JPH04320965A - Method for measuring small amount of constituent - Google Patents

Abstract

PURPOSE:To achieve a highly sensitive measurement in an extremely short time without interfering with an activity of beta-galactoxidase even under a high salt concentration by performing a hydrophobic chromatography using a traveling phase which is constituted by adding a salt between sulfuric acid ion and alkali metal. CONSTITUTION:In a method for measuring a small amount of constituent within a living body sample utilizing mutual operation of a substance to be measured and substances (connectable substances) with a connection capability for it, beta-galactoxidase is used as a labeled substance, separation of a complex (containing the labeled substance) between the substance to be measured and the connectable substance and a liberated connectable substance (containing the labeled substance) or the substance to be measured (containing the labeled substance) is performed by hydrophobic chromatography using a traveling phase which is made by adding a salt between sulfuric acid ion and alkali metal, and then analysis is made by the post column method.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is capable of quickly, easily and easily extracting trace components from living body fluids such as serum, blood, plasma, urine, lymphocytes, blood cells, various types of cells, etc. Concerning how to measure with high accuracy.

0002

[Prior art and its problems] Specific substances, such as antigens and antibodies, proteases and their proteinaceous protease inhibitors, sugar chains and lectins, enzymes and their substrates and coenzymes, physiologically active substances such as hormones, and their reactions It is known that receptors, transport proteins, a pair of polynucleotide chains of double-stranded DNA, and the like interact strongly with each other to form a strong complex.

One of the methods for measuring trace components in a sample that utilizes such interactions is a method described in Japanese Patent Application Laid-Open No. 2-28557, which was previously developed by the present inventors. The method described in the above publication is roughly as follows.

(1) Mixing a biological sample containing a substance to be measured with a substance that has the ability to bind to the substance to be measured (hereinafter abbreviated as a binding substance), which may or may not be labeled with a labeling substance. After the reaction, the complex of the target substance and the binding substance (hereinafter simply referred to as the complex) and the free binding substance are separated by high-performance liquid chromatography, and the components in the complex are separated. A measurement method characterized by measuring the amount of a substance to be measured in a sample by measuring the amount of a labeling substance or the amount of a binding substance.

(2) A biological sample containing a substance to be measured is mixed with a substance to be measured labeled with a labeling substance (hereinafter abbreviated as labeled substance to be measured) and a binding substance and reacted. After that, the complex of the labeled measurement substance and the binding substance (
Hereinafter, it will be abbreviated as a labeled complex. ) and the free labeled analyte by high-performance liquid chromatography, and the amount of the labeled substance in the labeled complex or the amount of the labeled analyte in the free labeled analyte is determined. A measurement method characterized by measuring the amount of a substance.

That is, the method described in the above publication involves separating the complex (or labeled complex) resulting from the interaction between the substance to be measured and the binding substance and the free binding substance (or labeled substance to be measured). This method is characterized in that it is carried out using high-performance liquid chromatography, and according to this method, trace components can be quantified using conventional EIA (enzyme immunoassay), RIA (radioimmunoassay) or FIA (fluorescence immunoassay). Since it can be performed easily, in a short time, and with high accuracy compared to other measurement methods such as 2012-2013, it is considered that this measurement method has great expectations for future development.

Among the various labels used in carrying out the above method, enzymes are the most common and preferably used because they are easy to handle and do not require special equipment. β-galactosidase is considered to be a more preferable labeling substance because it has higher sensitivity than other enzymes.

When the method described in the above publication is carried out using an enzyme as a labeling substance, there is a post in which a reagent for enzyme activity measurement is added to the effluent and reacted between the high performance liquid chromatography column and the detection section. If you use the column method,
This is more preferable because the analysis time is further reduced. When measuring enzyme activity using the post-column method, the reaction with the enzyme is mainly carried out under conditions of salt concentration in the column eluate.

On the other hand, it is more preferable to use a packing material for hydrophobic chromatography as the packing material used in the separation column because the separation ability is higher. Ammonium sulfate is the most common salt used in hydrophobic chromatography, and is usually used at a salt concentration of 0.1 to 2M. However, when attempting to carry out the above method using hydrophobic chromatography in a post-column method using β-galactosidase as a labeling substance, the enzymatic activity of β-galactosidase was significantly inhibited in ammonium sulfate at a salt concentration of 0.1 to 2M. It turned out that high-sensitivity measurements were not possible.

