JPH0544960B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel peptide. The peptide provided by the present invention can be easily immobilized on a carrier and then heat-treated, thereby achieving the ability to specifically react with human antibodies against nicotinic acetylcholine receptors and selectively adsorb the antibodies. Express. Therefore, the peptide provided by the present invention is believed to be the central cause of the pathology of neuromuscular transmission disorders caused by autoantibodies against nicotinic acetylcholine receptors present on the postsynaptic membrane of the neuromuscular junction. Useful in the treatment of myasthenia gravis. [Prior Art] Nature, Volume 299, Nos. 793-797
(1982) reported that the α-subunit precursor of the nicotinic acetylcholine receptor obtained from the electric organ of Torpedo californica, a species of ray, consists of 461 amino acids. It has been reported that the primary structure has been elucidated. According to this report, the amino acid sequence at positions 183 to 200 in the primary structure of the α-subunit precursor has the formula -Gly-
Trp−Lys−His−Trp−Val−Tyr−Tyr−Thr
−Cys−Cys−Pro−Asp−Thr−Pro−Tyr−Leu
Indicated by -Asp-. Proceedings
of the National Academy of Sciences of the United States
Proceedings of the National
Academy of Sciences of the United States
of America), Vol. 84, pp. 3633-3637 (1987
182 in the primary structure of the α-subunit of nicotinic acetylcholine receptor obtained from Torpedo californica electric organ.
A peptide corresponding to the amino acid sequence at position ~198 was synthesized, and this peptide was transferred to an agarose-based carrier [CNBr-activated Sepharose CL-4B (CNBr-
It has been reported that an adsorbent formed by immobilization on activated Sepharose CL-4B) binds to mouse and rabbit antibodies against nicotinic acetylcholine receptors. Biochemical and Biophysical Research Communications
Biophysical Research Communications), No.
Volume 135, pp. 82-89 (1986), Torpedo
A fragment with a molecular weight of 18 kilodaltons, which is thought to correspond to the amino acid sequence at positions 153 to 350 in the primary structure of the α-subunit, was obtained by degrading the α-subunit of the nicotinic acetylcholine receptor obtained from C. californica with protease. It has been reported that this fragment binds to a mouse monoclonal antibody directed against the ligand binding site of the nicotinic acetylcholine receptor and to α-bungarotoxin. [Problem to be solved by the invention] In the treatment of myasthenia gravis, it is desired to establish a means to remove human autoantibodies against nicotinic acetylcholine receptors, which are said to be the main causative agent of myasthenia gravis. The reality is that no practical means have yet been established. Therefore, an object of the present invention is to provide a novel peptide useful for efficiently producing an adsorbent capable of effectively adsorbing human antibodies against nicotinic acetylcholine receptors. [Means for Solving the Problem] According to the present invention, the above object is achieved by solving the general formula H-X
-Gly-A-Lys-His-B-Val-Tyr-Tyr-
Thf−Cys−Cys−Pro−Asp−Thr−Pro−Tyr−
Leu-Asp-Y-Z () [wherein A and B each represent Phe or Tyr, X and Y each represent a single bond, or Asp, Glu, Lys and the formula (In the formula, n represents an integer of 1 to 17.) 2 to 10 amino acid residues selected from the group consisting of divalent groups represented by or at least one type of amino acid residue selected from the group Represents a peptide residue formed by a peptide bond, Z represents a hydroxyl group or an amino group, and Cys-Cys of the above amino acid sequence
The mercapto groups of each Cys may be bonded to each other to form a disulfide bond. ] This is achieved by providing a peptide shown in the following. Various amino acid residues are described herein using conventional abbreviations. Abbreviations are well known in the technical field of the present invention, and those related to the present invention are listed below. Asp: L-aspartic acid residue Cys: L-cysteine residue Glu: L-glutamic acid residue Gly: Glycine residue His: L-histidine residue Leu: L-leucine residue Lys: L-lysine residue Phe: L-phenylalanine residue Pro: L-proline residue Thr: L-threonine residue Tyr: L-tyrosine residue Val: L-valine residue In addition, in this specification, the amino acid sequence is determined according to a conventional method. It is written so that the N-terminal amino acid is located on the left and the C-terminal amino acid is located on the right. X and Y in general formula () are defined as above, but peptides in which both X and Y are single bonds and amino acid residues or peptide residues in which either X or Y are different from those defined above. Not only may the base peptide not be immobilized efficiently on the carrier, but even when it is immobilized on the carrier and heat-treated, its ability to adsorb human antibodies against nicotinic acetylcholine receptors may not be sufficient. It may not occur. Examples of the peptide residues represented by X and Y in the general formula () include the following peptide residues.

