kyoritsu Shuppan Co., Ltd., issued on September 10, 1987, etc., and when the detection substance is a radioactive substance, the radiation emitted by the radioactive substance according to the RIA ordinary method Depending on the type and strength, measurement equipment such as an immersion type GM counter, liquid scintillation counter, well type scintillation counter, HPLC counter, etc. may be selected and used as appropriate (for example, a medicinal chemistry experiment course, Volume 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971 etc.). Further, when the property is fluorescent, FIA conventional methods using a measuring instrument such as a fluorimeter, such as “Illustrated Fluorescent Antibody, Akira Kawaio, First Edition, Soft Science Co., Ltd., 1983”, etc. Measurements may be carried out according to the method described in, and if the property is luminescent, a conventional method using a measuring instrument such as a photon counter, for example, “Enzyme Immunoassay, Protein Nucleic Acid Enzyme Separate Volume No. 31, Kitagawa Measurement may be carried out according to the method described in “Tsuneki, Toshio Minamihara, Akio Tsuji, Yuji Ishikawa, pp. 252-263, published by Kyoritsu Publishing Co., Ltd., September 10, 1987”. Furthermore, when the property has absorption in the ultraviolet region, measurement may be performed by a conventional method using a spectrophotometer or other measuring instrument. When the detection material is a material having a spin property, electron spin resonance is used. Conventional methods using a device, for example, “Enzyme immunoassay, protein, nucleic acid, enzyme, separate volume No.31, Kitagawa Tsuneo, Minamihara Toshio, Tsuji Akio, Ishikawa Shinji, pp. 264-271, Kyoritsu Shuppan Co., Ltd., September 1987 Measurements may be performed in accordance with the methods described in “Issuing on the 10th”.

  In the measurement method of the present invention, a substance to be measured in a biological sample and a conjugate of the present invention (including conjugates A, B, C, D, etc.), and if necessary, a binding ability substance labeled or not ( The reaction conditions when forming the complex by reacting with the substance to be bound, or the measurement target substance in the biological sample, the label measurement substance, and the conjugate of the present invention are reacted to form a label. The reaction conditions for forming the complex are not particularly limited as long as they do not interfere with the complex (or labeled complex) formation reaction, but conventional methods such as EIA, RIA, FIA, affinity chromatography, etc. The reaction may be carried out according to the reaction conditions for forming a complex or the like in a method known per se. For example, when a buffer solution is used during the reaction, the buffering agent and other reagents used may be appropriately selected from those used in a method known per se. The pH during the reaction is not particularly limited as long as it does not interfere with the formation reaction of the complex (or label complex), but it is usually in the range of 2 to 10, preferably 5 to 9. It is done. The temperature during the reaction is not particularly limited as long as it does not interfere with the formation reaction of the complex (or label complex), but it is usually 0 to 50 ° C, preferably 20 to 40 ° C. It is done. The reaction time is the time required for formation of the complex (or labeled complex), the reactivity between the substance to be measured in the biological sample and the binding substance of the present invention, the label binding substance, etc. Since it varies depending on the reactivity between the conjugate of the invention and the labeling substance, it may be appropriately reacted for several seconds to several hours depending on the respective properties.

  As the HPLC used for carrying out the anion exchange method in the measurement method of the present invention, the apparatus itself is usually used in the field of analysis and is particularly problematic if a constant flow rate can be obtained. Can be used.

  When the measurement method of the present invention is performed using HPLC, the measurement target substance may be measured using the peak area or the peak height.

  As an anion exchange method, more specifically, as a solvent (eluent) used for separation from a complex (or labeled complex) or a free label-binding substance by HPLC, the formed complex (Or a labeled complex) or the like is not decomposed again into a measurement target substance or a label binding ability substance in a biological sample, and the binding ability substance or the like contained in the complex (or label complex) The substance is not particularly limited as long as it does not lose the property of the detection substance that can be detected by any method, but is usually known per se such as EIA, RIA, FIA, affinity chromatography, etc. In this method, those used as buffer solutions are preferably used. Specific examples include phosphates, acetates, citrates, Good buffers, buffers such as tris (hydroxymethyl) aminomethane, salts such as sodium chloride, potassium chloride, ammonium sulfate, etc. Polar organic solvents such as methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran, and surfactants are appropriately selected according to the properties of the complex (or label complex) or free label-binding substance, and added. A buffer solution prepared by mixing and having a pH of 2 to 10 is preferably used.

  In addition, by appropriately adding an appropriate surfactant to the eluent, tailing of the peak of the complex (or label complex) is prevented, and the complex (or label complex) or free label binding substance It is possible to further freely adjust the elution position. The surfactant that can be used for such a purpose is not particularly limited as long as it is appropriately selected depending on the properties of the complex (or label complex) or the free label-binding substance. Cationic surfactants such as n-dodecyltrimethylammonium bromide and 1-laurinpyrimidinium chloride, such as lauryl betaine, lauryl amidopropyl betaine, coconut oil fatty acid amidopropyl betaine, 2-alkyl-N-carboxymethyl-N Preferred examples include amphoteric surfactants such as -hydroxyethylimidazolium betaine, 2-undecyl-N-carboxymethyl-N-hydroxyethylimidazolium betaine, and N-lauroyl-N-methyl-β-alanine sodium. In addition, the addition concentration in the case of adding a surfactant to the eluent for such purposes is not particularly limited as long as it is a concentration that produces the desired effect, but it varies slightly depending on the type of surfactant. It is appropriately selected from the range of 0.01 to 2%, preferably 0.05 to 1%.

  In the measurement method of the present invention, the measurement method after separation by HPLC is, for example, “Latest Liquid Chromatography, Shoji Hara and Akio Tsujio, 1st Edition, pp. 92-104, Nanzan-do, February 1978. The effluent from the HPLC column as described in “Issued on the 1st” is directly introduced to the detection section, and the detection substance (or binding ability) contained in the complex (or labeled complex) in the effluent The method of directly measuring the amount of the substance or the substance to be measured is more preferable because the measurement can be performed quickly. In this case, the binding substance (or measurement target substance) or the detection substance held in the label binding substance or the like (or label measurement substance) has a property that can be detected by any method. For example, in the case of enzyme activity, it goes without saying that it is necessary to provide a so-called post-column reaction part that adds a reagent for enzyme activity measurement and reacts with the effluent between the HPLC column and the detection part. When the property of the detection substance (or binding substance or measurement target substance) is enzyme activity, the reagent for enzyme activity measurement used in the reaction part is an ordinary method such as “enzyme immunoassay, Protein Nucleic Acid Enzyme Separate Volume No.31, edited by Kitagawa Tsuneo, Minamihara Toshio, Akio Ishikawa, Yuji Ishikawa, 51-63, Kyoritsu Publishing Co., Ltd., issued September 10, 1987 The prepared reagent may be used, or a commercially available reagent for a clinical test kit may be appropriately selected and used. In addition, even when the property of the detection substance (or binding ability substance or measurement target substance) is other than the enzyme activity, in order to add a predetermined reagent and react for the purpose of increasing the detection sensitivity, It is optional to provide an appropriate reaction part between the column and the detection part.

  When a plurality of HPLC eluents having different components are used in the measurement method of the present invention, the operation method may be the concentration gradient method (linear gradient method) or the stepwise method. Any of them may be used, but the stepwise method is more desirable because it has advantages such as simple operation, shortening the actual analysis time, and sharpening the target peak.

  In addition, the reagent for measuring a substance to be measured in the biological sample of the present invention includes a conjugate of the polypeptide of the present invention and a substance capable of binding to the substance to be measured in the biological sample. In addition, for example, a substance to be labeled, a substance capable of binding to a label, a buffering agent, a surfactant, and the like are appropriately contained as necessary. In addition, the preferable aspect of each component, a specific example, etc. are as having described above.

