JPH0731202B2 - New method for fractional analysis of trace components - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Use of the Invention] The present invention relates to biological fluids such as serum, blood, plasma and urine,
Two or more measurement target substances having the same action in a sample derived from a living body such as lymphocytes, blood cells, and various cells, or two or more measurement target substances having a similar structure but different actions, are chemically or And / or a method for performing a quick and easy and accurate fractional measurement according to a physical property.

[Background of the Invention] Among trace components contained in a biological sample, those having the same action but having chemically or / and physically different properties, or having a similar structure but having different actions Things exist. For example, an enzyme (isozyme) having a different protein or sugar chain structure or a physiologically active substance such as a hormone having a different sugar chain structure corresponds to the former, and steroid hormones, thyroid stimulating hormone (TSH), follicle stimulating hormone ( FSH), human chorionic gonadotropin (hCG), and other physiologically active substances such as hormones correspond to the latter. It is well known that a clinically effective index can be obtained if the amounts in these samples can be separately measured according to various properties.

As a general method for separately measuring these, for example, an electrophoresis method, an immunoassay method, a method utilizing inhibition of enzyme activity using an antibody or an inhibitor, and the like can be mentioned. However, these methods have problems such as time-consuming measurement and low quantification, and thus are not necessarily practical methods.

On the other hand, as a method for solving such a problem, a method for differentially measuring isozyme of lactate dehydrogenase by high performance liquid chromatography (HPLC) using a column packed with a packing material for ion exchange chromatography has been proposed. (J. Chromatogr., 374 , pp. 45-50, 1986, J. Chromatog.
r., 378 , pages 456-461, 1986). However, even in this method, there are some limits to what can be the target of separation measurement, and there is a problem that the measurement conditions for separation must be set according to the substance to be measured. Therefore, it is hard to say that this is a good method, and further improvements have been desired.

[Object of the Invention] The present invention has been made in view of the above situation, and has two or more substances to be measured having the same action in a sample derived from a living body or two substances having a similar structure but having different actions. It is an object of the present invention to provide a method capable of rapidly and easily and accurately and separately fractionally measuring the above-mentioned substance to be measured according to its chemical or / and physical properties.

[Structure of the Invention] In the present invention, two or more measurement target substances having the same action or two or more measurement target substances having a similar structure but different actions (hereinafter, simply referred to as measurement target substances). A sample containing a substance that has the ability to bind to all the substances to be measured, and that itself has the property of being detectable by some method, or is labeled with a substance that can be detected by some method (hereinafter , And binding substance A. The binding substance A is not fixed to a carrier.) And a substance that has a binding ability to at least one of the measurement target substances but does not bind to at least one ( Hereinafter, it will be abbreviated as a binding ability substance B. The binding ability substance B is not fixed to a carrier.) After being mixed and reacted, a complex of the measurement target substance and the binding ability substance A in a solution is obtained. Body (hereinafter, compound Abbreviated as “A”), a complex of the substance to be measured with the binding substance A and the binding substance B (hereinafter abbreviated as “complex B”), and the free binding substance A. And the binding ability substance A in the complex A
Of the binding ability substance A in the complex B and / or the amount of the binding substance A in the complex B to measure any amount of the substance to be measured in the sample.

To carry out the fractional measurement method of the present invention, for example, the following may be performed.

That is, first, a biological sample containing a substance to be measured, and the binding ability substance A and the binding ability substance B are added to and mixed in an appropriate buffer solution, if necessary, and reacted to give a complex A and a complex B. After formation of HPLC, a complex A, a complex B and a free binding ability substance A were attached to a column packed with a predetermined packing material, and the HPLC was installed.
To separate. Then, the amount of the binding ability substance A contained in the separated complex A or / and the amount of the binding ability substance A contained in the complex B is measured by a measuring method according to the property of the binding ability substance A. If so, the amount of any of the substances to be measured in the sample can be obtained.