[0010] Ammonium sulfate salting out method is generally employed for the purification of β-galactosidase, and in this case, almost no decrease in enzyme activity is observed. Therefore, the fact that the activity of β-galactosidase is reduced by ammonium sulfate in hydrophobic chromatography is probably due to the difference in its concentration, but in any case, this is completely unexpected and a solution is awaited. was.

[0011]

OBJECTS OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and it is possible to apply the method described in JP-A-2-28557 using β-galactosidase as a labeling substance, by a post-column method, and by hydrophobic chromatography. The purpose is to provide a method for implementing this method.

[Configuration of the Invention] The present invention provides a method for converting a biological sample containing a substance to be measured into a substance that is labeled with a labeling substance and has the ability to bind to the substance to be measured (hereinafter abbreviated as a labeled binding substance). ), and then the complex of the substance to be measured and the label-binding substance (hereinafter abbreviated as label complex A) and the free label-binding substance are separated by high-performance liquid chromatography. In a method for measuring the amount of a substance to be measured in a sample by separating and measuring the amount of a labeling substance in a labeled complex A or the amount of a labeling substance in a label-binding substance, β-galactosidase is used as a labeling substance. Using a mobile phase (eluent) containing a salt of sulfate ions and an alkali metal, the labeled complex A and the free form were separated by high-performance liquid chromatography equipped with a column packed with a hydrophobic chromatography packing material. The present invention is directed to the measurement method, which is characterized in that it separates a substance capable of binding to a label.

[0013] The present invention also provides a method in which a biological sample containing a substance to be measured is mixed with a substance to be measured labeled with a labeling substance (hereinafter abbreviated as a labeled substance to be measured) and a binding substance. After the reaction, the complex of the labeled analyte and the binding substance (hereinafter abbreviated as labeled complex B) and the free labeled analyte are separated by high performance liquid chromatography, and the labeled complex is separated. In a method for measuring the amount of a target substance in a sample by measuring the amount of a label substance in B or the amount of a label substance in a free labeled target substance, using β-galactosidase as a label substance, A mobile phase made by adding a salt of sulfate ions and an alkali metal (
An invention of the measuring method characterized in that the labeled complex B and the free labeled substance to be measured are separated by high-performance liquid chromatography equipped with a column packed with a hydrophobic chromatography packing material. be.

[0014] That is, the present inventors have
As a result of extensive research in search of a method that effectively implements the method described in Publication No. 7 using β-galactosidase as a labeling substance, post-column method, and hydrophobic chromatography, we have found that the mobile phase (eluent ) They have found that the above object can be achieved by using a salt of sulfate ion and an alkali metal instead of ammonium sulfate as the salt added to the solution, and have completed the present invention.

The measuring method of the present invention may be carried out, for example, as follows. That is, when carrying out the measurement method of the present invention based on the principle of so-called non-competitive reaction,
First, a biological sample containing the substance to be measured and a binding substance labeled with β-galactosidase (labeled binding substance)
If necessary, add and mix in an appropriate buffer solution and react to form a complex (labeled complex A), and then combine the complex and the binding substance with sulfate ion and an alkali metal. Separate by hydrophobic chromatography using a mobile phase (eluent) containing a salt of Next, the amount of β-galactosidase in the separated labeled complex A or the amount of β-galactosidase in the free label-binding substance is measured. Separately, measurements are performed using a similar method using a sample with a known concentration of the target substance to be measured, and the amount of the target substance to be measured is compared with the amount of β-galactosidase in the label complex A or the amount of β-galactosidase in the label-binding substance. Create a calibration curve that represents the relationship between For example, the amount of the substance to be measured in the sample is determined.

[0016] Furthermore, when carrying out the measurement method of the present invention based on the so-called competitive reaction principle, first, a biological sample containing the substance to be measured, a β-galactosidase-labeled substance to be measured, and a binding substance are, if necessary, Added to a suitable buffer,
After mixing and reacting to form a complex and a β-galactosidase labeled complex (labeled complex B), the labeled complex A and the labeled analyte are transferred by adding a salt of sulfate ion and an alkali metal. Separate by hydrophobic chromatography using the phase (eluent). Next, the amount of β-galactosidase in the separated labeled complex B or the amount of β-galactosidase in the free labeled substance to be measured is measured. Separately, measurements were performed using a similar method using a sample with a known concentration of the target substance, and the amount of the target substance and β in labeled complex B were determined.
-The amount of galactosidase or β in the labeled substance to be measured-
A calibration curve representing the relationship with the amount of galactosidase is created, and using this, the amount of the target substance to be measured corresponding to the amount of β-galactosidase in the labeled complex or the amount of β-galactosidase in the labeled target substance to be measured is determined. Once determined, the amount of the substance to be measured in the sample can be determined.