  The present invention includes a complex resulting from the interaction between a substance to be measured in a biological sample and a binding ability substance, a free binding ability substance, and a coexisting substance that may affect the detection of the complex. A novel polypeptide that can be used to more effectively separate the complex from a free binding substance or the like when separating using a method utilizing a negative charge, and a living body using the polypeptide The present invention provides a method for measuring a substance to be measured in a sample. When a trace component in a biological sample such as serum is measured using the measurement method of the present invention, a conventional method such as EIA or RIA is used. Compared with a measurement method or a method using a polymer having a carboxyl group as a separation-enhancing substance, there is a remarkable effect that a highly accurate measurement can be performed easily and in an extremely short time. To contribute to the industry Is a filtration size becomes invention.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

The abbreviations used in the following examples are as follows.
Ala; alanine, βAla; β-alanine, Ser; serine, Tyr; tyrosine, Asp; aspartic acid, Fmoc; 9-fluorenylmethoxycarbonyl group, Alko; p-alkoxybenzyl alcohol, tBu; t-butyl group, TFA Trifluoroacetic acid, BOP; benzothiazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate, HOBt; N-hydroxy-1H-benzotriazole, DMF; N, N-dimethylformamide, DIEA; N , N-diisopropylethylamine.

<Synthesis of Ala- (Ser (SO ₃ H)) ₅ -βAla (Polypeptide 3)>
(1) Synthesis of Fmoc-Ala- (Ser) ₅ -βAla
Fmoc-βAla-Alko resin (100-200mesh, manufactured by Watanabe Chemical Co., Ltd.) 2.3 g (1.5 mmol as βAla) was used as a starting material, and literature (J. Org. Chem., 53, 617-624 (1988) ) According to the method described (BOP / HOBt method).
That is, first, Fmoc-βAla-Alko resin was treated with piperidine to remove the Fmoc group. A DMF solution containing 3 times equivalent of a given amino acid with respect to βAla on the resin, 3 times equivalent of BOP, 3 times equivalent of HOBt and 5.3 times equivalent of DIEA were added and coupled at room temperature for 2 hours ( The amino acids were sequentially introduced by the operation of (twice). The order of the introduced amino acids is Fmoc-Ser (tBu) (manufactured by Wako Pure Chemical Industries, Ltd.), Fmoc-Ser (tBu), Fmoc-Ser (tBu), Fmoc-Ser (tBu), Fmoc-Ser (tBu), Fmoc-Ala (manufactured by Wako Pure Chemical Industries, Ltd.).
After all amino acids have been introduced, the resin is washed with MeOH, 20 ml of a TFA: anisole (95: 5) mixture is added and allowed to react with stirring at room temperature for 1 hour to cleave the desired polypeptide from the resin and at the same time the tBu group (Ser hydroxyl group). The protecting group) was cleaved. After completion of the reaction, the resin was removed by filtration, and the filtrate was concentrated under reduced pressure, and ether was added to precipitate the reaction product. The precipitate was collected and dried with a desiccator to obtain 0.84 g of Fmoc-Ala- (Ser) ₅ -βAla.

(2) Synthesis of Ala- (Ser (SO ₃ H)) ₅ -βAla (polypeptide 3) Fmoc-Ala- (Ser) ₅ -βAla 547 mg (0.67 mmol) synthesized in (1) was dissolved in 40 ml of DMF, Further, 30 ml of DMF / SO ₃ solution [2.57 g of DMF · SO ₃ (manufactured by Fluka) dissolved in 30 ml of DMF: pyridine (4: 1)] was added, and the mixture was reacted at 4 ° C. overnight. The same amount of DMF / SO ₃ solution was further added to the reaction solution, and further reacted at 4 ° C. overnight. After completion of the reaction, the reaction solution was added to 400 ml of acetone, and the resulting precipitate was collected by filtration. The precipitate was dissolved in 40 ml of DMF, added with 8 ml of piperidine, and stirred at room temperature for 40 minutes to cleave the Fmoc group. The reaction solution was added to 500 ml of acetone, and the resulting precipitate was collected by filtration, washed with acetone and ether, and then dried with a desiccator. The obtained precipitate was subjected to anion exchange chromatography and then gel filtration to obtain 210 mg of Ala- (Ser (SO ₃ H)) ₅ -βAla (polypeptide 3).
Table 1 shows the results of amino acid analysis and ion chromatography of the reaction product.
In addition, amino acid analysis was performed by Wako PTC-amino acid analysis system (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter the same).

<Synthesis of Ala-Tyr (SO ₃ H) ₃ -βAla (polypeptide 11)>
Fmoc-βAla-Alko resin 0.77 g (0.5 mmol) was used as a starting material, and the same reagent was used as in Example 1 (1) to carry out the same procedure to introduce a predetermined amino acid. The order of the introduced amino acids was Fmoc-Tyr (SO ₃ Na) (manufactured by Bachem), Fmoc-Tyr (SO ₃ Na), Fmoc-Tyr (SO ₃ Na), and Fmoc-Ala.
After completion of the reaction, the resin was washed with MeOH, 50 ml of a DMF: piperidine (4: 1) mixture was added, and the reaction was stirred at room temperature for 1 hour to cleave the Fmoc group. The solvent was removed by filtration, the resin was washed with MeOH, TFA: H ₂ O: m-cresol (45: 5: 2) was added, and after substitution with nitrogen gas, the mixture was allowed to react with stirring at 4 ° C. for 16 hours. Was cut from the resin. The resin was removed from the reaction solution by filtration, the filtrate was concentrated under reduced pressure, and the reaction product was precipitated with ether. The precipitate was subjected to anion exchange chromatography and then gel filtration to obtain 180 mg of Ala-Tyr (SO ₃ H) ₃ -βAla (polypeptide 11).
Table 1 also shows the results of amino acid analysis and ion chromatography of the reaction product.

<Synthesis of Ala- (Tyr (SO ₃ H)) ₅ -βAla (polypeptide 14)>
(1) Synthesis of Fmoc-Ala- (Tyr) ₅ -βAla
Fmoc-βAla-Alko Resin 2.3 g (1.5 mmol) was used as a starting material, the same reagent was used as in Example 1 (1), and the same operation was performed to introduce a predetermined amino acid. The order of the introduced amino acids is Fmoc-Tyr (tBu) (Watanabe Chemical Co., Ltd.), Fmoc-Tyr (tBu), Fmoc-Tyr (tBu), Fmoc-Tyr (tBu), Fmoc-Tyr (tBu), Fmoc -It was Ala.
After completion of the reaction, the resin was washed with MeOH, mixed with TFA: thioanisole: 1,2-ethanedithiol (95: 5: 1) and stirred at room temperature for 1 hour to cleave the polypeptide from the resin and simultaneously remove the tBu group. (Tyr's hydroxyl protecting group) was cleaved. After completion of the reaction, the resin was removed by filtration, and the resulting filtrate was concentrated under reduced pressure, and ether was added to precipitate the reaction product. The precipitate was collected by filtration and dried with a desiccator to obtain 1.71 g of Fmoc-Ala- (Tyr) ₅ -βAla.

(2) Synthesis of Ala- (Tyr (SO ₃ H)) ₅ -βAla (polypeptide 14) Fmoc-Ala- (Tyr) ₅ -βAla 0.5g obtained in (1) was mixed with ₃ ml of DMF. Add 15 ml of DMF / SO ₃ solution (3.2 g of DMF / SO ₃ dissolved in 15 ml of DMF: pyridine (4: 1)), react at 4 ° C. overnight, then add ether to precipitate the reaction product. It was. The precipitate was dissolved in 10 ml of DMF, 2.5 ml of piperidine was added, and the mixture was stirred at room temperature for 1 hour. After adding 150 ml of ether to the reaction solution, the resulting precipitate was collected by filtration. The obtained precipitate was dissolved in 4 ml of water, and the reaction product was fractionated by ODS column liquid chromatography [column: Wakosil ₁₀ C ₁₈ (2.0φx25 cm) (manufactured by Wako Pure Chemical Industries, Ltd.), elution condition: 10 mM. AcONa (pH 6.0), 2 → 60% acetonitrile]. The obtained fraction containing the desired product was further processed by gel filtration to obtain 203 mg of Ala- (Tyr (SO ₃ H)) ₅ -βAla (polypeptide 14).
Table 1 also shows the results of amino acid analysis and ion chromatography of the reaction product.