【table】 ‖
O
(-Asp)- ₁₀ , (-Glu)- ₁₀ , (-Lys)- ₁₀ , (-Gl
y) _-10 ,
〓-NH(CH ₂ ) ₁₁ C〓- ₁₀ , 〓-NH(CH ₂ ) ₁₇ C〓- _1
0 , −Lys−Glu
‖ ‖
OO
-Gly-NH( _CH2 ) _11C- ,Asp-Asp-Lys-Lys-Glu
‖
O
−Gly−, −Lys−Glu−Glu−Asp−Asp−Lys−Lys
−Gly−Gly−
The synthesis of the peptide represented by the general formula () is carried out by methods commonly used in peptide synthesis, such as solid phase synthesis; or by liquid phase synthesis such as stepwise elongation and fragment condensation; It is operationally simple to carry out the phase synthesis method [for example, the Journal of the American
Chemical Society (Journal of the
American Chemical Society), Volume 85, No. 2149
〜2154 pages (1963); Edited by the Japanese Biochemical Society, "Biochemistry Experiment Course 1: Chemical Modification of Proteins and Peptide Synthesis" (published by Tokyo Kagaku Dojin Co., Ltd. on November 15, 1971), pp. 207-495; Japan Edited by the Biochemical Society, “Continued Biochemistry Experiment Course 2 Chemistry of Proteins (Part 2)” (May 20, 1988, published by Tokyo Kagaku Dojin Co., Ltd.), No. 641-
See page 694, etc.] The production of the peptide represented by the general formula () by solid-phase synthesis involves, for example, removing a hydrogen atom from the α-carboxyl group or α-carbamoyl group of the amino acid or amino acid amide corresponding to the C-terminus of the target peptide, respectively. The corresponding amino acid is added to a reaction solvent-insoluble polymer such as a styrene-divinylbenzene copolymer to which an acyloxy group or an acylamino group is bonded, in the direction of the N-terminus of the desired peptide. A process of protecting a functional group such as an α-amino group other than the α-carboxyl group, and then condensing and bonding it, and a protective group of an amino group forming a peptide bond such as an α-amino group in the bound amino acid. By sequentially repeating the removal operation, the peptide chain is elongated to form a peptide chain corresponding to the desired peptide, and then the peptide chain is detached from the polymer and removed from the protected functional group. This is carried out by removing the protecting group to obtain the desired peptide and then purifying it. Here, it is preferable to simultaneously remove the peptide chain from the polymer and remove the protecting group using hydrogen fluoride from the viewpoint of suppressing side reactions. Furthermore, it is effective to purify the obtained peptide by reverse phase liquid chromatography. The peptide represented by the general formula () can be efficiently immobilized on a carrier. When a peptide represented by the general formula () immobilized on a carrier is subjected to heat treatment, it significantly exhibits the ability to adsorb human antibodies against nicotinic acetylcholine receptors in body fluids such as serum. The carrier used to immobilize the peptide represented by the general formula () includes amino groups, carboxyl groups, Those having a reactive functional group such as a hydroxyl group are preferred. Furthermore, when the peptide is used as an adsorbent for adsorbing human antibodies against nicotinic acetylcholine receptors in body fluids, the carrier is preferably insoluble in body fluids and porous. Since porous carriers have a large effective surface area for adsorbing human antibodies against nicotinic acetylcholine receptors, such carriers should have an exclusion limit protein molecular weight in the range of approximately 10 ⁶ to 10 ⁹ or an average pore diameter. is preferably within the range of about 50 to 1000 nanometers. The carrier can be in any shape such as particulate, fibrous, sheet, or hollow fiber. Examples of such carriers include cellulose-based carriers such as CM-Cellulofine CH (exclusion limit protein molecular weight: approximately 3×10 ⁶ ; sold by Seikagaku Corporation); CM-Toyopearl 650C (exclusion limit protein molecular weight: 5×
10 ⁶ ; Polyvinyl alcohol carrier such as Tosoh Corporation); CM-Tris Acrylic M (CM-
Trisacryl M) [Exclusion limit protein molecular weight: 1
×10 ⁷ ; Sweden - Pharmacia - LKB
Polyacrylamide-based carriers such as (Pharmacia-LKB); Sepharose CL-4B (Sepharose CL-4B);
CL-4B) [Exclusion limit protein molecular weight: 2×
10 ⁷ ; Pharmacia, Sweden - LKB
Organic carriers such as agarose-based carriers such as (Pharmacia-LKB); and CPG-10-1000
