JP3102611B2 - Complex separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a trace component in a biological sample such as a biological fluid such as serum, blood, plasma and urine, a lymphocyte, a blood cell and various cells, and a specific binding thereto. The present invention relates to a method for separating a complex with a substance having an ability from other substances and a method for measuring a trace component using the separation method.

[0002]

2. Description of the Related Art Bioactive substances such as antigens and antibodies, proteases and their proteinase inhibitors, sugar chains and lectins, enzymes and their substrates, coenzymes, hormones and the like. It is known that a receptor, a transport protein, a pair of polynucleotide chains of double-stranded DNA, and the like have a strong interaction with each other and form a strong complex.

As a method of measuring a trace component in a sample utilizing such an interaction, Japanese Patent Application Laid-Open Nos. 2-28557, 3-206964 and 3-206964 previously developed by the present inventors have been proposed. There is a method described in JP-A-221865. JP-A-2-2-2855
The method described in Japanese Patent Publication No. 7 is roughly described below, for example, in the case of utilizing the interaction between an antigen and an antibody. (1) In the measurement of a measurement target substance in a sample derived from a living body, an antibody prepared using the measurement target substance as an antigen is labeled with a detectable substance (hereinafter simply referred to as a detection substance) by any method ( After reacting by mixing with the sample, a complex of the substance to be measured (antigen) and the labeled antibody,
High-performance liquid chromatography (H
A method of measuring the amount of a substance to be measured in a sample by measuring the amount of a substance to be detected in the sample by separation by PLC). (2) In measuring a target substance in a sample derived from a living body, the target substance to which the target substance is bound and the antibody prepared using the target substance as an antigen are mixed with the sample and allowed to react. A complex of the target substance (antigen) labeled with the substance and the antibody and the target substance (antigen) to which the free detection substance is bound are separated by HPLC, and the amount of the detection substance in the complex is separated. Alternatively, a measurement method characterized by measuring the amount of a substance to be measured in a sample by measuring the amount of the substance to be measured (antigen) to which a free-type substance to be detected is bound.

[0004] The invention disclosed in Japanese Patent Application Laid-Open No. 3-206964 discloses that two or more isozymes or hormones having different sugar chain structures have the same action and the same detectable chemical properties. This is a measurement method in which the "substance to be measured" is used as the substance to be measured. This method will be described as follows, taking as an example the case where salivary α-amylase and pancreatic α-amylase are used as the substances to be measured. That is, the above 2
After mixing and reacting an anti-saliva-type α-amylase (mouse) monoclonal antibody with a sample containing various types of α-amylase, a reaction between the saliva-type α-amylase and the anti-saliva type α-amylase (mouse) monoclonal antibody was performed. The complex and free pancreatic α-amylase are separated by HPLC, and the amount or / and / or amount of salivary α-amylase contained in the complex of salivary α-amylase and anti-salivary α-amylase (mouse) monoclonal antibody This is a measuring method characterized in that the amount of salivary α-amylase and / or the amount of pancreatic α-amylase in a sample is separately measured by measuring the amount of free pancreatic α-amylase.

The invention disclosed in Japanese Patent Application Laid-Open No. 3-221865 discloses "two or more substances having the same action" such as isozymes and hormones having different sugar chain structures, or steroid hormones and human. This is a measurement method using “two or more substances to be measured having similar structures but different actions” such as chorionic gonadotropin as the substances to be measured. This method was performed by using hCG derived from placental villi and hC derived from choriocarcinoma.
The following is an example of the case where G is the substance to be measured. That is, in a sample containing the above two types of hCG, an anti-hCG-
a β-chain monoclonal antibody labeled with a detection substance,
Placental villi-derived hC that has binding ability only to choriocarcinoma-derived hCG
After mixing and reacting with a lectin that does not bind to G,
Placental villi-derived hCG and anti-hC labeled with a detection substance
A complex of a G-β chain monoclonal antibody, a complex of a choriocarcinoma-derived hCG and an anti-hCG-β chain monoclonal antibody and a lectin labeled with a detection substance, and an anti-hCG- labeled with a free detection substance. The β-chain monoclonal antibody was separated by HPLC, and the
This is a measuring method characterized by measuring the amount of two kinds of hCG in a sample by measuring the amount of quality.