<Synthesis of sulfated polypeptides (polypeptides 1, 2, 4-9, 18 and 20-22)>
Using the same reagent as in Example 1, the same operations were performed to synthesize polypeptides 1, 2, 4 to 9, 18, and 20 to 22 shown in Table 1. Moreover, the same reagent as Example 2 was used and the same operation was performed and the polypeptide 10 of Table 1 was synthesize | combined.
In addition, Fmoc-Ser (tBu) -O-polymer (manufactured by Kokusan Chemical Co., Ltd.) is used as a starting material for the synthesis of polypeptide 6, and Fmoc is used as a starting material for the synthesis of polypeptides 21 and 22. -Tyr (tBu) -Alko Resin (100-200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd.) was used.
Table 1 also shows the results of amino acid analysis and ion chromatography of the various polypeptides obtained.

<Synthesis of sulfated polypeptides (polypeptides 12, 13, 15-17 and 19)>
Using the same reagents as in Example 3, the same operations were performed to synthesize polypeptides 12, 13, 15 to 17 and 19 listed in Table 1. As starting materials for the synthesis of polypeptides 12, 16, Fmoc-βAla-Alko resin (100-200mesh, manufactured by Watanabe Chemical Co., Ltd.) was used to synthesize polypeptides 13, 15, 17, 19 Fmoc-Tyr (tBu) -Alko Resin (100-200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd.) was used as a starting material for this.
Table 1 also shows the results of amino acid analysis and ion chromatography of the reaction product.

<Synthesis of Ala- (Tyr (PO ₃ H ₂ )) ₅ -βAla (Polypeptide 23)>
Fmoc-βAla-Alko Resin 780 mg (0.5 mmol) was used as a starting material, the same reagent was used as in Example 1 (1), and the same operation was performed to introduce a predetermined amino acid. The amino acid sequences introduced were Fmoc-Tyr (PO ₃ H ₂ ) (Nova biochem), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Ala.
After completion of the reaction, the resin was washed with MeOH, and then 50 ml of a DMF: piperidine (4: 1) mixture was added, followed by stirring at room temperature for 1 hour to cleave the Fmoc group. After completion of the reaction, the resin was collected by filtration, washed with MeOH, and then added with 20 ml of TFA: phenol: H ₂ O: thioanisole: ethanediol (33: 2: 2: 2: 1) and stirred at room temperature for 1 hour. The polypeptide was cleaved from the resin. After completion of the reaction, the resin was removed by filtration, and ether was added to the filtrate to precipitate the reaction product. The obtained precipitate was subjected to anion column chromatography and gel filtration to obtain 760 mg of Ala- (Tyr (PO ₃ H ₂ )) ₅ -βAla (polypeptide 23).
Table 1 also shows the results of amino acid analysis and ion chromatography of the reaction product.

<Synthesis of 4-maleimidobutyryl-Ala- (Tyr (PO ₃ H ₂ )) ₅ -βAla (polypeptide 24)>
Fmoc-βAla-Alko Resin 780 mg (0.5 mmol) was used as a starting material, the same reagent was used as in Example 1 (1), and the same operation was performed to introduce a predetermined amino acid. The amino acid sequences introduced were Fmoc-Tyr (PO ₃ H ₂ ) (Nova biochem), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Ala, and maleimidobutyric acid.
After completion of the reaction, the resin was washed with MeOH, and this was treated with a TFA: anisole (95: 5) mixture to cleave the polypeptide from the resin. The resin was removed by filtration, and ether was added to the resulting filtrate to cause precipitation. The obtained precipitate was subjected to ODS column [column: Wakosil ₅ C ₁₈ (2.0φx25cm) (manufactured by Wako Pure Chemical Industries, Ltd.), elution condition: 0.1% TFA, 0 → 10% acetonitrile], and then subjected to gel filtration. 820 mg of 4-maleimidobutyryl-Ala- (Tyr (PO ₃ H ₂ )) ₅ -βAla (polypeptide 24) was obtained.
Table 1 also shows the results of amino acid analysis and ion chromatography of the reaction product.

<Examination of elution position of anionic polypeptide on anion exchange column>
(sample)
An aqueous solution containing 5 mg / ml of each of polypeptides 1 to 24 obtained in Examples 1 to 7 was used as a sample.
As a control, (Asp) ₉ -βAla (hereinafter abbreviated as “Asp9”; self-produced by the BOP / HOBt method), polyaspartic acid having an average molecular weight of 6000 (hereinafter abbreviated as “Asp6K”, manufactured by Sigma) ) Or polyaspartic acid having an average molecular weight of 50,000 (hereinafter abbreviated as “pAsp”; manufactured by Sigma) was also prepared as a sample.
(Analysis conditions)
Column: POROS-DEAE (4.6φx10mm, manufactured by Perceptive).
Eluent A: 50 mM phosphate buffer (pH 7.6).
Eluent B: 50 mM phosphate buffer (pH 7.6, containing 5 M NaCl).
Flow rate: 1ml / min.
Detection: For polypeptides containing sulfated serine residues; UV 220 nm.
For sulfated tyrosine residue-containing polypeptides; UV 260 nm.
For polypeptides containing phosphorylated tyrosine residues; UV 260 nm.
Gradient condition: 0 → 5min A = 100%
5 → 30min B = 0 → 100%
30 → 35min B = 100%
(Measurement operation)
A 20 μl sample was analyzed by HPLC under the above conditions.

(result)
The salt concentration required for elution of each polypeptide (hereinafter abbreviated as “eluting salt concentration”) is shown in FIG. In FIG. 1, ● represents the salt concentration at the peak of the elution peak, and I represents the range of salt concentration at the beginning and end of elution, that is, the peak width. In addition, each symbol and number on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1).
The following can be seen from the results of FIG.
(1) It can be seen that increasing the number of acid residues (sulfuric acid groups, phosphoric acid groups) derived from strong acids increases the elution salt concentration.
(2) Polypeptides 3 (sulfuric acid residue number: 5) and 11 (sulfuric acid residue number: 3) show elution salt concentrations equivalent to Asp9, and polypeptides 6 (sulfuric acid residue number: 10) and 8 (sulfuric acid residue) Residue number: 9), 9 (sulfuric acid residue number: 10), 12,13 (sulfuric acid residue number: 4) and 14,15 (sulfuric acid residue number: 5) are the same elution salt concentration as Asp6K and pAsp In other words, the polypeptide of the present invention has a shorter chain length (a smaller number of amino acid residues) than a conventional polypeptide having a carboxylic acid residue, and has the same effect (anion exchange column). It can be seen that the effect that the degree of strength held in the head is equal is obtained.
(3) The degree of strength retained in the anion exchange column varies depending on the type and number of acid residues derived from strong acids, the types of amino acid residues into which the acid residues are introduced, the properties of the column filler, etc. . For example, when the results for polypeptides 2-9 having sulfated serine residues are compared with the results for polypeptides 10-20 having sulfated tyrosine residues, the number of acid residues derived from strong acids is the same. It can be seen that the polypeptide having a sulfated tyrosine residue has a higher elution salt concentration. This result shows that the column packing material used this time is slightly hydrophobic, so that the polypeptide having a tyrosine residue having a benzene ring is more likely to be retained in the column, and as a result It is thought that the elution salt concentration of was increased. In addition, from the comparison of the results for polypeptides 3 and 14 having 5 sulfate residues and the results for polypeptides 23 and 24 having 5 phosphate residues, the polypeptide having sulfate residues is more eluted. It can be seen that the concentration increases. From these results, it is possible to select a polypeptide having an arbitrary elution salt concentration by appropriately selecting the type and number of acid residues to be introduced, the type of constituent amino acid residues, the type of column filler, and the like. I understand.