In the measurement method of the present invention, the mobile phase (
Examples of salts of sulfate ions and alkali metals added to the eluent include Na2SO4, K2SO4, Li2SO
4, Rb2SO4, etc., particularly preferably Na2
Examples include SO4 and Li2SO4. These salts of sulfate ions and alkali metals are usually used alone or in an appropriate mixture of two or more, but if necessary, salts of chlorine ions and alkali metals can also be used in combination. That is, like salts of sulfate ions and alkali metal ions, salts of chloride ions and alkali metals do not reduce the activity of β-galactosidase at the concentrations used in hydrophobic chromatography. However, in the case of a salt of chlorine ion and an alkali metal, if it is added alone to the mobile phase (eluent) for hydrophobic chromatography without using it together with a salt of sulfate ion and an alkali metal, measurement will not be possible. It cannot be used for this purpose because target substances and binding substances pass through without being adsorbed to the column.

[0018] The dissolving solution for dissolving each salt is a p-containing solution containing a buffer substance such as phosphate, acetate, citrate, Good's buffer, or tris(hydroxyethyl)aminomethane.
Preferred examples include solutions having a pH of 2 to 10, preferably 4 to 9.

In the measurement method of the present invention, the HPLC used for B/F separation is one that is normally used in the field of analysis and can be used without any problems as long as it has a constant flow rate. column packed with a hydrophobic chromatography packing material, such as TSKgel Bu
tyl-NPR (manufactured by Tosoh Corporation), MCI GEL CQ
H3BS (manufactured by Mitsubishi Chemical Industries, Ltd.), MCI GEL
CQH3ES (manufactured by Mitsubishi Chemical Industries, Ltd.), Protein Pack G-Butyl (manufactured by Waters Corporation), CY
PRESS PolyLC Poly Propyl (
(manufactured by CYPRESS), CYPRESS PolyLC
Poly Ethyl (manufactured by CYPRESS), CYP
RESS PolyLC Poly Methyl(C
YPRESS), YMC-Pack HIS-Pr
It may be used by attaching opyl (manufactured by YMC) or the like.

The substance to be measured that can be measured by the measurement method of the present invention includes: i) a bond that can have a strong interaction (affinity) with the substance to be measured and form a strong complex; or ii) the substance to be measured is itself labelable by β-galactosidase and has a strong interaction with the substance to be measured. Examples include serum, blood, plasma, etc., but are not particularly limited to those in which there is a binding substance that can interact with each other and form a strong labeling complex. ，
Typical examples include proteins, peptides, nucleic acids, sugar chains, lipids, hormones, drugs, etc. contained in biological body fluids such as urine, lymphocytes, blood cells, various types of cells, and other biological samples. More specifically, for example, α-fetoprotein (
AFP), CA19-9, prostate specific antigen (PSA),
Cancer markers such as carcinoembryonic antigen (CEA), substances with special sugar chains produced by cancer cells, such as immunoglobulin A
Serum proteins such as (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), β2-microglobulin, albumin, ferritin, e.g. C-peptide,
Peptides such as angiotensin I, e.g. amylase,
Enzyme proteins such as alkaline phosphatase and γ-glutamyltransferase (γ-GTP), antiviral antibodies against viruses that have received clinical attention such as rubella virus, herpes virus, hepatitis virus, ATL virus, and AIDS virus; Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) of pathogens, single-stranded polynucleotides that constitute these nucleic acids, antigenic substances derived from pathogens such as viruses, and allergens such as pollen from cedar and other plants and indoor dust. Antibodies that react with lipids such as lipoproteins, proteases such as trypsin, plasmin, elastase I, serine proteases, such as insulin, human chorionic gonadotropin (hCG), thyroxine (T4), triiodothyronine (T3), Prolactin, thyrotropin (T)
Examples include hormones such as SH), drugs such as digoxin, phenytoin, morphine, and nicotine.

[0021] The substances capable of binding to these analyte substances according to the present invention include substances that have a strong interaction (affinity) with these analyte substances and form a strong complex, Any substance that can be labeled with β-galactosidase (this does not apply if the substance to be measured itself can be labeled with β-galactosidase) may be mentioned without particular limitation, but for example, it has antigenicity. Antibodies to substances (including haptens), antigens to antibodies, concanavalin A, etc. that have the ability to bind to sugar chains with specific structures,
Lectins such as lentil lectin, kidney bean lectin, Datura lectin, and wheat germ lectin, inhibitors for specific enzymes such as α1 antitrypsin for trypsin, α2 macroglobulin for plasmin, and α2 macroglobulin for serine protease, and substances to be measured. Examples include polynucleotide chains complementary to single-stranded polynucleotides.