As is apparent from the above description, each of the measuring methods described in the above publications relates to a substance to be measured (or a substance to be measured labeled with a detection substance) and a substance capable of binding thereto (hereinafter referred to as binding ability). Separation of a complex (or a complex labeled with a detection substance) resulting from an interaction with a free binding substance (or a free measurement target substance labeled with a detection substance) The HPLC
According to this method, trace components can be quantified by conventional EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or FIA (fluorescent immunoassay). Since the method can be performed easily and extremely accurately in a short time as compared with the measuring method described above, it is considered that the measuring method is expected to be greatly developed in the future.

[0007] Further, in the above publication, when a complex is formed, two or more kinds of binding substances (specifically, two or more binding substances each having a property of binding to a different site on a substance to be measured).
Or more than one kind of binding ability substance), furthermore, a method of using two or more kinds of binding ability substances labeled with a detection substance, and a result of using such two or more kinds of binding ability substances. The molecular weight of the complex becomes large,
Since the fluctuation range of the isoelectric point of the complex becomes large, the separation of the complex and the binding ability substance becomes easier, and the measurement accuracy can be improved, and each binding ability substance is labeled with a detection substance. It is disclosed that the measurement sensitivity can be increased by doing so.

However, the method using these two types of binding substances (including labeled substances) cannot be used when there is only one binding ability substance binding site in the substance to be measured. In addition, the method can cause a phenomenon in which the properties (molecular weight, hydrophobicity, ionicity, etc.) of the complex change as a result and the elution position shifts, but the above-described properties of the complex can be obtained. It was impossible to adjust it freely.

Therefore, the HPL in the method as described above
In the separation operation using C, the mere use of two kinds of binding substances may result in a complex (or a complex labeled with a detection substance) and a free binding substance (or a measurement labeled with a detection substance). Or the elution position of the complex (or the complex labeled with the detection substance) may overlap with the elution position of the biological component in serum or urine depending on the target substance. Problems such as a decrease in measurement accuracy may occur, and further improvements have been desired.

[0010]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and is intended to detect a complex resulting from the interaction between a substance to be measured and a binding substance and a complex such as a free binding substance. When using HPLC to separate coexisting substances that may affect the method, a method that can more effectively separate the complex from free binding substances and the like and trace components using this separation method Its purpose is to provide a measurement method.

[0011]

The present invention provides a measurement kit in which the substances to be measured are two or more substances having the same action as each other and having the same detectable chemical properties,
And although at least one specific binding of (a) analyte, the at least one of these having the property of not binding (hereinafter, abbreviated as affinity substance A _2.),
(B) Modified with a separation improving substance capable of changing the properties of the complex formed by binding of the measurement target substance and the binding ability substance A ₂ and binding to the complex of the measurement target substance and the binding ability substance A ₂ It is an invention of a kit comprising a substance having the following properties (hereinafter abbreviated as modified binding substance B1).

Further, the present invention provides a measurement kit in which the substance to be measured is two or more substances having the same action or two or more substances having a similar structure but different actions, a ') labeled material by and detectable substance has a binding capacity for all analyte (hereinafter abbreviated as affinity substance a _3.) and, (b' binding to) a particular analyte ability to have and the analyte and the affinity substance a ₃ and complex nature modified material by the separation improving substance capable of changing (hereinafter abbreviated as modified affinity substance C.) and the include The invention of a kit consisting of:

Furthermore, the present invention relates to a biological sample containing two or more substances to be measured having the same action and the same detectable chemical property, a binding ability substance A ₂ and a modified binding ability substance and B1 is reacted, to form a complex of the specific analyte and the affinity substance a ₂ and modified affinity substance B1, after separation of the analyte of free and complex, the complex An invention of a measurement method, characterized in that the amount of a substance to be measured in a sample is determined by measuring the amount of a substance to be measured contained therein and / or the amount of a free substance to be measured.