(4) The type and number of amino acid residues into which acid residues derived from strong acids are introduced, and even if the type and number of acid residues are the same, the presence of other amino acid residues in the polypeptide The degree of strength retained on the ion exchange column varies. For example, from the results of polypeptides 12 to 19 having sulfated tyrosine residues, even if the number of sulfated tyrosine residues is the same, the elution salt concentration varies depending on the presence or absence of β-alanine residues (β-alanine residues It can be seen that the concentration of the eluted salt is reduced by the introduction. From this result, it is understood that a polypeptide having an arbitrary elution salt concentration can be prepared by introducing an appropriate amino acid residue in addition to the amino acid residue into which the acid residue has been introduced.
(5) From the results obtained with pAsp-1 and pAsp-2 of comparative examples (pAsps with different production lots), in the conventional polypeptide having a carboxylic acid residue, the elution salt concentration varies depending on the production lot, In other words, it can be seen that the tailing situation of the target peak changes, that is, it is difficult to obtain a polypeptide having a uniform molecular weight. On the other hand, since the polypeptide of the present invention can be easily obtained by peptide synthesis, such a problem does not occur in pAsp.

<Preparation of antibody-sulfated polytyrosine conjugate>
(1) 4- (p- maleimidophenyl) butyryl _{Ala- (Tyr (SO 3 H)} ) 8 prepared in Preparation Example 4 of _{-βAla Ala- (Tyr (SO 3 H} )) 8 -βAla 1mg of 0.1M Dissolved in 500 μl of phosphate buffer (pH 7.0), 1.2 mg of sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (Pierce Chemical Co.) was added and reacted at 37 ° C. for 3 hours. . This reaction solution was treated with a Superdex peptide column (16 mm ID × 30 cm: manufactured by Pharmacia) to remove excess reagent, and 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃ H)) ₈ − An aqueous solution of βAla 0.84 mg was obtained (yield 75%).
(2) Preparation of Fab ′ Fragment Anti-AFP monoclonal antibody A4-4 (hereinafter abbreviated as “AFP-A4-4”, manufactured by Wako Pure Chemical Industries, Ltd.) ab ′) ₂ fragment (5 mg, yield 80%), which was further treated by a conventional method to obtain 3.1 mg of Fab ′ fragment (hereinafter abbreviated as “AFP-A4-4 • Fab ′”). (Yield 62%).
(3) Preparation of Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A4-4 • Fab ′ 4- (p-maleimidophenyl) butyryl Ala- () obtained in (1) above Tyr (SO ₃ H)) ₈ -βAla 3.1 mg and AFP-A4-4 • Fab ′ 3.1 mg obtained in (2) above were added at 4 ° C. in 0.1 M phosphate buffer (pH 7.0). Reacted for 16 hours. This reaction solution was applied to a Superdex 200 pg column (26 mm ID × 60 cm: manufactured by Pharmacia) to remove excess 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃ H)) ₈ -βAla. Treatment with DEAE TOYOPEARL column (10mmID × 2cm: manufactured by Tosoh Corporation) collects the adsorbed fraction and binds Ala- (Tyr (SO ₃ H)) ₈ -βAla to AFP-A4-4 • Fab ' 1 mg of the product was obtained (yield 15%).

<Preparation of antibody-sulfated polytyrosine conjugate (1) Preparation of 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃ H)) ₈ >
25 mg of Ala- (Tyr (SO ₃ H)) ₈ (polypeptide 19) prepared in Example 4 was dissolved in 3 ml of DMF, and sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (Pierce (Chemical Co., Ltd.) 10 mg was added and reacted at room temperature for 1 hour. This reaction mixture was treated with an ODS column [column: Wakosil ₅ C ₁₈ (2.0φx25cm) (manufactured by Wako Pure Chemical Industries, Ltd.), elution condition: 50 mM ammonium acetate pH 6, 2 → 60% acetonitrile], and obtained. The fraction containing the desired product was concentrated to dryness to obtain 26.5 mg of 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃ H)) ₈ (yield 95%).
The NMR data of the obtained 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃ H)) ₈ are shown below.
1H-NMR (270 MHz, DMSO-d6) δppm: 7.16 (s, 2H maleimide proton)
Further, from the comparison with the result of Example 9 (1), it can be seen that this method can yield a polypeptide having a maleimide group introduced at the N-terminus in a high yield.
Furthermore, it was also found that 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃ H)) ₈ obtained by this method can be stably stored without decomposition if stored at 15 ° C. or lower. Therefore, it was also found that it was easier to use than the one obtained by the method of Example 9 (1) (in the form of an aqueous solution. Almost decomposes in about one day).
(2) Preparation of Fab ′ Fragment Anti-AFP monoclonal antibody A4-4 (hereinafter abbreviated as “AFP-A4-4”, manufactured by Wako Pure Chemical Industries, Ltd.) ab ′) ₂ fragment (16 mg, yield 80%), and further treated by a conventional method to obtain 11.1 mg of Fab ′ fragment (hereinafter abbreviated as “AFP-A4-4 • Fab ′”). (Yield 70%).
(3) Preparation of conjugate of Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4 • Fab ′ 4- (p-maleimidophenyl) butyryl Ala- (Tyr () obtained in (1) above SO ₃ H)) ₈ 1 mg and AFP-A4-4 • Fab′11.1 mg obtained in (2) above were reacted with 50 mM phosphate buffer pH 6.5 at 4 ° C. for 16 hours. This reaction solution was fractionated from the combined product of Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4 • Fab ′ using a POROS DEAE column (6 mm ID × 1 cm: manufactured by Perceptive) to obtain 6 mg. (Yield 60%).

From the comparison between this result and the result of Example 9 (3), the use of the polypeptide of the present invention obtained by the method of Example (1) with the maleimide group introduced at the N-terminus, It can be seen that a conjugate with Fab ′ can be obtained efficiently.
Although the reason for this is not clear, in Example 9 (1), free sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate was removed using a Superdex peptide column. In this example (1), it is considered that this is because an ODS column is used. That is, since the difference in molecular weight between the peptide of the present invention having a maleimide group introduced and free sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate is small, the Superdex peptide column can sufficiently separate them. In the reaction with Fab ′, free sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate reacted with Fab ′, resulting in Ala- (Tyr (SO ₃ H)) ₈ − It can be considered that the yield of the conjugate of βAla and AFP-A4-4 • Fab ′ was lowered.

<Examination of anion exchange chromatographic elution position of the conjugate of an anionic polypeptide and antibody>
(sample)
Using the same reagent as in Example 9, the same operation was performed to prepare conjugates of various anionic polypeptides listed in Table 1 and AFP-A4-4 • Fab ′, and an aqueous solution containing 1 mg / ml of this. Was used as a sample. However, since polypeptide 24 has a maleimide group bonded to the N-terminus, it was directly bonded to AFP-A4-4 • Fab ′ without performing 4- (p-maleimidophenyl) butyrate treatment.
As a control, commercially available pAsp (2Lot) was also subjected to the same operation using the same reagent as in Example 9 to obtain a conjugate with AFP-A4-4 • Fab ′, and an aqueous solution containing 1 mg / ml thereof. Was prepared as a sample.
Regarding polypeptide 22, the target AFP-A4-4 • Fab ′ conjugate was not obtained. This is presumably because the presence of an anionic amino acid residue at the N-terminus of the polypeptide reduces the efficiency of binding to the antibody (more precisely, the binding between the polypeptide and the cross-linking agent) due to charge.
(Analysis conditions and measurement operation)
The same operation as in Example 8 was performed. However, detection was performed at UV 280 nm in all cases.

(result)
FIG. 2 shows the elution salt concentration and peak width of the conjugate of various polypeptides with AFP-A4-4 • Fab ′. In FIG. 2, ● indicates the salt concentration at the peak of the elution peak, and I indicates the range of salt concentration at the beginning and end of the elution (that is, peak width). Each symbol on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1).
The following can be seen from the results of FIG.