Inorganic supports such as porous glass such as [Exclusion limit protein molecular weight: 1×10 ⁸ ; average pore diameter: 100 nm; manufactured by Electro-nucleonics, Inc.] may be used. Immobilization of the peptide represented by the general formula () onto a carrier is performed according to a method generally employed when immobilizing a peptide or protein onto a carrier. As an immobilization method, for example, the carboxyl group of the carrier is reacted with N-hydroxysuccinimide to convert it into a succinimidoxycarbonyl group, and then the peptide represented by the general formula () is added to the amino group. A method of reacting with a carboxyl group or an amino group of a peptide represented by the general formula ( condensation method), and a method of crosslinking a carrier and a peptide represented by the general formula () using a compound having two or more functional groups such as glutaraldehyde (carrier crosslinking method). An adsorbent obtained by immobilizing a peptide represented by the general formula () on a carrier using an active ester method has the highest ability to adsorb human antibodies to nicotinic acetylcholine receptors. General formula ()
The amount of peptide immobilized on the carrier, ^represented by In order for the peptide represented by the general formula () immobilized on the carrier to be effectively utilized for the adsorption of human antibodies, approximately 1 × 10 ^-7 to 2 × 10 ^-6 mol/g (carrier ) is preferably within the range. The peptide represented by the general formula () immobilized on a carrier exhibits high activity for adsorbing human antibodies against nicotinic acetylcholine receptors when subjected to heat treatment as described above. The heat treatment temperature is preferably 60°C or higher; however, if the heat treatment temperature is too high, the peptide and/or the carrier may decompose, so the heat treatment temperature is preferably kept at 180°C or lower. Further, the heat treatment time is preferably about 5 minutes or more,
If the heat treatment time is too long, the peptide and/or
Otherwise, the carrier may decompose, so the heat treatment time is preferably within about 1 hour. The heat treatment can be carried out by heating the peptide immobilized on the carrier to a predetermined temperature in water or an aqueous solution. It is preferable to carry out the heat treatment in an aqueous buffered salt solution such as a phosphate buffer containing sodium chloride, since decomposition of the peptide can be suppressed. Antibodies against nicotinic acetylcholine receptors can be removed by contacting an adsorbent obtained by immobilizing a peptide represented by the general formula () on a carrier with body fluids such as blood, plasma, and serum containing the antibody.
This is carried out by adsorbing the antibody onto an adsorbent. For example, the adsorbent is used by filling a column. The column used for this purpose has an inlet and an outlet shaped so that it can be easily connected to the blood circuit, and a material such as polyester is used between the inlet and the adsorbent layer and between the outlet and the adsorbent layer. It is desirable to have a filter made of the same material. Examples of the material for the column include polyethylene, polypropylene, polycarbonate, polyester, and polymethyl methacrylate. Of these, polypropylene and polycarbonate require autoclave sterilization and γ
- It is particularly suitable in that it can be subjected to sterilization such as radiation sterilization. Removal of antibodies against nicotinic acetylcholine receptors from a patient's body fluids using the above-described packed columns takes place, for example, in an extracorporeal blood circulation system. The following two types of extracorporeal blood circulation systems can be exemplified. (1) Blood taken from the patient's blood vessel is sent to a column filled with an adsorbent, where antibodies against nicotinic acetylcholine receptors are removed from the blood by adsorption, and the treated blood that has passed through the column is then circulated to the patient's blood vessel. do. (2) Blood taken from a patient's blood vessel is first separated into blood cells and plasma components, and the separated plasma components are sent to a column filled with an adsorbent, where antibodies against nicotinic acetylcholine receptors are removed from the plasma components by adsorption. The treated plasma components that have passed through the column are then mixed with the separated blood cell components, and the resulting mixture is then circulated into the patient's blood vessels. [Examples] The present invention will be explained below with reference to Examples, but the present invention is not limited by the Examples. Example 1