The measurement target substance that can be measured by the fractional measurement method of the present invention is a substance that binds to all of the measurement target substances, and that itself has a property that can be detected by any method, or that can be detected by any method ( (Hereinafter, abbreviated as “detection substance”), there is a substance that can be labeled, and further exerts a strong interaction (affinity) with at least one of the measurement target substances to form a strong complex. There is no particular limitation as long as there is a substance that forms but does not bind to at least one of them. For example, biological fluids such as serum, blood, plasma, urine, etc., lymphocytes, blood cells, various cells Typical examples include enzymes, physiologically active substances, cancer-related antigens, substances having sugar chains, etc. contained in biological samples such as genus. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyl transferase (γ-GTP), lipase, creatine kinase (C
K), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase and other enzymes such as steroid hormones and human chorionic gonadotropin (hCG) , prolactin, thyroid stimulating hormone (TSH), physiologically active substances, such as luteinizing hormone (LH), such as prostate specific antigen (PSA), α ₂ - macroglobulin, carcinoembryonic antigen (CE
Cancer-related antigens such as A) and α-fetoprotein are preferably exemplified.

The binding substance A according to the present invention is not particularly limited as long as it is a substance that binds to all of the substances to be measured and has a property that can be detected by some method or is labeled with a detection substance. Can be listed without doing. Specific examples of the substance having the binding ability with respect to all the measurement target substances related to the binding substance A include, for example, an antibody against a specific partial structure of a substance having an antigenicity (including a hapten) or an antigen-determining site, For example, concanavalin A, lentil lectin, kidney bean lectin, Datsura lectin, Hiirochawantake lectin, Himaame lectin, peanut lectin, which has the ability to bind to sugar chains of a specific structure.
Examples include lectins such as wheat germ lectins, or inhibitors against enzymes such as amylase, creatine kinase (CK), glutamate oxaloacetate transaminase (GOT), and the like. Preferable examples are given. Examples of the detection substance include alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase used in enzyme immunoassay (EIA). , Used in enzymes such as luciferase, eg radioimmunoassay (RIA)
Fluorescence of radioisotopes such as ⁹⁹ mTc, ¹³¹ I, ¹²⁵ I, ¹⁴ C and ³ H, such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or their derivatives used in fluorescence immunoassay (EIA) Substances, such as luciferin, isoluminol, luminol, bis (2,4,6-trifluorophenyl) oxalate, and other luminescent substances, such as phenol, naphthol, anthracene, or their derivatives, which absorb in the ultraviolet. , For example 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- Examples thereof include substances having properties as spin labeling agents represented by compounds having an oxyl group such as cyclohexadiene-1-ylidene) -p-tolyloxyl, but it goes without saying that the substances are not limited to these. Yes.

As a method for preparing the binding ability substance A by labeling the substance as described above with the detection substance as described above, EI known per se can be used.
Known labeling methods generally used in A, RIA, FIA, etc. (eg, Medical Chemistry Laboratory, Volume 8, edited by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Kawa Ikuaki, First Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, Second Edition.
Medical School, 1982, etc.) are all cited without exception, and the procedure may be carried out in accordance with these. Further, it goes without saying that a conventional method utilizing the reaction of avidin (or streptavidin) and biotin may be used as the labeling method.

As the binding substance A according to the present invention, in addition to the substance labeled with the detection substance as described above, a substance having the property of being detectable by some method itself can be mentioned. Examples of the property of the binding substance A according to the present invention that can be detected by any method include, for example, enzyme activity, fluorescence,
Examples thereof include luminescent property and absorption property in ultraviolet region.

The binding substance B according to the present invention has a strong interaction (affinity; affinity or affinity) with at least one of the substances to be measured to form a strong complex. As long as it is a substance that does not bind to one, it can be mentioned without particular limitation. Specifically, for example, concanavalin A, lentil lectin, which has the ability to bind to a specific partial structure of a substance having an antigenicity (including a hapten) or a specific antibody against an antigenic determinant site or a sugar chain of a specific structure. , Kidney bean lectin,
Preferable examples include lectins such as Datsura lectin, Hiirochawantake lectin, Himaima lectin, peanut lectin, wheat germ lectin, and specific inhibitors for enzymes such as amylase, creatine kinase (CK), and glutamate oxaloacetate transaminase (GOT). To be

If necessary, the binding substance B may be labeled with the detection substance described in the binding substance A by the method as described above. In this case, if the same substance as the detection substance held by the binding ability substance A is labeled, the detection sensitivity of the complex B becomes high, which facilitates the detection. However, when the fractionation measuring method of the present invention is carried out using the labeled binding substance B, the free binding substance B has a property that it can be separated from the complex A and the complex B by HPLC. It goes without saying that you have to do it.