(1) Although the elution salt concentration is slightly lower than that of the polypeptide alone by binding to the antibody, the relative relationship between the various polypeptides and the elution salt concentration of pAsp is the same as that obtained in Example 8. It turns out that it is the same.
(2) Although not shown in FIG. 2, elution of coexisting substances in serum when the serum components were analyzed under the analysis conditions of Example 8 using POROS-DEAE (4.6φ × 10 mm, anion exchange column) The salt concentration at the peak apex was around 0.3M. Therefore, when the conjugate of polypeptide 1 (number of sulfate residues: 1) and antibody is used as a separation-enhancing substance, the peak of interest overlaps with the peak of coexisting substances in serum (analysis). It was found that the accuracy decreased. From the results of FIG. 2, although not as much as polypeptide 1, polypeptide 2 (sulfuric acid residue number: 3), polypeptide 10 (sulfuric acid residue number: 1) and polypeptide 11 (sulfuric acid residue number: It was suggested that even when a conjugate of 3) and the antibody was used as a separation-enhancing substance, the accuracy of measurement (analysis) slightly decreased due to the influence of coexisting substances in serum.

In addition, since POROS-DEAE, which is the filler used this time, has a slightly hydrophobic base material, the conjugate of the polypeptide 10-20 having sulfated tyrosine residues and the antibody is more sulfated. The apparent elution salt concentration is higher than the conjugate of the polypeptide having a serine residue and the antibody. Therefore, the apparent elution salt concentration of the conjugate of the polypeptide 10 having only one sulfate residue but having the tyrosine residue and the antibody is polypeptide 2 having 3 sulfated serine residues. The same as that of the conjugate of antibody and antibody.
However, when the same experiment as described above was performed by changing the filler used to have a hydrophilic base material, a conjugate of polypeptide 10 and antibody having only one sulfated tyrosine residue. The apparent elution salt concentration of this protein is similar to that of the conjugate of antibody 1 and polypeptide 1 having only one sulfated serine residue, and it is overlapped with the peak of coexisting substances in serum (analysis). The accuracy of is expected to decline.
From these results, it can also be seen that a polypeptide having at least 3, preferably 4 or more, more preferably 5 or more acid residues derived from a strong acid is desirable as the separation enhancing substance.

<Examination of the stability of sulfated serine-containing polypeptides and sulfated tyrosine-containing polypeptides in aqueous solutions>
Ala- (Ser (SO ₃ H)) ₈ -βAla (polypeptide 4) or Ala- (Tyr (SO ₃ H)) ₅ -βAla (polypeptide 14) at 40 ° C. in a pH 6-10 buffer. Stored and examined for stability.
(sample)
A sample was prepared by dissolving polypeptide 4 or 14 in the following buffer solution to a final concentration of 1 mg / ml.
Buffer: pH 6.0 2-morpholinoethanesulfonic acid (MES).
pH 7.0 3-morpholinopropane sulfonic acid (MOPS).
pH 8.0 N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS).
pH 9.0 TAPS.
pH 10.0 N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO).
The buffer solution concentration was all 50 mM.
(Preservation method)
Each sample was stored at 40 ° C. for a predetermined number of days.

(result)
The polypeptide residual ratio (%) in the sample on the 6th and 19th days of storage was determined by the analysis conditions and operation method of Example 8, and the peak area of the polypeptide in the sample immediately after preparation and the predetermined number of days It calculated | required based on the peak area of the polypeptide in the sample after a preservation | save.
The measurement result for polypeptide 4 is shown in FIG. 3, and the measurement result for polypeptide 14 is shown in FIG. In each figure, the black bars show the results obtained for the sample on the 6th day of storage, and the white bars show the results obtained for the sample on the 19th day of storage.
From the results shown in FIG. 3, with respect to polypeptide 4, degradation is accelerated as the pH during storage increases, and more than 10% is degraded after storage for 16 days even at pH 6, which was the most stable. I understand that.
On the other hand, it can be seen from the results of FIG. 4 that the polypeptide 14 is stable at all pHs.
From these results, the sulfate group introduced into the tyrosine residue is more stable and less susceptible to pH than the sulfate group introduced into the serine residue. It turns out that it has.

<Measurement of TSH (Thyroid stimulating hormone)>
(Preparation of peroxidase-labeled anti-TSH antibody Fab ′ fragment)
An anti-TSH antibody (hereinafter abbreviated as “TSH-1”, manufactured by Wako Pure Chemical Industries, Ltd.) is treated by a conventional method to produce a Fab ′ fragment, and peroxidase (manufactured by Toyobo Co., Ltd.) is bound thereto by a conventional method. Peroxidase-labeled anti-TSH antibody Fab ′ fragment (hereinafter abbreviated as “TSH-1 • Fab′-POD”) was prepared.
(Antibody solution 1)
An antibody solution 1 was prepared by preparing 50 mM MOPS buffer (pH 7.5) containing 5 nM of TSH-1 • Fab′-POD.
(Antibody solution 2)
An anti-TSH monoclonal antibody (hereinafter abbreviated as “TSH-2”, manufactured by Wako Pure Chemical Industries, Ltd.), which has been confirmed to have a different recognition site from TSH-1, was treated with a Fab ′ fragment ( Hereinafter, it is abbreviated as “TSH-2 • Fab ′”), and this, and Ala- (Tyr (SO ₃ H)) ₅ -βAla (polypeptide 14) or Ala- (Ser (SO ₃ H)) ₅ A conjugate with -βAla (polypeptide 3) was prepared in the same manner as in Example 9 (3).
A 50 mM MOPS buffer (pH 7.5) containing 50 nM of any of these was prepared and used as antibody solution 2.
(sample)
A sample prepared by adding commercially available TSH (manufactured by genzyme) to 50 mM MOPS buffer (pH 7.5, containing 0.5% bovine serum albumin) to 70 pM was used as a sample.
(HPLC usage conditions)
Column: 0.46 ¢ x1.0cm.
Filler: POROS-DEAE gel (trade name of Perceptive).
Eluent A: 50 mM MOPS buffer pH 7.5.
Eluent B: 50 mM MOPS buffer pH 7.5, 3M sodium chloride.
Substrate solution: 25 mM 4-N-acetylaminophenol (manufactured by Dojindo Laboratories) aqueous solution.
Flow rate: eluent A + eluent B; 1.0 ml / min, substrate solution; 0.1 ml / min.
Reaction part: 0.025 ¢ x1000cm (heated at 60 ° C).
Detection: Measurement was performed at an excitation wavelength of 328 nm and a fluorescence wavelength of 432 nm.
Gradient condition: 0 → 10min B = 0 → 100%
(Measurement operation)
100 μl of antibody solution 1, 50 μl of sample and 50 μl of antibody solution 2 were mixed and allowed to stand at 25 ° C. for 30 minutes, and 10 μl of the mixture solution was measured (analyzed) by HPLC under the above conditions.

(result)
As a result of HPLC analysis, the elution salt concentration (sodium chloride concentration) of various substances was as follows.
TSH-1 • Fab′-POD and a complex of TSH-1 • Fab′-POD and TSH: 0 to 0.1M.
Complex of TSH-1 • Fab′-POD, TSH, and TSH-2 • Fab ′ and Ala- (Tyr (SO ₃ H)) ₅ -βAla (polypeptide 14): 0.5 to 1.2 M .
Complex of TSH-1 • Fab′-POD, TSH and TSH-2 • Fab ′ and a conjugate of Ala- (Ser (SO ₃ H)) ₅ -βAla (polypeptide 3): 0.25 to 0.45M .
From the above results, it becomes possible to more clearly separate the free POD-labeled antibody that coexists and the complex containing the substance to be measured by using a conjugate of the sulfated polypeptide and the antibody. It can also be seen that the elution position of the target complex can be freely adjusted by appropriately selecting the type of sulfated polypeptide.
It has also been found that by using the peak of the complex to which sulfated polypeptide is bound, a good calibration curve can be obtained for TSH in the sample, in other words, the amount of TSH can be quantified.