The peptides shown in [Table] were synthesized using an automatic peptide synthesizer [Applied Biosystems (USA)].
Biosystems) Model 430A]
It was synthesized by solid phase synthesis using. 4-[N-
(t-butoxycarbonyl)glycyloxymethyl]phenylacetamidomethyl Styrene-divinylbenzene copolymer having groups at a ratio of 0.78 mmol/g (resin) [component ratio (mole ratio) of styrene and divinylbenzene: 99:1]
Using 0.64 g of granular resin [PAM Glycine, t-Boc-Gly, manufactured by Applied Biosystems, USA],
Then, according to the series of operations shown in Table 1, the corresponding L-
Aspartic acid, L-cysteine, glycine, L
-Histidine, L-leucine, L-lysine, L-
Proline, L-threonine, L-phenylalanine, L-tyrosine and L-valine were sequentially combined. In the condensation reaction, the above amino acids are N-(t-butoxycarbonyl) ^-O4 -benzyl-L-aspartic anhydride, N-(t-butoxycarbonyl)-S-(p-methoxybenzyl), respectively.
-L-cysteine anhydride, N-(t-butoxycarbonyl)glycine anhydride, N ^a -(t-butoxycarbonyl)-N ^Im -tosyl-L-histidine anhydride, N-(t-butoxycarbonyl)-L -Leucine anhydride, ^N2- (t-butoxycarbonyl)-
N ⁶ -benzyloxycarbonyl-L-lysine anhydride, N-(t-butoxycarbonyl)-L-proline anhydride, N-(t-butoxycarbonyl)-
O ³ -benzyl-L-threonine anhydride, N ^a -(t
-butoxycarbonyl)-L-phenylalanine anhydride, N-(t-butoxycarbonyl)-O ⁴ -
Benzyl-L-tyrosine anhydride and N-(t-
(butoxycarbonyl)-L-valine anhydride, and the amount used was approximately twice the molar amount relative to the substrate. The condensation reaction is carried out at room temperature, and the reaction time varies depending on the type of amino acid to be condensed.
It was within 30 minutes. In addition, since the conversion rate is low in the condensation reaction using N ^a -(t-butoxycarbonyl)-N ^Im -tosyl-L-histidine anhydride, after completing the series of operations shown in Table 1, The condensation reaction was carried out again by repeating steps 4 to 6.