β- used in the measurement method of the present invention
Galactosidase may be derived from any source, but β-galactosidase derived from Escherichia coli, which has a high specific activity, is more preferably used. Substrates for β-galactosidase include, but are not particularly limited to, synthetic substrates with clear structures, such as 4-methylumbelliferyl-β-D-galactopyranoside, 2-nitrophenyl-β-D-galactoside, etc. Preferably used.

[0023] As a method for binding β-galactosidase to a binding substance or a substance to be measured, the known EI method can be used.
Labeling methods commonly used in A, etc. (
For example, Medical Chemistry Experiment Course, Volume 8, supervised by Yuichi Yamamura, Volume 1.
Edition, Nakayama Shoten, 1971; Enzyme immunoassay, Eiji Ishikawa,
(edited by Tadashi Kawai and Kiyoshi Miyai, 2nd edition, Igaku Shoin, 1982, etc.) are mentioned without exception, and it is sufficient to follow these guidelines.

[0024] It goes without saying that the labeling substance may be bound to the binding substance or the substance to be measured by a conventional method utilizing a reaction between avidin (or streptavidin) and biotin.

In the measurement method of the present invention, the reaction conditions for forming a complex by reacting the substance to be measured with a labeled substance capable of binding, or the reaction conditions for forming a complex between the substance to be measured and the labeled substance to be measured and the bond Reaction conditions for forming a labeled complex by reacting with a binding substance (type of buffer used, concentration of the binding substance used, concentration of the labeled substance to be measured, pH during the reaction,
Temperature, time, etc.) may be carried out in accordance with those described in JP-A No. 2-28557.

In the present invention, it is essential to use a post-column method that allows rapid measurement, and the post-column method may be carried out, for example, as follows. That is, labeled complex A (
Or, when measuring the labeling substance contained in the labeling complex B) or the labeling substance contained in the free label-binding ability substance (or the labeling substance contained in the free labeled substance to be measured), for example, As described in "Latest Liquid Chromatography, edited by Shoji Hara and Akio Tsuji, 1st edition, pp. 92-104, published by Nanzando, February 1, 1978," A reaction section for the post-column method is installed in the
After a reagent for enzyme activity measurement is added to the effluent and reacted, it is introduced into the detection section and the amount of labeling substance contained in labeled complex A (or labeled complex B) in the effluent is measured. The amount of the labeling substance in the free label-binding ability substance (or the amount of the labeling substance in the free labeled substance to be measured) may be directly measured. Measurement of enzyme activity in the reaction zone can be carried out using conventional methods, for example,
Enzyme immunoassay, protein, nucleic acid, enzyme separate volume No.
．． 31, edited by Tsunehiro Kitagawa, Toshio Minamibara, Akio Tsuji, and Eiji Ishikawa,
pp. 51-63, Kyoritsu Shuppan Co., Ltd., September 10, 1987
The measurement may be carried out according to the method described in the Japanese publication, etc., or may be carried out by appropriately selecting and using reagents from commercially available clinical test kits.

[0027] In either case of the measurement method based on the principle of non-competitive reaction or the measurement method based on the principle of competitive reaction of the present invention, when an antibody is used as the binding substance, it can be used depending on the purpose. F(ab')2, Fab' or Fab is digested with appropriate enzymes such as pepsin and papain.
It is desirable to use it as b.

[0028] The antibody used as the binding substance used in the present invention may be either a polyclonal antibody or a monoclonal antibody, and these may be used alone or in an appropriate combination.

In the method of the present invention, when forming a complex (or labeled complex), if necessary, two or more types of binding substances (specifically, each substance is added to a different site on the substance to be measured). If two or more types of binding substances with binding properties are used, the hydrophobicity of labeled complex A (or labeled complex B) will change as a result, and the molecular weight will increase. The labeling complex) and the binding substance (or the labeled substance to be measured) can be more easily separated, and measurement accuracy can be improved. Furthermore, in this case, it goes without saying that if a labeling substance is bound to each binding substance, the measurement sensitivity can be increased.

[0030] Furthermore, in a measurement method based on the principle of non-competitive reaction, it is disclosed in Japanese Patent Application Laid-Open No. 2002-11991 that the measurement sensitivity may be adjusted by using a labeled binding substance and a simple binding substance in combination.
As described in Japanese Patent No. 28557.