In the differential measurement method of the present invention, the reaction conditions when the substance to be measured is reacted with the binding ability substance A and the binding ability substance B to form the complex A and the complex B are the complex A There is no particular limitation as long as the conditions are such that the formation of the complex B and the formation of the complex B are prevented or the substance to be measured, the binding ability substance A and the binding ability substance B are altered.
Generally, it is carried out according to the reaction conditions for forming the complex or the like employed in the methods known per se such as EIA, RIA, FIA and affinity chromatography. For example, when a buffer solution is used in the reaction, the buffer agent and other reagents used may be appropriately selected from those used in the methods known per se.

In the fractionation measuring method of the present invention, the complex A and the complex B
The use concentrations of the binding ability substance A and the binding ability substance B at the time of forming the compound may be appropriately set depending on the calibration limit and the measurement sensitivity of the measurement target substance, and are not particularly limited. As the binding substance A and the binding substance B, it is usually sufficient to use one type each, but if necessary, two or more types may be used in combination for each. In this case, two or more kinds of binding ability substances A having the property of respectively binding to different sites on the measurement target substance, and two or more types of binding ability substances having the property of respectively binding to different sites on the arbitrary measurement target substance When B and the like are used in combination, as a result, the complex A and the complex B are obtained.
Has a large molecular weight, and the isoelectric point also varies depending on the case. Therefore, it becomes easier to separate the complex A, the complex B and the free binding ability substance A, and it is possible to improve the measurement accuracy. it can. The substance to be measured is, for example, X,
In the case of a mixture of Y and Z, for example, if the binding substance B for X and the binding substance B for Y are used together,
It is also possible to separately measure X, Y and Z simultaneously.

In the fractional measurement method of the present invention, the pH during the reaction is not particularly limited as long as it does not prevent the formation of the complex A and the complex B, but usually 2 to 10, The range of 5 to 9 is preferable. The temperature during the reaction is not particularly limited as long as it does not prevent the formation of the complex A and the complex B, but it is usually 0 to 70 ° C, preferably 20 to 40 ° C. To be The reaction time varies depending on the properties of the substance to be measured and the binding ability substance A and the binding ability substance B since the time required for forming the complex A and the complex B is several seconds to several hours depending on each property. It may be appropriately reacted.

In the measuring method of the present invention, as the HPLC used for separating the complex A, the complex B and the free binding substance A, the apparatus itself is one that is usually used in the field of analysis and has a constant flow rate. Any of these can be used without any particular problem, but the packing material used for the separation column has any difference in properties between the complex A, the complex B and the free binding substance A. It goes without saying that various types must be appropriately selected and used depending on the situation. That is, for example, the molecular weight of the complex B is about 1.2 times or more the molecular weight of the complex A,
It is preferably 1.5 times or more, more preferably 2 times or more,
Moreover, the molecular weight of the complex A is about 1.2 times or more, preferably 1.5 times or more, more preferably 2 times the molecular weight of the free binding substance A.
If more than twice, gel filtration (gel chromatography)
Fillers for use are suitable. Further, for example, when the isoelectric points of the complex A, the complex B and the free binding ability substance A are different from each other, the difference between the respective isoelectric points is 0.05 or more at pH, preferably
When it is 0.2 or more, a packing material for ion exchange chromatography or isoelectric point chromatography is suitable, and for example, when there is a clear difference in the hydrophobicity of the complex A, the complex B and the free binding substance A. Suitable for this purpose are packing materials for hydrophobic chromatography, packing materials for reverse phase chromatography, hydroxyapatite and the like.