【table】

[Table] After the reaction operations for all amino acids were completed, the obtained resin was sequentially washed with diethyl ether, dichloromethane and methanol on a glass filter, and then vacuum dried to obtain 2.1 g of dry resin. I got it. In a reaction vessel made of polytrifluoromonochloroethylene (HF-Reactor Type I manufactured by Peptide Institute Co., Ltd.), 1 g of dry resin was mixed with 1.5 ml of anisole and 0.25 ml of ethyl methyl sulfide, and this mixture was mixed with -20 ℃
10 ml of hydrogen fluoride was added at a temperature of 0.degree. C., and the mixture was stirred at the same temperature for 30 minutes and then at 0.degree. C. for 30 minutes. Hydrogen fluoride, anisole and ethyl methyl sulfide were removed from the resulting reaction mixture under reduced pressure, and the residue was thoroughly washed with diethyl ether on a glass filter. The obtained residue was extracted with a 2N acetic acid aqueous solution, and the extract was freeze-dried to obtain 0.5 mg of a crude peptide. The obtained crude product was subjected to preparative reverse-phase high-performance liquid chromatography [Column: octadecylated silica gel (particle size: 5 μm) packed column (inner diameter: 10 mm, length:
300mm) (Develosil ODS10mmφ x 300mm manufactured by Kemco Co., Ltd.); Mobile phase: mixed solvent of acetonitrile and water containing 0.05% trifluoroacetic acid (concentration of acetonitrile increased from 20% to 35% by volume in 20 minutes) 50 mg of the purified peptide of interest was obtained by purifying the target peptide using 50 mg of purified peptide. The obtained purified product was subjected to analytical reverse phase high performance liquid chromatography [Column: octadecylated silica gel (particle size: 5 μm) packed column (inner diameter: 4 mm, length:
150mm) (TSKgel ODS− manufactured by Tosoh Corporation)
80TM 4mmφ×150mm); Mobile phase: mixed solution of acetonitrile and water containing 0.05% trifluoroacetic acid (the concentration of acetonitrile was gradually changed from 5% by volume to 50% by volume in 30 minutes); flow rate : 1 ml/min; detection method: absorbance at a wavelength of 210 nm], a single sharp peak was observed at 18.1 minutes. The molecular weight of the purified product determined by FAB (fast atom bombardment) mass spectrum is
It was 2737 (theoretical value: 2737.15). Further, the product obtained by hydrolyzing the purified product using hydrochloric acid was subjected to amino acid composition analysis, and the results were as follows (numbers in parentheses indicate theoretical values). lysine:
5.22(5), glycine: 1.95(2), phenylalanine:
2.01(2), histidine: 0.97(1), valine: 0.93(1),
Tyrosine: 3.70(3), Threonine: 2.05(2), Cystine: 0.87(1), Proline: 2.13(2), Aspartic acid: 2.10(2), Leucine: 1.01(1). Examples 2-16 The peptides shown in Table 2 were obtained by solid-phase synthesis and purification of peptides in the same manner as in Example 1. However, as a solid phase resin,
In Example 2 and Example 10, a styrene-divinylbenzene copolymer [styrene and divinylbenzene composition ratio (mole ratio): 99:1] [Applied Biosystems (USA)]
Biosystems) PAM Glycine, t
-Boc-Gly], and in Example 3, Example 5, Example 8, and Example 12, 4-[N-(t-butoxycarbonyl)-O ⁴ -benzyl-α-L-aspartyloxymethyl ] Granular resin consisting of a styrene-divinylbenzene copolymer having phenylacetamidomethyl groups at a ratio of 0.78 mmol/g (resin) [component ratio (mole ratio) of styrene and divinylbenzene: 99:1] [U.S. Applied Co., Ltd.] Applied Biosystems PAM Aspartic acid, t-Boc-L-
Asp(OBzl)], Example 4 and Example 6
Then 4-[N-(t-butoxycarbonyl)-O ⁵ −
0.78 mmol/g of benzyl-α-L-glutamyloxymethyl]phenylacetamidomethyl group
(resin) [Mole ratio of styrene and divinylbenzene: 99:1]
Biosystems) PAM Glutamic acid (Glutamic acid), t-Boc-L-Glu (OBzl)]
In Examples 7, 9 and 11, 4-[ ^N2- (t-butoxycarbonyl) ^-N6- (chlorobenzyloxycarbonyl)-L-lysyloxymethyl]phenylacetamidomethyl group was used.
Granular resin made of styrene-divinylbenzene copolymer [component ratio (mole ratio) of styrene and divinylbenzene: 99:1] at a ratio of 0.78 mmol/g (resin) [Applied Biosystems, USA] In Examples 13 to 16, α-amino-p-methylbenzyl group was added at a rate of 0.78 mmol/g (resin). A granular resin consisting of a styrene-divinylbenzene copolymer [component ratio (molar ratio) of styrene and divinylbenzene: 99:1] [Applied Biosystems, USA]
p-Methyl BHA resin (p-Methyl BHA)
Resin)] was used. Also, in the condensation reaction, L-
Glutamic acid, 12-aminododecanoic acid and 18-
Aminooctadecanoic acid is N-(t-butoxycarbonyl) ^-O5 -benzyl-L-glutamic anhydride, 12-(t-butoxycarbonylamino)dodecanoic anhydride, and 18-(t-butoxycarbonylamino)octadecane, respectively. It was used as an acid anhydride. When the purified peptides obtained were subjected to analytical reverse phase high performance liquid chromatography, a single peak was observed in each case. Table 3 shows the molecular weights of these purified products determined by FAB mass spectroscopy and the amino acid composition analysis values of the products obtained by hydrolysis with hydrochloric acid.