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

[0032]

【Example】

Experimental example 1. Effect of each salt on β-galactosidase activity (Reaction buffer 1) Dissolve 4.2 g of monosodium phosphate and 80.0 g of disodium phosphate in purified water to make a total volume of 5 liters, and mix with reaction buffer 1. did. (Reaction buffer 2) (NH4)2SO4 was dissolved in the reaction buffer to give a concentration of (NH4)2SO4 of 0.11,0.
22, 0.33, 0.44, 0.55, 0.66, 0.
Solutions of 77, 0.88, 0.99, and 1.10 M were prepared and adjusted to pH 7.5 with 4N NaOH. Similarly, Li2SO4, Na2SO4, K2SO4,
Rb2SO4, NaCl, CH3COONH4, K3P
Dissolve each O4 in reaction buffer 1 and add reaction buffer 2.
And so. Incidentally, K2SO4 was not dissolved above 0.55M. (Enzyme solution) β-galactosidase (derived from Escherichia coli, manufactured by Oriental Yeast Co., Ltd.) was dissolved in reaction buffer 1 to a concentration of 20 nM to obtain an enzyme solution. (Substrate solution) 1 mg of 4-methylumbelliferyl-β-D-galactopyranoside was dissolved in 30 ml of DMF,
Reaction buffer 1 was added to make the total volume 100 ml, which was used as a substrate solution. (Conditions for use of HPLC) An outline of the system is shown in FIG. Column: Not used. Flow rate: reaction buffer 2 1ml/min, substrate solution
0.1ml/min. Detection: Excitation wavelength 350nm, fluorescence wavelength 460n
Fluorescence was measured at m.

(Measurement procedure) 10 μl of the enzyme was injected into reaction buffer 2 containing each salt, and the activity was determined from the height of the β-galactosidase activity peak in each reaction buffer. (Results) The relationship between each salt concentration and β-galactosidase activity is shown in FIG. 6. As shown in Figure 6, in the case of (NH4)2SO4, it is almost 100% deactivated at a concentration of 0.4M, but N
For a2SO4 and NaCl, even at a concentration of 1M, it is about 15%
All I can see is the deactivation of . It is also seen that the enzyme activity of salts of sulfate ions and alkali metals decreases less with increasing concentration than other salts (excluding NaCl).

Experimental example 2. Calibration curve for β-galactosidase (reaction buffer 1) 4.2 g of monosodium phosphate and 80.0 g of disodium phosphate were dissolved in purified water to make a total volume of 5 liters, and reaction buffer 1 was prepared. (Reaction Buffer 2) (NH4)2SO4 was dissolved in a reaction buffer to prepare a solution with a (NH4)2SO4 concentration of 1.10M, and the pH was adjusted to 7.5 with 4N NaOH. Similarly, Na2SO4 and NaCl were each dissolved in reaction buffer 1 to prepare reaction buffer 2. (Antibody solution) Anti-human α-fetoprotein (mouse) monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was treated with a conventional method to obtain Fab', which was then labeled with β-galactosidase using a conventional method to obtain β- Galactosidase-labeled anti-human α-fetoprotein-Fab' was dissolved in reaction buffer 1 to prepare an antibody solution of 20, 16, 12, 8, 4, 2 nM. (Substrate solution) 1 mg of 4-methylumbelliferyl-β-D-galactopyranoside was dissolved in 30 ml of DMF,
Reaction buffer 1 was added to make the total volume 100 ml, which was used as a substrate solution. (Conditions for use of HPLC) An outline of the system is shown in Fig. 1. Column: TSKgel Butyl-NPR (4.6m
m x 3.5 cm, Tosoh Corporation) Flow rate: Reaction buffer 2 1 ml/min, substrate solution
0.1ml/min. Gradient pattern: linear gradient from reaction buffer 2 containing each salt to reaction buffer 1 (10 minutes) (FIG. 7). Detection: Excitation wavelength 350nm, fluorescence wavelength 460n
Fluorescence was measured at m.

(Measurement procedure) 10 μl of antibody solution of each concentration was injected using reaction buffer 2 containing each salt, and the activity was determined from the height of the eluted β-galactosidase activity peak. (Results) When NaCl was used, β-galactosidase-labeled anti-human α-fetoprotein-Fab' was not adsorbed to the column and passed through. After 9.5 minutes when using Na2SO4, after 9.5 minutes when using (NH4)2SO4
．． It eluted after 8 minutes. Figure 8 shows the relationship between each enzyme concentration and activity.
Shown below. As is clear from FIG. 8, the calibration relationship was a straight line passing through the origin no matter which salt was used. However, the measurement sensitivity is
In the case of Na2SO4 and NaCl, the sensitivity was more than 20 times higher than that of (NH4)2SO4.