Complex A, complex B and free binding substance A by HPLC
As the solution (eluent) used when separating
The formed complex A and complex B are not decomposed into the measurement target substance and the binding substance A or the measurement target substance and the binding substance B again, and the complex A and the complex B are formed. The binding ability substance A contained therein or the detection substance held by the binding ability substance A has, and is not particularly limited unless it loses the property detectable by any method. It can be mentioned without any mention, but usually EIA,
Those used as buffers in methods known per se such as RIA, FIA and affinity chromatography are preferably used. Specific examples include, for example, phosphates, acetates, citrates, Good's buffers, buffers such as tris (hydroxyethyl) aminomethane, salts such as sodium chloride, potassium chloride, ammonium sulfate, etc. Polar organic solvents such as methanol, ethanol, isopropyl alcohol, acetonitrile, and tetrahydrofuran, and surfactants are appropriately selected according to the properties of the complex A, the complex B, and the free binding substance A, added, and mixed. A buffer solution having a pH of 2 to 10 prepared according to the above is preferably used.

In the fractionation measuring method of the present invention, the amount of the binding ability substance A contained in the complex A and the complex B separated by HPLC is measured by determining whether the binding ability substance A or the binding ability substance A is retained. Depending on the properties of the substance to be detected, which can be detected by any method, the detection substance is subjected to a predetermined method.
For example, when the property is enzymatic activity, a conventional EIA method such as “enzyme immunoassay, protein / nucleic acid / enzyme separate volume No. 3” is used.
1, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51-6
Page 3, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 ”, etc., and when the substance to be detected is a radioactive substance, the radioactive substance shall be Immersion type GM counter according to the type and intensity of radiation emitted by
Liquid scintillation counters, well scintillation counters, HPLC counters, and other measuring devices can be selected and used as appropriate (for example, medical chemistry experiment course, Volume 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten). , 1971
See also. ). When the property is fluorescent, a conventional FIA method using a measuring instrument such as a fluorometer, for example, “illustration
Fluorescent antibody, Akira Kawao, 1st edition, Soft Science Co., Ltd., 1983 ”, etc. may be used for measurement, and when the property is luminescent, measurement with a photon counter, etc. A conventional method using an instrument, for example, "enzyme immunoassay, protein / nucleic acid / enzyme separate volume No.31, Tsunehiro Kitagawa / Toshio Minamihara
Edited by Akio Tsuji and Eiji Ishikawa, pages 252-263, Kyoritsu Publishing Co., Ltd.
The measurement may be carried out according to the method described in “September 10, 1987”. Further, when the property is that which has absorption in the ultraviolet, the measurement may be carried out by a conventional method using a measuring device such as a spectrophotometer, and when the substance to be detected has a spin property, electron spin resonance Conventional method using equipment,
For example, "enzyme-linked immunosorbent assay, protein / nucleic acid / enzyme separate volume N
o.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 26
4 to 271 pages, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 "and the like.

Further, in the fractionation measuring method of the present invention, as the measuring method after separation by HPLC, for example, “Latest Liquid Chromatography, Shoji Hara and Akio Tsuji, First Edition, pages 92 to 104, Nanzandou, 1978 2 Issued on the 1st of the month ", etc.,
A method in which the effluent from the HPLC column is directly guided to the detection unit and the amounts of the binding ability substance A contained in the complex A and the complex B in the effluent are directly measured is more preferable because the measurement can be performed quickly. . In this case, if the binding substance A or the detection substance retained by the binding substance A has a property that can be detected by any method, for example, enzyme activity, then the HPLC column and the detection unit are used. Needless to say, a so-called post-column method reaction section for adding a reagent for measuring enzyme activity and reacting with the effluent must be provided between and. When the property of the engaging substance A is an enzyme activity, the reagent for measuring the enzyme activity used in the reaction part is a conventional method, for example, "enzyme immunoassay, protein nucleic acid enzyme separate volume No. 3".
1, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51-6
3 page, Kyoritsu Shuppan Co., Ltd., published September 10, 1987 "may be used, or the reagents of commercially available clinical test kits may be appropriately selected. You may use it. In addition, even when the property of the binding substance A is other than enzyme activity, a suitable reagent is added between the HPLC column and the detection section in order to add and react a predetermined reagent for the purpose of increasing the detection sensitivity. Providing a reaction part is optional.
When the binding substance B is labeled with the same detection substance as that retained by the binding substance A, the amount of the binding substance A in each complex is measured to obtain the complex. It goes without saying that the amount of the binding substance B in B is also measured.