【table】

【table】

【table】

[Table] Numbers in parentheses indicate theoretical values.
Examples 17-32 The peptides shown in Table 4 were obtained by solid-phase synthesis and purification of peptides in the same manner as in Example 1. However, as a solid phase resin,
In Example 17, Example 18 and Example 26, 4-[N
Styrene-divinylbenzene copolymer having -(t-butoxycarbonyl)glycyloxymethyl]phenylacetamidomethyl groups at a ratio of 0.78 mmol/g (resin) [composition ratio (molar ratio) of styrene and divinylbenzene: 99 Example 19 using a granular resin [PAM Glycine, t-Boc-Gly manufactured by Applied Biosystems, USA]
In Example 21, Example 24 and Example 28, 4-[N
-(t-butoxycarbonyl-O ⁴ -benzyl-α
-L-aspartyloxymethyl] styrene-divinylbenzene copolymer containing phenylacetamidomethyl copolymer at a ratio of 0.78 mmol/g (resin) [composition ratio (molar ratio) of styrene and divinylbenzene: 99:1 ] [Applied Biosystems (U.S.)
Biosystems) PAM Aspartic acid (Aspartic acid), t-Boc-L-Asp (OBzl)]
In Example 20 and Example 22, 4-[N-
(t-butoxycarbonyl)-O ⁵ -benzyl-α
-L-glutamyloxymethyl] phenylacetamidomethyl group at a ratio of 0.78 mmol/g (resin), consisting of a styrene-divinylbenzene copolymer [component ratio (mole ratio) of styrene and divinylbenzene: 99:1] Granular resin [Applied Biosystems, USA]
PAM Glutamic acid, t-
Boc-L-Glu(OBzl)], and in Example 23, Example 25 and Example 27, 4-[ ^N2- (t-butoxycarbonyl) ^-N6- (chlorobenzyloxycarbonyl)-L-lysyl [U.S. Applied Biosystems
Biosystems) PAM Lysine, t-
Boc-L-Lys(Cl-Z)] and Example 24
~32 has an α-amino-p-methylbenzine group.
Granular resin consisting of styrene-divinylbenzene copolymer [component ratio (mole ratio) of styrene and dihinylbenzene: 99:1] at a ratio of 0.78 mmol/g (resin)] Manufactured by Applied Biosystems, USA p-methyl BHA
resin (p-Methyl BHA Resin)] was used.
In addition, in the condensation reaction, L-glutamic acid, 12-aminododecanoic acid and 18-aminooctadecanoic acid are each converted to N-(t-butoxycarbonyl)-
O ⁵ -benzyl-L-glutamic anhydride, 12-
(t-butoxycarbonylamino)dodecanoic anhydride and 18-(t-butoxycarbonylamino)
It was used as octadecanoic anhydride. When the purified peptides obtained were subjected to analytical reverse phase high performance liquid chromatography, a single peak was observed in each case. Table 5 shows the molecular weights of these purified products determined by FAB mass spectroscopy and the amino acid composition analysis values of the products obtained by hydrolysis with hydrochloric acid.

【table】

【table】

【table】

【table】

[Table] Numbers in parentheses indicate theoretical values.
Examples 33 and 34 By performing solid phase synthesis and purification of peptides in a manner similar to that in Example 1, the formula

The peptide (Example 34) shown in [Table] was obtained. However, as a solid phase resin, 4-[N-(t-butoxycarbonyl)-O ⁴ -benzine-α-L-aspartyloxymethyl]phenylacetamidomethyl group is used at a ratio of 0.78 mmol/g (resin). Styrene-divinylbenzene copolymer having [component ratio (mole ratio) of styrene and divinylbenzene: 99:1]
A granular resin consisting of [PAM Aspartic acid, t-Boc-L-Asp manufactured by Applied Biosystems, USA]
(OBzl)] was used. When the purified peptides obtained were subjected to analytical reverse phase high performance liquid chromatography, a single peak was observed in each case. Table 6 shows the molecular weights of these purified products determined by FAB mass spectroscopy and the amino acid composition analysis values of the products obtained by hydrolysis with hydrochloric acid.