Example 1. α-fetoprotein (AFP)
) Measurement (Reaction Buffer 1) 4.2 g of monosodium phosphate and 80.0 g of disodium phosphate were dissolved in purified water to make a total volume of 5 liters, and reaction buffer 1 was prepared. (Reaction Buffer 2) Na2SO4 was dissolved in Reaction Buffer 1 to prepare a solution with a Na2SO4 concentration of 1.10M, and the pH was adjusted to 7.5 with 4N NaOH to obtain Reaction Buffer 2A. Similarly, Li2SO4 was dissolved in reaction buffer 1 to make it 1.10M (pH 7.5), and Li2SO4 was dissolved in reaction buffer 2B.
And so. (Reaction Buffer 3) 0.2% bovine serum albumin was dissolved in Reaction Buffer 1 to obtain Reaction Buffer 3. (Antibody solution 1) Anti-human α-fetoprotein (mouse)
Monoclonal antibody 1 (clone No. 10-1) (manufactured by Wako Pure Chemical Industries, Ltd.) was treated with a conventional method to obtain Fab'
Then, β-galactosidase-labeled anti-human α-fetoprotein-Fab′ obtained by labeling β-galactosidase by a conventional method was dissolved in reaction buffer 3 to give 100%
Antibody solution 1 was prepared to have a concentration of nM. (Antibody solution 2) Anti-human α-fetoprotein (anti-human α-fetoprotein), which was confirmed to have a different epitope from the above-mentioned anti-human α-fetoprotein antibody (clone No. 10-1) by a conventional method.
Mouse) Monoclonal Antibody 2 (Clone No. 2-1
) (manufactured by Wako Pure Chemical Industries, Ltd.) in the same manner as in the case of antibody solution 1 to obtain β-galactosidase-labeled anti-human α-fetoprotein-Fab'. The labeled antibody was dissolved in reaction buffer 3 to a concentration of 100 nM, which was used as antibody solution 2. (Substrate solution) 1 mg of 4-methylumbelliferyl-β-D-galactopyranoside was dissolved in 30 ml of DMF,
Reaction buffer 1 was added to make the total volume 100 ml, which was used as a substrate solution. (Sample) Human placenta α-fetoprotein (manufactured by Wako Pure Chemical Industries, Ltd.) was mixed with reaction buffer 3 at 10, 20, 40,
The concentrations were adjusted to 60, 80, and 100 ng/ml. (Conditions for use of HPLC) An outline of the system is shown in Figure 1. Column: TSKgel
Butyl-NPR (4.6 mm x 3.5 cm, manufactured by Tosoh Corporation) Flow rate: Reaction buffer 1 + reaction buffer 2 1 m
l/min, substrate solution 0.1ml/min. Gradient pattern: Elution was performed for 5 minutes at a ratio of reaction buffer 1 and reaction buffer 2 of 3:7, and for 5 minutes at a ratio of 9:1 (Figure 2). Detection: Excitation wavelength 350nm, fluorescence wavelength 460n
Fluorescence was measured at m.

(Measurement procedure) 50 μl of antibody solution 1 and 50 μl of antibody solution 2 were mixed and reacted at 37° C. for 30 minutes, and 50 μl of this reaction solution was injected into a column. (Results) After 2.5 minutes, the labeled antibody that is not bound to α-fetoprotein forms an immune complex (antibody 1, antibody 2 and α-fetoprotein).
Fetoprotein) eluted after 7.7 minutes. The relationship between each α-fetoprotein concentration and the peak height at 7.7 minutes is shown in FIG. As is clear from FIG. 3, good calibration relationships were obtained in all cases. In addition, in the case of Na2SO4, L
The sensitivity was approximately 1.8 times that of i2SO4.