In the fractionation measurement method of the present invention, the binding ability substance A is
When an antibody is used as the substance capable of binding to all the substances to be measured and / or the substance B having binding ability, the antibody to be used according to the purpose is appropriately digested with an enzyme such as pepsin or papain to obtain F ( It can also be used as ab ') ₂ , Fab' or Fab. Especially when Fab ′,
On the other hand, there is an advantage that the detection substance can be easily labeled. Also, when used as Fab 'or Fab,
One binding ability substance A or one binding ability substance B per one measurement target substance having binding ability (per monomer when the measurement target substance is a dimer or trimer) It is more preferable because the complex A and the complex B are eluted by HPLC at a substantially constant time since they are bound to each other. Also, when a monoclonal antibody having the property of binding to only one antigen recognition site is used as the antibody, one per measurement target substance (the measurement target substance is a dimer or trimer) In this case, since one monoclonal antibody binds to each monomer), Complex A and Complex B
It is more preferable because the time of elution by HPLC becomes almost constant. Also in this case, this is digested and Fab ′ or F
Needless to say, it may be used as ab.

In the present invention, an antibody as a substance having a binding ability to all the measurement target substances or / and an antibody as a binding ability substance B, which is related to the binding ability substance A, is used in a conventional method, for example, “Introduction to Immunological Experiments, 2nd edition, Nao Matsuhashi et al., Academic Publishing Center, 19
81 ”etc. according to the method described in“ Horse, cow, sheep, rabbit, goat,
A polyclonal antibody prepared by immunizing an animal such as rat or mouse with a substance to be measured, or a conventional method, that is, G.Khler and C. Milstein; Natur
e, 256, 495, 1975) according to the cell fusion method established by
Any monoclonal antibody produced by a hybridoma obtained by fusing cells from a mouse tumor line with splenocytes of a mouse previously immunized with a substance to be measured may be used alone or Are used in an appropriate combination, and so on.

According to the differential measurement method of the present invention, the time required for measurement is from several minutes to several hours, and the necessary measurement operation itself is after mixing the sample containing the substance to be measured, the binding ability substance A and the binding ability substance B. , The complex A, the complex B and the free binding substance A are separated by HPLC, and the amount of the binding substance A in the complex A and / or the amount of the binding substance A in the complex B is detected. Only. As is clear from these, the fractional measurement method of the present invention can perform the desired measurement more easily and rapidly than the conventional fractional measurement method for the same purpose.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[Examples] Example 1. Fractional measurement of human chorionic gonadotropin (hCG) having different sugar chain structures (eluent) monosodium phosphate 3.9 g, disodium phosphate (12-hydrate) 81 g, sodium chloride 44 g and 3 After dissolving 8.3 g of-(p-hydroxyphenyl) -propionic acid in ion-exchanged water and adding a 1N NaOH solution so as to have a pH of 7.5, the total amount was 5 l.
Was used as the eluent.

(Substrate solution) Dilute 30% hydrogen peroxide solution with ion-exchanged water and add 20 mM H ₂ O ₂ .
A solution was prepared and used as a substrate solution.

(Antibody solution) Anti-hCG-β chain monoclonal antibody (Wako Pure Chemical Industries, Ltd.)
Fab) was treated with a conventional method to give Fab ′, which was labeled with horseradish peroxidase (POD) by a conventional method to obtain P ′.
OD-labeled anti-hCG-β chain-Fab ′ was added to 50 mM phosphate buffer (pH 7.
(0, 150 mM sodium chloride) to give an antibody solution at a protein concentration of 95 ng / ml.