[Table] Numbers in parentheses indicate theoretical values.
Reference Example 1 (a) Suspend 10 g of cellulose particles (CM-Cellulofine CH, sold by Seikagaku Corporation) in 50 ml of dioxane obtained by distillation in the presence of metallic sodium, and suspend the resulting suspension. Add 0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide to the liquid,
The mixture was shaken and stirred at room temperature overnight. The resulting mixture was diluted with 0.02 mol/phosphate buffer (PH:
7.4) and aspirated. The obtained particles were added to a 0.02 mol/phosphate buffer (PH: 7.4) containing 20 mg of the peptide obtained in Example 1.
20 ml and the mixture was stirred overnight at a temperature of 4°C. The resulting mixture was filtered under suction.
The solution was subjected to analytical reverse-phase high-performance liquid chromatography, but no residual unreacted peptide was observed (immobilization rate of peptide on the carrier: approx.
100%). In this way, about 10 g of cellulose particles on which 20 mg of the peptide obtained in Example 1 was immobilized were obtained. (b) Suspend 1 g each of the cellulose particles on which the peptides obtained as described above are immobilized in 5 ml of 0.02 mol/phosphate buffer (PH: 7.4) containing 0.15 mol/sodium chloride. cloudy,
Heat treated in an autoclave sterilizer under pressure at a temperature of 121° C. for 20 minutes. An adsorbent was thus obtained. Reference Example 3 (a) Porous glass particles [manufactured by Electro-nucleonics, USA]
CPG-10-1000] was reacted in 100 ml of a toluene solution containing 5 ml of γ-aminopropyltriethoxysilane under heating under reflux for 24 hours.
The resulting mixture was washed with dioxane obtained by distillation in the presence of metallic sodium and filtered off with suction. The particles obtained were suspended in 100 ml of dioxane obtained by distillation in the presence of sodium metal, 3 g of succinic anhydride was added to this suspension, and the mixture was stirred overnight at room temperature. The resulting mixture was washed with dioxane obtained by distillation in the presence of metallic sodium and filtered off with suction. The obtained particles were suspended in 50 ml of dioxane obtained by distillation in the presence of metallic sodium, 0.5 g of N-hydroxysuccinimide and 1.0 g of dicyclohexylcarbodiimide were added to this suspension, and the mixture was was stirred at room temperature overnight. The resulting mixture was washed with 0.02 mol/phosphate buffer (PH: 7.4) and filtered by suction.
The obtained particles were mixed with the peptide obtained in Example 3.
20 ml of 0.02 mol/phosphate buffer (PH: 7.4) containing 20 mg and the mixture was stirred overnight at a temperature of 4°C. The resulting mixture was filtered by suction to obtain about 10 g of porous glass particles on which 20 mg of the peptide obtained in Example 3 was immobilized (peptide immobilization rate: about 100%). (b) Same procedure as in Reference Example 2(b) except that 1 g of the porous glass particles on which the peptide was immobilized obtained as described above was used instead of 1 g of the polyvinyl alcohol particles on which the peptide was immobilized. According to the method, an adsorbent was obtained. Reference Examples 4 to 16 (a) Peptides were prepared in the same manner as in Reference Example 1(a), Reference Example 2(a), or Reference Example 3(a) except that 20 mg of the peptide shown in Table 7 was used. A particulate carrier on which was immobilized was obtained. Table 7 shows the particulate carrier used and the immobilization rate of the peptide on the carrier. (b) Reference except that 1 g of the particulate carrier on which the peptide obtained as above was immobilized was used instead of 1 g of polyvinyl alcohol particles on which the peptide obtained in Reference Example 2(a) was immobilized. The adsorbents were each obtained by a method similar to that in Example 2(b).