<AFP measurement using a conjugate of Fab 'and an anionic polypeptide>
(Preparation of peroxidase-labeled anti-AFP antibody Fab ′ fragment)
An anti-AFP antibody / WA-1 (hereinafter abbreviated as “AFP-WA-1”, manufactured by Wako Pure Chemical Industries, Ltd.) that recognizes an epitope different from AFP-A4-4 was treated with a conventional method to produce Fab. A fragment is combined with peroxidase (manufactured by Toyobo Co., Ltd.) by a conventional method to produce a peroxidase-labeled anti-AFP antibody Fab ′ fragment (hereinafter abbreviated as “AFP-WA-1 • Fab′-POD”). Prepared.
(reagent)
Among the conjugates of various anionic polypeptides described in Table 1 and AFP-A4-4 • Fab ′ prepared in Example 11, 200 nM, AFP-WA-1 • Fab′-POD A MOPS buffer solution (pH 7.5) containing 100 nM and 0.2 (W / V)% polyvinyl alcohol (Aldrich) was prepared and used as a reagent.
(sample)
A sample prepared by adding commercially available AFP to a 50 mM MOPS buffer (pH 7.5, containing 0.2 (W / V)% polyvinyl alcohol) at 100 ng / ml and dissolving was used as a sample.
(HPLC conditions)
Column: POROS-DEAE (4.6φX10mm).
Eluent A: 50 mM MOPS buffer (pH 7.5).
Eluent B: 50 mM MOPS buffer (containing 3M NaCl, pH 7.5).
Substrate solution: 50 mM MOPS buffer (containing 90 mM 4-N- (4-carbobutyryl) aminophenol and 20 mM H ₂ O _2, pH 7.5).
Flow rate: A + B solution 1 ml / min (substrate solution 0.1 ml / min).
Reaction part: 0.025φX1000 cm.
Temperature: 60 ° C.
Detection: excitation wavelength 328 nm / fluorescence wavelength 432 nm.
Gradient conditions: the same as in Example 8.
(Measuring method)
100 μl of the reagent and 10 μl of the sample were mixed and reacted at 8 ° C. for 10 minutes, and then 20 μl of the mixed solution was analyzed by HPLC under the above conditions.

(result)
Figure 5 shows the elution salt concentration and peak width of conjugates of various polypeptides and AFP-A4-4 • Fab ', and complexes (antigen-antibody complexes) of AFP-WA-1, Fab'-POD and AFP. Show. In FIG. 5, ● represents the salt concentration at the peak of the elution peak, and I represents the range of salt concentration at the beginning and end of the elution (that is, the peak width). Each symbol on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1).
The following can be understood from the results of FIG.
(1) The elution salt concentration (elution position) of the antigen-antibody complex varies depending on the type of polypeptide bound to AFP-A4-4 • Fab ′. The order of elution of each antigen-antibody complex was the same as the order of elution of the conjugate of AFP-A4-4 • Fab ′ and the polypeptide (see Example 11 and FIG. 2). It can also be seen from this result that the elution salt concentration of the target antigen-antibody complex can be appropriately set by appropriately selecting the type of polypeptide.
(2) Since the elution salt concentration of free AFP-WA-1 • Fab′-POD was 0 to 0.1 M, free AFP-WA-1 • Fab′- POD and antigen-antibody complex could be completely separated. In particular, when polypeptides 4 to 9 and 11 to 21 are used, the elution salt concentration of the antigen-antibody complex can be set to 0.3 M or higher. The influence of the coexisting substances on the measurement can be avoided more reliably.
As already mentioned, POROS-DEAE, which is the filler used this time, has a slightly hydrophobic base material, so it binds polypeptides 10-20 with sulfated tyrosine residues to antibodies. Since the antigen-antibody complex formed from the product has a higher apparent elution salt concentration compared to those formed from the conjugate of the polypeptide having a sulfated serine residue and the antibody, the sulfate The apparent elution salt concentration of an antigen-antibody complex formed from a conjugate of a polypeptide 10 having only one residue but having a tyrosine residue and an antibody has a sulfated serine residue of 3 It is about the same as that of the antigen-antibody complex formed from the conjugate of the polypeptide 2 and the antibody.
However, when the same experiment as described above was performed by changing the filler used to have a hydrophilic base material, a conjugate of polypeptide 10 and antibody having only one sulfated tyrosine residue. The apparent elution salt concentration of the antigen-antibody complex formed from is about the same as that of the antigen-antibody complex formed from the conjugate of polypeptide 1 having only one sulfated serine residue and the antibody. Therefore, it is expected that the accuracy of measurement (analysis) overlapped with the peak of coexisting substances in serum will be reduced.
(3) From the above results, in order to use a polypeptide as a substance for improving separation, the number of acid residues derived from a strong acid in the polypeptide is 3 to 20, preferably 4 to 30, more preferably 5 to 15 (If the number of acid residues is increased, the elution salt concentration from the column becomes too high, which is not preferable for the measurement operation.).

(4) A combination of two or more polypeptides having different elution salt concentrations (for example, any one of polypeptides 4-7, 8, 9, 11-14 and any combination of polypeptides 7, 16-21). Are combined with an appropriate binding ability substance, so that two or more substances having similar structures (for example, two or more substances having different structures such as sugar chain structures, isozymes, and different for one antigen) It is possible to carry out differential measurement of two or more antibodies that recognize epitopes.
(5) From the results when pAsp-1 and pAsp-2 (pAsps with different production lots) were used as polypeptides, the elution salt concentration varied depending on the production lot in the conventional polypeptide having a carboxylic acid residue, It was suggested that the tailing situation of the target peak changes, in other words, it is difficult to obtain a polypeptide having a uniform molecular weight. On the other hand, since the polypeptide of the present invention can be easily obtained by peptide synthesis, such a problem does not occur in pAsp.
In addition, 100 μl of antibody solution 1 prepared in Example 11, 50 μl of sample and antibody solution 2 [TSH-2 • Fab ′ and Ala- (Ser (SO ₃ H)) ₅ -βAla (polypeptide 3) -containing conjugate When 50 μl was mixed and 20 μl of the mixture obtained by standing at 25 ° C. for 30 minutes was measured (analyzed) by HPLC under the above conditions, AFP was measured (analyzed) using polypeptide 3 The target antigen-antibody complex was eluted with the elution salt concentration of the antigen-antibody complex (data of the eluted salt concentration is also shown in the section of “polypeptide abbreviation TSH” in FIG. 5). From this result, it can be seen that by using the polypeptide of the present invention as a separation-enhancing substance, the target measurement (analysis) can be carried out using HPLC under certain analytical conditions even when the measurement target substance is different. .