(Lectin solution) Lentil bean lectin (LCA-A) was added to 50 mM phosphate buffer (pH 7.0) at a protein concentration of 1.5 mg / ml to prepare a lectin solution.

(Sample) Commercially available hCG (placenta villi-derived, manufactured by Sigma) or hCG derived from choriocarcinoma purified from serum of choriocarcinoma was dissolved in ion-exchanged water to prepare solutions with hCG concentrations of 100, 200, 300, 400 or 500 mIU / ml, respectively. Then, it was used as a sample.

(HPLC usage conditions) The outline of the system is as shown in Fig. 2.

・ Column and packing material: 0.46φ × 60 cm. YMC-Pack Diol-
200 (trade name of Yamamura Chemical Laboratory Co., Ltd.).

Flow rate: eluent: 0.5 ml / min., Substrate solution: 0.05 ml / min.

・ Reaction part: 0.04φ x 900 cm (40 ℃ insulation).

-Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm.

(Measurement procedure) 40 μl of antibody solution, 40 μl of lectin solution and 20 μl of sample were mixed and allowed to stand at 30 ° C. for 30 minutes, and then 20 μl of the mixed solution was subjected to HPLC.
Was analyzed by.

(Result) As a result of analysis by HPLC, POD-labeled anti-hCG-β chain-Fab ′ was 1
After 2.5 minutes, the complex of POD-labeled anti-hCG-β chain-Fab 'and placental villus-derived hCG (complex A) was analyzed after 11.0 minutes.
It was found that the complex of hCG-β chain-Fab 'with LCA-A and hCG derived from choriocarcinoma (complex B) was eluted after 10.2 minutes. As is clear from this result, POD-labeled anti-hCG-β chain-
Fab ′ and LCA-A are bound to substance A and substance B, respectively.
It can be seen that hCG with different sugar chain structure can be separated by using

Example 2. Fractional measurement of hCG and thyroid stimulating hormone (TSH) (eluent) The same as in Example 1.

(Substrate solution) The same as in Example 1.

(Antibody solution 1) Anti-hCG-α chain monoclonal antibody (Wako Pure Chemical Industries, Ltd.)
Fab) was treated with a conventional method to give Fab ′, which was labeled with horseradish peroxidase (POD) by a conventional method to obtain P ′.
OD-labeled anti-hCG-α chain-Fab 'was added to 50 mM phosphate buffer (pH 7.
Antibody solution 1 was prepared by adding it to a protein concentration of 70 ng / ml in 0.10 mM sodium chloride).

(Antibody solution 2) Anti-hCG-β chain monoclonal antibody (Wako Pure Chemical Industries, Ltd.)
Antibody solution 2) was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM NaCl) at a protein concentration of 950 μg / ml.
And

(Hormone solution) Commercially available hCG (placenta villi-derived, manufactured by Sigma) and TSH (UCB
BioProduct SA Co., Ltd.) 0.04,0.08,0.12,
50 mM phosphate buffer (pH 7.
It was dissolved in 5, 150 mM NaCl) to obtain a hormone solution.

(HPLC usage conditions) The outline of the system is the same as in Fig. 2.

・ Column and packing material: 0.46φ × 60 cm. YMC-Pack Diol-
200 (trade name of Yamamura Chemical Laboratory Co., Ltd.).

Flow rate: eluent: 0.5 ml / min., Substrate solution: 0.05 ml / min.

・ Reaction part: 0.04φ x 900 cm (55 ° C heating).

-Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm.

(Measurement procedure) Antibody solution 1 40 μl, hormone solution 20 μl and antibody solution 2 40 μl
After mixing and leaving at 30 ℃ for 30 minutes, add 15 μl of the mixed solution.
It was analyzed by HPLC.