[Table] Reference Example 17 (a) In Reference Example 1(a), the peptide obtained in Example 33 was used instead of 20 mg of the peptide obtained in Example 1.
Example 2 was carried out in the same manner except that 20 mg was used.
Approximately 10 g of cellulose particles on which 15.0 mg of the peptide obtained in step 33 was immobilized were obtained (peptide immobilization rate: approximately 75%). (b) By the same method as in Reference Example 2(b), except that 1 g of the cellulose particles on which the peptide was immobilized obtained as described above was used instead of 1 g of the polyvinyl alcohol particles on which the peptide was immobilized. , an adsorbent was obtained. Reference Example 18 (a) In Reference Example 2(a), the peptide obtained in Example 34 was used instead of 20 mg of the peptide obtained in Example 2.
Example 2 was carried out in the same manner except that 20 mg was used.
Approximately 10 g of polyvinyl alcohol particles on which 15.2 mg of the peptide obtained in step 34 was immobilized were obtained (peptide immobilization rate: approximately 76%). (b) Reference Example 2 except that 1 g of the polyvinyl alcohol particles obtained by the above immobilization procedure was used instead of 1 g of the polyvinyl alcohol particles on which the peptide obtained in Reference Example 2 (a) was immobilized. An adsorbent was obtained by the same method as in (b). Reference Examples 19-34 Peptides were immobilized by the same method as in Reference Example 1(a), Reference Example 2(a), or Reference Example 3(a) except that 30 mg of the peptide shown in Table 8 was used. A particulate carrier was obtained. Table 8 shows the particulate carrier used and the immobilization rate of the peptide on the carrier. (b) Reference except that 1 g of the particulate carrier on which the peptide obtained as above was immobilized was used in place of 1 g of polyvinyl alcohol particles on which the peptide obtained in Reference Example 2(a) was immobilized. The adsorbents were each obtained by a method similar to that in Example 2(b).

[Table] Test Example 1 50 mg of the adsorbent obtained in Reference Example 1 was added to 0.5 ml of serum from a myasthenia gravis patient and suspended at a temperature of 37°C for 3 hours. The resulting suspension was centrifuged to obtain a supernatant. The concentration of human antibody against nicotinic acetylcholine receptor in the obtained supernatant was determined.
Con A method [Protein Nucleic Acid Enzyme, Volume 26, No. 1578~
1591 (1981) etc.]. That is, the test solution was sequentially brought into contact with nicotinic acetylcholine receptor and radiolabeled α-bungarotoxin, and the resulting treated solution was applied to a column filled with Sepharose immobilized with concanavalin A (Con A). By measuring the radioactivity of the column after passing through the column, the amount of human antibody that inhibits the binding of α-bungarotoxin contained in the sample solution to nicotinic acetylcholine receptors can be determined by measuring the toxin binding inhibitory activity. It was quantified as degree (rate of decrease in column radioactivity). The results are shown in Table 9. For comparison, Reference Example 1
The results obtained when using cellulose particles (without heat treatment) on which the peptide obtained in (a) was immobilized, and Reference Example 1 except that glycine was used instead of the peptide obtained in Example 1. Reference Example 1 except that cellulose particles on which glycine was immobilized obtained by the same method as in (a) and cellulose particles on which glycine was immobilized were used instead of cellulose particles on which peptide was immobilized. Table 9 also shows the results obtained when using an adsorbent heat-treated at 121° C. in the same manner as in b).

[Table] Without
Glycine 121 45

Claims (1)

[Claims] 1 General formula H-X-Gly-A-Lys-His-B-Val-Tyr
−Tyr−Thr−Cys−Cys−Pro−Asp−Thr−Pro
-Tyr-Leu-Asp-Y-Z [wherein A and B each represent Phe or Tyr, X and Y each represent a single bond, or Asp, Glu, Lys and the formula (In the formula, n represents an integer of 1 to 17.) 2 to 10 amino acid residues selected from the group consisting of divalent groups represented by or at least one type of amino acid residue selected from the group Represents a peptide residue formed by a peptide bond, Z represents a hydroxyl group or an amino group, and Cys-Cys of the above amino acid sequence
The mercapto groups of each Cys may be bonded to each other to form a disulfide bond. ] Peptide indicated by.