<A fractional measurement method for AFPs having different sugar chain structures using the polypeptide of the present invention>
(Reagent 1)
As an antibody, anti-AFP monoclonal antibody WA-2 (hereinafter abbreviated as “AFP-WA-2”; manufactured by Wako Pure Chemical Industries, Ltd.) The antigen recognition sites of this antibody are AFP-WA-1 and AFP -A4-4.), And Ala- (Tyr (SO ₃ H)) ₅ -βAla was used as the polypeptide, and the same procedure as in Example 9 was used. The combined product of AFP-WA-2 • Fab ′ and Ala- (Tyr (SO ₃ H)) ₅ -βAla prepared in the above manner is abbreviated as 139 nM, lentil lectin (hereinafter “LCA”. Wako Pure Chemical Industries, Ltd.) A 50 mM MES buffer solution (pH 6.5) containing 1 mg / ml), 1 mM magnesium chloride and 1 mM calcium chloride was prepared as Test Solution 1.
(Reagent 2)
Binding of AFP-WA-1 • Fab′-POD prepared in Example 12 to 147 nM and Ala- (Tyr (SO ₃ H)) ₈ -βAla prepared in Example 9 to AFP-A4-4 • Fab ′ Test solution 2 was a 156 nM product and a 50 nM MOPS buffer (pH 7.5) containing 0.2 (W / V)% polyvinyl alcohol.
(sample)
Human hepatocellular carcinoma-derived AFP was fractionated into LCA-unbound AFP and LCA-bound AFP using an LCA-immobilized column. Any one of these was added to human serum not containing AFP at 100 ng / ml to obtain Sample 1 (containing LCA-unbinding AFP) and Sample 2 (containing LCA-binding AFP).
(HPLC conditions)
Column: POROS-DEAE (4.6mmID × 10mm).
Buffer A: 50 mM TAPS buffer (pH 8.5, containing 0.25 M NaCl).
Buffer B: 50 mM TAPS buffer (pH 8.5, containing 3M NaCl).
-Substrate solution: 50 mM MOPS buffer (pH 7.5, containing 90 mM 4-N- (4-carbobutyryl) aminophenol and 20 mM H2O2).
Gradient conditions: Buffer A + B, flow rate 2 ml / min.
0 → 2 minutes B = 0%
2 → 4.5 minutes B = 13%
4.5 → 8 minutes B = 100%
8 → 8.5 minutes B = 0%
-Post column: POD substrate added (substrate solution, 0.1 ml / min).
Reaction at 60 ° C. for 30 seconds • Detection: excitation wavelength 328 nm / fluorescence wavelength 432 nm.
(Measurement operation)
100 μl of sample solution 1 and 10 μl of sample 1 or sample 2 were mixed and reacted at 8 ° C. for 10 minutes. Next, 10 μl of the test solution 2 was added thereto, and the mixture was further reacted for 20 minutes. 80 μl of this reaction mixture was measured (analyzed) by HPLC under the above conditions.

(result)
The measurement results are shown in FIG. In the figure, the solid line (-) indicates the data obtained for the sample 1, and the dotted line (----) indicates the data obtained for the sample 2.
From the results shown in FIG. 6, the conjugate of AFP, AFP-WA-2 • Fab ′ and Ala- (Tyr (SO ₃ H)) ₅ -βAla and the antigen-antibody complex of AFP-WA-1 • Fab′-POD (Complex 1) is located at 2.9 minutes, and the complex 1 is further bound with a conjugate of Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A4-4 • Fab ′. It can be seen that the complex (complex 2) elutes at the position of 5.8 minutes, and each is clearly separated.
Further, from the results of FIG. 6, in the sample 1 containing LCA non-binding AFP, the antigen-antibody complex produced is Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A4-4 · It can also be seen that the complex 2 to which the conjugate with Fab ′ is bound is mainly, and in the sample 2 containing the LCA-binding AFP, the complex 1 is the antigen-antibody complex to be generated. This result indicates that the conjugate of Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A4-4 • Fab ′ inhibits the reaction with AFP by the competitive reaction with LCA. .
Further, when the complex 1 amount ratio (complex 1 ratio) relative to the amount of both complexes produced was determined (see Table 2), this value reflects the LCA binding AFP ratio in the sample, in other words, this It was also found that the LCA-binding AFP ratio in the sample can be measured by using the value.

Comparative example
Instead of the conjugate of AFP-WA-2 • Fab ′ and Ala- (Tyr (SO ₃ H)) ₅ -βAla, a conjugate of AFP-WA-2 • Fab ′ and an aspartic acid polymer having an average molecular weight of 6,000, In addition, instead of the combination of Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A4-4 • Fab ′, the combination of AFP-A4-4 • Fab ′ and an aspartic acid polymer having an average molecular weight of 28,800 Except that was used, the same experiment as in Example 15 was performed to determine the complex 1 ratio.
The same experiment as described above was performed to determine the ratio of complex 1 even when the sample solution 1 used here was added γ-polyglutamic acid having an average molecular weight of 550,000 to 100 μg / ml.
The results are also shown in Table 2.

From the results of Table 2, it can be seen that in the method of the comparative example (method using polyaspartic acid), the ratio of the complex 1 tends to increase in the sample 1, in other words, a nonspecific reaction tends to occur. It can be seen that it is necessary to add an anionic additive such as γPGA in order to suppress the above.
On the other hand, when the polypeptide of the present invention is used, such an additive is unnecessary. Therefore, the measurement method using the polypeptide of the present invention as a separation-enhancing substance is superior to this in comparison with the measurement method using a polymer having a carboxyl group such as polyaspartic acid, which has been conventionally used as a separation-enhancing substance. It can be seen that this is a measurement method.
Although the reason why such a phenomenon occurs is not clear, for example, it is estimated as follows.
That is, since an anionic polymer such as polyaspartic acid has a relatively large molecule, the antigen-antibody reaction is inhibited due to charge, steric hindrance, etc., and the above phenomenon occurs. It is presumed that since the molecule is small, it hardly affects the antigen-antibody reaction.

<Examination of addition effect of surfactant to eluent>
(Reagent for measurement)
50 mM MOPS buffer (pH 7) containing 200 nM of the conjugate of polypeptide 18 prepared in Example 9 and AFP-A4-4 Fab ′ and 100 nM of AFP-WA-1 • Fab′-POD prepared in Example 14 .5) was used as a measuring reagent.
(sample)
Same as Example 14.
(HPLC conditions)
Column: POROS-DEAE (4.6mmID × 10mm).
Buffer A: 50 mM MOPS buffer (pH 7.5, containing 0.1 (W / V)% of a predetermined surfactant).
Buffer B: 50 mM MOPS buffer (pH 7.5, 3M NaCl and 0.1% (W / V) containing a predetermined surfactant).
-Substrate solution: 50 mM MOPS buffer (pH 7.5, containing 90 mM 4-N- (4-carbobutyryl) aminophenol and 20 mM H ₂ O ₂ ).
Gradient conditions: Buffer A + B, flow rate 1 ml / min.
0 → 5min A = 100%
5 → 30min B = 0 → 100%
30 → 35min B = 100%
-Post column: POD substrate added (substrate solution, 0.1 ml / min).
Reaction at 60 ° C for 30 seconds • Detection: excitation wavelength 328 nm / fluorescence wavelength 432 nm.
Table 3 shows the types of surfactants added to the eluent.

(Measurement operation)
100 μl of the reagent for measurement and 10 μl of the sample were mixed and reacted at 8 ° C. for 10 minutes. Next, 20 μl of this reaction solution was measured (analyzed) by HPLC under the above conditions.

(result)
FIG. 7 shows the elution salt concentration and peak width of the AFP antigen-antibody complex obtained for the eluent to which various surfactants were added. In FIG. 7, ● indicates the salt concentration at the peak of the elution peak, and I indicates the range of salt concentration at the beginning and end of the elution (that is, peak width). In addition, each symbol on the horizontal axis of the figure indicates the type of surfactant (surfactant No. in Table 3).
The following can be understood from the results of FIG.
(1) It can be seen that by adding a surfactant, the peak width can be narrowed, in other words, peak tailing during elution can be prevented, and in other words, the measurement accuracy can be improved. . Moreover, it turns out that this effect appears notably with a cationic surfactant and an amphoteric surfactant.
(2) The elution salt concentration (elution position) fluctuates by adding a surfactant, in other words, the elution salt concentration of the target antigen-antibody complex is appropriately adjusted by appropriately selecting the type of surfactant. It turns out that it can be set.

<Separation by electrophoresis>
(sample)
AFP-A4 • Fab ′ prepared in Example 10, Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4 • Fab ′, AFP-WA2 • Fab ′ prepared in Example 15, A sample prepared by diluting the conjugate of Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 • Fab ′ with 50 mM MOPS buffer (pH 7.5) to 1 mg / ml each was used.
(Measurement operation)
Apply 4 μl of each sample to the sample groove of 1% agarose gel, perform electrophoresis at a voltage of 200 V for 30 minutes using the sample groove side as a cathode, and then use Quick-CBB (trade name of Wako Pure Chemical Industries, Ltd.) The protein was stained and the Rf value of each sample was measured.
(result)
Each Rf value is AFP-A4 • Fab ′ 0.38, Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4 • Fab ′ combined 0.66, AFP-WA2 • Fab ′ 0.06, Ala- ( Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 • Fab ′ were combined at 0.22.
From this result, it can be seen that by binding the sulfated peptide, the negative charge increases and the mobility increases. It can also be seen that the mobility increases as the number of sulfate residues in the sulfated peptide increases.