(Result) As a result of analysis by HPLC, POD-labeled anti-hCG-α chain-Fab ′ was 1
After 2.5 minutes, the complex of POD-labeled anti-hCG-α chain-Fab 'and TSH (Complex A) was 11.7 minutes later, and POD-labeled anti-hCG-α chain-Fa.
It was found that the complex of b ′ with the anti-hCG-β chain monoclonal antibody and hCG (complex B) was eluted after 10.2 minutes. As is clear from this result, POD-labeled anti-hCG-α chain-
By using Fab ′ and an anti-hCG-β chain monoclonal antibody as the binding substance A and the binding substance B, respectively, hCG and T
It turns out that SH can be separated.

Further, FIG. 1 shows a calibration curve showing the relationship between the hCG concentration (nM) or TSH concentration (nM) of each sample and the peak height value (μV) of the complex, which was obtained as a result of the HPLC analysis. . still,
In Figure 1,-○-is the calibration curve for hCG, and-●-
Shows the calibration curves for TSH, respectively.

Example 3. Differential measurement of hCG, luteinizing hormone (LH) and thyroid stimulating hormone (TSH) (eluent) The same as in Example 1.

(Substrate solution) The same as in Example 1.

(Antibody solution 1) The same as in Example 2.

(Antibody solution 2) Anti-hCG-β chain monoclonal antibody (Wako Pure Chemical Industries, Ltd.)
Fab) and an anti-LH-β chain monoclonal antibody (Bioclon Australia)
Antibody solution 2 was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM NaCl) so that each protein concentration was 2 μg / ml.

(Hormone solution) Commercially available hCG (derived from placenta villi, manufactured by Sigma), LH (manufactured by UCB Bioproduct SA) and TSH (UCB bioproduct)
A solution prepared by dissolving SA (manufactured by SA Co.) in 50 mM phosphate buffer (pH 7.5, containing 150 mM NaCl) was used as a hormone solution so that each contained 1 nM.

(HPLC usage conditions) The outline of the system is the same as in Fig. 2.

・ Column and packing material: 0.46φ × 60 cm. YMC-Pack Diol-
200 (trade name of Yamamura Chemical Laboratory Co., Ltd.).

Flow rate: eluent: 0.5 ml / min., Substrate solution: 0.05 ml / min.

・ Reaction part: 0.04φ x 900 cm (55 ° C heating).

-Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm.

(Measurement procedure) Antibody solution 1 40 μl, hormone solution 30 μl and antibody solution 2 40 μl
After mixing and leaving at 30 ℃ for 30 minutes, add 15 μl of the mixed solution.
It was analyzed by HPLC.

(Result) As a result of analysis by HPLC, POD-labeled anti-hCG-α chain-Fab ′ was 1
After 2.5 minutes, the complex of POD-labeled anti-hCG-α chain-Fab 'with anti-hCG-β chain-Fab and hCG was 10.0 minutes later, and POD-labeled anti-hCG-Fab.
The complex of α chain-Fab 'with anti-LH-β chain-Fab and LH is 11.0
After a minute, the complex of POD-labeled anti-hCG-α chain-Fab 'and TSH was
After 11.9 minutes, each peak eluted.

As is clear from this result, it can be understood that each of the hormones in the sample in which hCG, LH and TSH are mixed can be quantified by the fractional measurement method of the present invention.

Comparative Example 1. (Eluent) The same as in Example 1.

(Antibody solution 1) The same as in Example 1.

(Hormone solution) Same as in Example 3.

(Use condition of HPLC) The conditions were the same as in Example 3.

(Measurement procedure) 40 μl of antibody solution, 30 μl of hormone solution and 40 μl of 50 mM phosphate buffer solution (pH 7.5, containing 150 mM NaCl) were mixed and mixed at 30 ° C. for 3 days.
After standing for 0 minutes, 15 μl of the mixture was analyzed by HPLC.

(Results) As a result of the analysis by HPLC, only one broad peak was observed except that the POD-labeled anti-hCG-α chain-Fab ′ peak was observed after 12.5 minutes, and the POD-labeled anti-hCG-α chain was observed. −Fa
b′-hCG complex, POD-labeled anti-hCG-α chain-Fab ′ and LH
The peaks of the complex with TSH and the complex with POD and anti-hCG-α chain-Fab ′ with TOD could not be identified.