Example 2. Measurement of human chorionic gonadotropin (hCG) (Reaction buffer 1) 4.2 g of monosodium phosphate and 80.0 g of disodium phosphate were dissolved in purified water to a total volume of 5 liters, which was used as reaction buffer 1. . (Reaction Buffer 2) Na2SO4 was dissolved in Reaction Buffer 1 to prepare a solution with a Na2SO4 concentration of 1.10M, and the pH was adjusted to 7.5 with 4N NaOH to obtain Reaction Buffer 2. (Reaction Buffer 3) 0.2% bovine serum albumin was dissolved in Reaction Buffer 1 to obtain Reaction Buffer 3. (Antibody solution 1) Anti-hCG α chain (mouse) monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was treated with a conventional method to obtain Fab', which was then labeled with β-galactosidase using a conventional method to obtain β- Galactosidase-labeled anti-hCG-
Fab' was dissolved in reaction buffer 3 to give a concentration of 50 nM, which was used as antibody solution 1. (Antibody solution 2) Anti-hCG β chain (mouse) monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was treated in the same manner as in antibody solution 1, and β-galactosidase-labeled anti-hCG-
Fab' was obtained. The labeled antibody was dissolved in reaction buffer 3 to a concentration of 150 nM, which was used as antibody solution 2. (Substrate solution) 1 mg of 4-methylumbelliferyl-β-D-galactopyranoside was dissolved in 30 ml of DMF,
Reaction buffer 1 was added to make the total volume 100 ml, which was used as a substrate solution. (Sample) Commercially available hCG (manufactured by Sigma) was added to reaction buffer 3
10, 20, 40, 60, 80, 100 mIU/ml
It was prepared as follows. (Conditions for use of HPLC) An outline of the system is shown in Figure 1. Column: TSKgel
Butyl-NPR (4.6 mm x 3.5 cm, manufactured by Tosoh Corporation) Flow rate: Reaction buffer 1 + reaction buffer 2 1 m
l/min, substrate solution 0.1ml/min. Gradient pattern: Elution was performed for 5 minutes at a ratio of reaction buffer 1 and reaction buffer 2 of 3:7, and for 5 minutes at a ratio of 9:1 (Figure 2). Detection: Excitation wavelength 350nm, fluorescence wavelength 460n
Fluorescence was measured at m.

(Measurement procedure) Mix 50 μl of antibody solution 1, 50 μl of antibody solution 2, and 50 μl of sample, and heat at 37°C.
, for 30 minutes, and 100 μl of this reaction solution was injected into the column. (Results) The labeled antibody not bound to hCG was eluted after 2.5 minutes, and the immune complex (antibody 1, antibody 2, and hCG) was eluted after 7.8 minutes. Figure 4 shows the relationship between each hCG concentration and the peak height at 7.8 minutes.
Shown below. As is clear from FIG. 4, a good calibration relationship was obtained in this case as well.

[0040]

As described above, the present invention provides a method for measuring trace components in a sample by utilizing the interaction between a target substance and a substance capable of binding thereto, in which β-galactosidase is used as a labeling substance. The label complex A (or label complex B) produced as a result of the interaction and the free label-binding substance (or the target substance to be measured) are separated by adding a salt of sulfate ion and an alkali metal. Mobile phase (eluent) consisting of
The present invention provides a method for performing high-performance liquid chromatography using a post-column method using a column packed with a hydrophobic chromatography packing material, in which a mobile phase (eluent) containing the salt according to the present invention is used. When used, β-galactosidase activity is not inhibited even under high salt concentrations, and it has a remarkable effect in that highly sensitive measurements can be performed, and it is easier and more effective than conventional measurement methods such as EIA and RIA. This is a great invention that will contribute to this industry because highly accurate measurements can be made in an extremely short period of time.

[Brief explanation of the drawing]

FIG. 1 shows a schematic diagram of the HPLC system used in Experimental Example 2, Example 1, and Example 2.

[Figure 2] Figure 2 shows HPL in Example 1 and Example 2.
This shows the gradient pattern of C. The vertical axis represents the concentration (w/w%) of reaction buffer 1 in the eluate (reaction buffer 1 + reaction buffer 2), and the horizontal axis represents time ( minutes) respectively.

[Fig. 3] Fig. 3 shows the calibration curve of α-fetoprotein obtained in Example 1, and the immune complex obtained for the α-fetoprotein concentration (ng/ml) of each sample on the horizontal axis. It connects the points where the peak height (μV) of is plotted along the vertical axis. However, -□- uses reaction buffer 2A (Na2SO4), and -○- uses reaction buffer 2B (Na2SO4).
The results obtained when using Li2SO4) are shown.

FIG. 4 shows the calibration curve of human chorionic gonadotropin obtained in Example 2, and the immunity obtained against the human chorionic gonadotropin concentration (mIU/ml) of each sample on the horizontal axis. The peak height (μV) of the complex is plotted along the vertical axis and the points are connected.

FIG. 5 shows a schematic diagram of the HPLC system used in Experimental Example 1.