<Separation of sulfated peptide-bound antibody and antigen reaction product>
(sample)
Example 50 Apl of AFP prepared to 50 mg MOPS buffer (pH 7.5) to 0.5 mg / ml was added to Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4 prepared in Example 17. • 50 μl of Fab ′ conjugate (1 mg / ml), or 50 μl of Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 • Fab ′ conjugate (1 mg / ml), or MOPS buffer (pH 7) .5) A sample prepared by adding 50 μl and reacting at 37 ° C. for 30 minutes was used.
(Measurement operation)
Apply 4 μl of each sample to the sample groove of 1% agarose gel, perform electrophoresis for 30 minutes at a voltage of 200 V with the sample groove side as the cathode, and then antibody affinity transfer (blotting) with nitrocellulose membrane bound with anti-AFP antibody For 30 minutes at room temperature. The membrane was washed twice with a washing solution (0.9% NaCl). This membrane was immersed in an anti-AFP antibody solution at 37 ° C. for 30 minutes, and then washed twice with a washing solution. Next, this membrane was immersed in a POD-labeled anti-IgG antibody solution at 37 ° C. for 30 minutes, and then washed twice with a washing solution. Further, this membrane was developed in a coloring solution (0.37 mM nitrotetrazolium blue, 2.6 mM β-nicotinamide adenine dinucleotide reduced type, 0.01% hydrogen peroxide), and the Rf value of the antigen-antibody reaction product was measured.
(result)
The Rf value of the conjugate of Ala- (yr (SO ₃ H)) ₈ and AFP-A4Fab 'and the antigen-antibody reaction of AFP is 0.63, Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP- The Rf value of the conjugate of WA2 • Fab ′ and the antigen-antibody reaction product of AFP was 0.20, and the Rf value of only AFP was 0.85.
As is apparent from the above results and the results of Example 17, the Rf value of the Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4 • Fab ′ conjugate and AFP antigen-antibody reaction product, and Ala -(Tyr (SO ₃ )) ₈ -Rf values of the conjugate of βAla and AFP-WA2 ・ Fab 'and the antigen-antibody reaction of AFP are Ala- (Tyr (SO ₃ )) ₈ and AFP-A4- The Rf value of the conjugate of 4 • Fab ′ and the Rf value of the conjugate of Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 • Fab ′ were almost the same. Thus, the negative charge of AFP is the Rf value of the conjugate of Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4Fab 'and the antigen-antibody reaction of AFP, and Ala- (Tyr (SO ₃ H )) Rf values of _5- βAla-AFP-WA2 • Fab ′ conjugate and AFP antigen-antibody reaction are hardly affected.

The elution position of the anionic polypeptide by the high performance liquid chromatography (HPLC) using the anion exchange column obtained in Example 8 is shown. The elution position of the antibody Fab ′ fragment and the anionic polypeptide conjugate obtained by HPLC using the anion exchange column obtained in Example 11 is shown. The data of the storage stability in various pH solutions of the polypeptide which has a sulfated serine residue obtained in Example 12 are shown. The data of the storage stability in various pH solutions of the polypeptide which has the sulfated tyrosine residue obtained in Example 12 are shown. The elution positions of various antigen-antibody complexes by HPLC using an anion exchange column obtained in Example 14 are shown. The measurement (analysis) elution pattern of the sample obtained in Example 15 by HPLC is shown. The elution positions of various antigen-antibody complexes obtained by HPLC using an anion exchange column obtained in Example 16 are shown.

Explanation of symbols

  1, FIG. 2 and FIG. 5, ● indicates the salt concentration at the peak of the elution peak, and I indicates the range of salt concentration at the beginning and end of the elution (that is, peak width). Each symbol on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1).

  3 and 4, the black bars show the results obtained for the sample on the 6th day of storage, and the white bars show the results obtained for the sample on the 19th day of storage.

  In FIG. 6, the solid line (-) indicates the data obtained for the sample 1, and the dotted line (----) indicates the data obtained for the sample 2.

  In FIG. 7, ● indicates the salt concentration at the peak of the elution peak, and I indicates the range of salt concentration at the beginning and end of the elution (that is, peak width). In addition, each symbol on the horizontal axis of the figure indicates the type of surfactant (surfactant No. in Table 3).

Claims (10)

Binding ability to a polypeptide having 3 to 30 acid residues derived from a strong acid selected from sulfuric acid and phosphoric acid and having a total number of amino acid residues of 3 to 30 and a substance to be measured in a biological sample Combined with a substance having The conjugate according to claim 1, wherein the polypeptide is a polypeptide having 4 to 20 sulfated tyrosine residues. The polypeptide has the following general formula [1]
^{A- (R 4) j-B} [1]
(Wherein, j is 4-20 of .j number of R ⁴ represents an integer of 4 to 20 are sulfated tyrosine residues, and the remaining R ⁴ may be the same or different, and sulfuric acid It represents an amino acid residue in which an acid residue derived from a strong acid selected from phosphoric acid has not been introduced, and a protective group may be introduced into the reactive group of the side chain of each amino acid residue, where A is a hydrogen atom, sulfuric acid Represents a protecting group for the N-terminal amino group of an amino acid into which a tyrosine residue or an acid residue derived from a strong acid selected from sulfuric acid and phosphoric acid has not been introduced, and B represents a protecting group for the hydroxyl group or the C-terminal carboxyl group of an amino acid .)
The conjugate | bonded_body of Claim 1 which is polypeptide represented by these. The polypeptide has the following general formula [2]
^{A- (R 5) k-B} [2]
(Wherein R ⁵ represents an amino acid residue into which an acid residue derived from a strong acid selected from sulfuric acid and phosphoric acid has been introduced, and may be the same or different. K represents an integer of 4 to 20, and A represents Represents an acid residue derived from a strong acid selected from a hydrogen atom, sulfuric acid and phosphoric acid, or a protecting group for the N-terminal amino group of an amino acid, and B represents a protecting group for a hydroxyl group or a C-terminal carboxyl group of an amino acid, provided that k 4-20 of R ⁵ are sulfated tyrosine residues.)
The conjugate | bonded_body of Claim 1 which is polypeptide represented by these. The polypeptide has the following general formula [3]
^{A- (R 5) j- (R} 6) l-B [3]
(Wherein R ⁵ represents a sulfated tyrosine residue and j represents an integer of 4 to 20. R ⁶ represents an amino acid residue into which an acid residue derived from a strong acid selected from sulfuric acid and phosphoric acid has not been introduced. (J + 1) is 5 to 21. A is a hydrogen atom, a sulfated tyrosine residue, or an amino acid N-terminal amino group into which an acid residue derived from a strong acid selected from sulfuric acid and phosphoric acid has not been introduced. Represents a protecting group, and B represents a protecting group for a hydroxyl group or a C-terminal carboxyl group of an amino acid.)
The combination according to claim 3, represented by: A compound having a maleimide group bound to the N-terminus of a polypeptide having 3 to 30 acid residues derived from a strong acid selected from sulfuric acid and phosphoric acid and having 3 to 30 amino acid residues in total via a spacer . A reagent for measuring a substance to be measured in a biological sample, comprising the conjugate according to claim 1. A biological sample and a reagent comprising the conjugate according to claim 1 are reacted, and then the resulting complex is separated by a method using a negative charge, and the measurement target in the biological sample is based on the amount of the complex. A method for measuring a biological component, characterized in that a substance amount is obtained. The biological component measuring method according to claim 8, wherein the method using a negative charge is an anion exchange method. The biological component measurement method according to claim 9, wherein a surfactant is allowed to coexist in an eluent used for separation by an anion exchange method.

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