As is clear from this result, POD-labeled anti-hCG-α chain-Fa
It can be seen that hCG, LH, and TSH cannot be separately detected when only b ′ is used.

[Effects of the Invention] As described above, the present invention provides rapid, easy, and accurate differential measurement of a substance to be measured in a biological sample according to its chemical or / and physical properties. It provides a method of obtaining. According to the method of the present invention, the time required for measurement is about several minutes to several hours, and the necessary measurement operation itself is that after mixing the sample containing the substance to be measured with the binding ability substance A and the binding ability substance B, The complex A, the complex B and the free binding substance A are separated by HPLC, and the amount of the binding substance A in the complex A and / or the binding substance A in the complex B is separated.
It is an invention having a remarkable effect in that the target measurement can be carried out easily and quickly, as compared with the conventional similar fractionation measurement method because it only detects the amount. It is a great invention to contribute.

[Brief description of drawings]

FIG. 1 shows the calibration curve obtained in Example 2. FIG. 2 shows the HP used in Examples 1, 2, 3 and Comparative Example 1.
1 is a schematic diagram of an LC system.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication location G01N 33/543 D 9217-2J (56) References JP-A-2-221860 (JP, A) JP-A-2 -266263 (JP, A) JP-A-3-218463 (JP, A) JP-A-57-45454 (JP, A) JP-A-55-126856 (JP, A) JP-A-61-120058 (JP-A) ) International Publication 89/8263 (WO, A) J. Am. Pharmacol. Exp. Ther. , 206 (1), P158-66, (1978) Methodol. Anal. Toxi col. , 3, P. 135-46, (1985)

Claims (6)

[Claims] 1. A sample containing two or more measurement target substances having the same action or two or more measurement target substances having a similar structure but different actions (hereinafter, simply referred to as measurement target substance). , A substance that has the ability to bind to all the substances to be measured, and that itself has the property of being detectable by some method, or is labeled with a substance that can be detected by some method (hereinafter referred to as a substance having a binding ability. It is abbreviated as A. The binding substance A is not fixed to a carrier) and a substance that has a binding ability to at least one of the measurement target substances but does not bind to at least one (hereinafter, binding ability). It is abbreviated as substance B. Incidentally, the binding ability substance B is mixed with a carrier and allowed to react, and then a complex of the measurement target substance and the binding ability substance A in the solution (hereinafter, Abbreviated as Complex A ), A complex of the substance to be measured with the binding ability substance A and the binding ability substance B (hereinafter abbreviated as the complex B) and the free binding ability substance A are separated by high performance liquid chromatography to form a complex. Binding A in Body A
And / or the amount of the binding substance A in the complex B is measured to measure any amount of the substance to be measured in the sample. 2. The method for differential measurement according to claim 1, wherein the substance to be measured is an enzyme, a physiologically active substance, a cancer-associated antigen or a substance having a sugar chain. 3. The substance having a binding ability to all the substances to be measured, which is related to the binding substance A, is an antibody or a lectin,
The differential measuring method according to claim 1, wherein the binding substance B is an antibody or a lectin that specifically binds to at least one of the substances to be measured, but does not bind to at least one of them. 4. The method for differential measurement according to claim 3, wherein the antibody is a monoclonal antibody. 5. The lectin is concanavalin A, lentil lectin, kidney bean lectin, datura lectin,
The fractionation measuring method according to claim 3, wherein the method is a Hiirochawantake lectin, a chickpea lectin, a peanut lectin, or a wheat germ lectin. 6. Separation of the complex A, the complex B and the free binding substance A is performed by using a packing material for gel filtration (gel chromatography), a packing material for ion exchange chromatography, a packing material for hydrophobic chromatography, an isoelectric focusing chromatography. The method for fractionation measurement according to any one of claims 1 to 5, which is carried out by high performance liquid chromatography equipped with a column packed with a packing material for packing, a packing material for reverse phase chromatography or a hydroxyapatite.