FIG. 6 shows the relationship between the salt concentration of each salt in the reaction buffer 2 and the activity of β-galactosidase in Experimental Example 1, and shows the relationship between the salt concentration (M) of each salt on the horizontal axis - Galactosidase activity peak height (μV) plotted along the vertical axis. However, -●- is (NH4)2SO4
, -x- is Li2SO4, -○- is Na2SO4, -+
- is K2SO4, -□- is Rb2SO4, -◇- is Na
Cl, -▽- is CH3COONH4, -△- is K3PO
4 are shown respectively.

[Fig. 7] Fig. 7 shows the HPLC gradient pattern in Experimental Example 2, and the vertical axis indicates the concentration of reaction buffer 1 in the eluate (reaction buffer 1 + reaction buffer 2). The concentration (w/w%) and the horizontal axis represent time (minutes), respectively.

[Fig. 8] Fig. 8 shows a calibration curve of β-galactosidase obtained in Experimental Example 2, in which β-galactosidase in each antibody solution on the horizontal axis is shown.
The height (μV) of the β-galactosidase activity peak obtained against the galactosidase concentration (nM) is plotted along the vertical axis, and the points are connected. However, -○- is obtained when (NH4)2SO4 is dissolved in reaction buffer 1, -□- is obtained by dissolving Na2SO4 in reaction buffer 1, and -△- is used by dissolving NaCl in reaction buffer 1. The results are shown below.

Claims (13)

[Claims] Claim 1: A biological sample containing a substance to be measured is mixed with a substance labeled with a labeling substance and has the ability to bind to the substance to be measured (hereinafter abbreviated as labeled binding substance) and reacted. After that, the complex of the substance to be measured and the label-binding substance (hereinafter abbreviated as label complex A) and the free label-binding substance are separated by high performance liquid chromatography, and the label complex A In the method of measuring the amount of a target substance in a sample by measuring the amount of a labeling substance in a sample or the amount of a labeling substance in a substance with label binding ability, β-galactosidase is used as a labeling substance, and sulfate ion and alkali are used. Using a mobile phase (eluent) containing a salt with a metal, the label complex A and the free label-binding substance are separated by high-performance liquid chromatography equipped with a column packed with a hydrophobic chromatography packing material. The measuring method is characterized in that: [Claim 2] The substance to be measured is a protein, a peptide,
The measuring method according to claim 1, which is a nucleic acid, a sugar chain, a lipid, a drug, or a hormone. [Claim 3] The substance to be measured is an antigen or an antibody,
2. The measuring method according to claim 1, wherein the binding substance is an antibody or an antigen against the substance to be measured. 4. The measurement method according to claim 3, wherein the antibody against the substance to be measured is a labeled monoclonal antibody. 5. The measurement method according to claim 3, wherein the antibody against the substance to be measured is a labeled Fab or Fab' derived from a monoclonal antibody. 6. The measurement method according to claim 1, wherein the substance to be measured is a sugar chain and the binding substance is a lectin. 7. The measuring method according to claim 6, wherein the lectin is concanavalin A, lentil lectin, kidney bean lectin, Datura lectin, or wheat germ lectin. 8. The measuring method according to claim 1, wherein the substance to be measured is a single-stranded polynucleotide constituting deoxyribonucleic acid, and the binding substance is a polynucleotide chain complementary to the polynucleotide chain. 9. After mixing and reacting a biological sample containing a substance to be measured with a substance to be measured labeled with a labeling substance (hereinafter abbreviated as a labeled substance to be measured) and a binding substance; The complex of the labeled target substance to be measured and the binding substance (hereinafter abbreviated as labeled complex B) and the free labeled target substance to be measured are separated by high-performance liquid chromatography, and the label in the labeled complex B is separated. In a method for measuring the amount of a target substance in a sample by measuring the amount of a substance or the amount of a labeling substance in a free labeled target substance, β-galactosidase is used as a labeling substance, and sulfate ion and alkali are used. Using a mobile phase (eluent) containing a salt with a metal, the labeled complex B and the free labeled analyte are separated by high performance liquid chromatography equipped with a column packed with a hydrophobic chromatography packing material. The measuring method is characterized in that: Claim 10: Claim 9 wherein the substance to be measured is a protein, peptide, nucleic acid, sugar chain, lipid, drug, or hormone.
Measurement method described in. 11. The measurement method according to claim 9, wherein the substance to be measured is an antigen or an antibody, and the binding substance is an antibody or antigen to the substance to be measured. 12. The measurement method according to claim 11, wherein the antibody against the substance to be measured is a monoclonal antibody. 13. The measurement method according to claim 11, wherein the antibody against the substance to be measured is Fab or Fab' derived from a monoclonal antibody.