Further, the present invention relates to a biological sample containing two or more substances to be measured having the same action or two or more substances to be measured having similar structures but different actions, ₃ and the modified binding substance C to react with each other to form a complex (complex A) between the substance to be measured and the binding substance A ₃ and a specific substance to be measured, the binding substance A ₃ and the modified binding substance C After forming the complex (complex B) and separating the complex A and the complex B, the amount of the detection substance contained in the complex A or / and the amount of the detection substance contained in the complex B are determined. An invention of a measurement method characterized in that any amount of a substance to be measured in a sample is obtained by measuring.

Further, the present invention is an invention of a complex between analyte and the affinity substance A ₂ and modified affinity substance B1.
Furthermore, the present invention includes a substance to be measured, an invention of a complex between affinity substance A ₃ and modified affinity substance C.

That is, the present inventors have conducted intensive studies on a method for easily and efficiently fractionating and measuring two or more substances to be measured using a binding substance having a binding ability to these substances. When a separation improving substance having more appropriate properties is bound to the complex of the substance to be measured and the binding ability substance, and the separation operation of the complex is performed based on the properties of the separation improving substance, the separation is improved. By appropriately selecting and using the substance, the elution position in the column chromatography of the complex can be freely adjusted,
In other words, it has been found that by binding the separation-enhancing substance to the complex, the complex can be clearly separated from a coexisting substance that may affect the detection of the complex. Reached.

As the separation improving substance used in the method of the present invention, for example, a property of a complex of the substance to be measured and the binding substance A ₂ or A ₃ such as molecular weight, hydrophobicity and isoelectric point is changed. As long as it is a substance that can be obtained, it is not particularly limited, and specifically, for example, α-chymotrypsinogen,
β-galactosidase, lysozyme, cytochrome C,
Proteins such as trypsin inhibitors, for example, peptides containing amino acids such as phenylalanine, proline, arginine, lysine, aspartic acid, glutamic acid, etc., for example, halogen atoms such as bromine, chlorine, iodine, etc., synthetic polymers such as polyethylene glycol, for example, polyglutamic acid, Polyamino acids such as polyaspartic acid, polylysine, polyarginine, polyphenylalanine, and polytyrosine; alkyl chains having 3 to 10 carbon atoms, such as fatty acids such as palmitic acid, oleic acid, and stearic acid;
(ε-maleimidocaproyloxy) succinimide [N-
(ε-maleimidocaproyloxy) succinimide, hereinafter EMC
Abbreviated as S. ], N-Succinimidyl-6-maleimidohexanoat
e), Bismaleimidohexane,
Hereinafter, it is abbreviated as BMH. ), A chemical substance having a reactive group capable of binding to a substance capable of binding to a complex of a substance to be measured and a binding substance A ₂ or A ₃ , such as octylamine, and having hydrophobicity or ionicity. Preferred are mentioned.

A substance having a binding ability to a complex of a specific substance to be measured and a binding ability substance A ₂ (hereinafter, referred to as a substance to be used for preparing the modified binding ability substance B1 according to the present invention)
Abbreviated as binding substance B '. The), there is it does not inhibit the reaction to detect the analyte or the free analyte in the complex formation reaction and the complex with the affinity substance A ₂ with a specific analyte, the measurement object materials include but are not particularly limited as long as it is a substance capable of binding to affinity substance a ₂ or complex. In particular,
For example such as an antibody capable of binding to the measurement substance, for example concanavalin A, lentil lectin, Phaseolus vulgaris lectin, Datura stramonium lectin, lectin such as wheat germ lectin (where the binding site is different from the affinity substance A _2. ) and the like, or for example, an antibody capable of binding to the binding affinity substance a _2, for example concanavalin a, lentil lectin, Phaseolus vulgaris lectin, Datura stramonium lectin, lectin such as wheat germ lectin, and the like preferably. Also used to prepare the modified affinity substance C according to the present invention, substances capable of binding to the complex of the affinity substance A ₃ with a specific analyte (hereinafter affinity substance C ' abbreviated as. the), it was as it does not inhibit the reaction for detecting the detectable substance complex formation reaction and in complex with the analyte and the free affinity substance a ₃ (or affinity substance a ₃₎ Te, particular analyte, as long as it has a binding affinity substance capable of binding to affinity substance a ₃ or complexes thereof, or the detection substance bound to the affinity substance a ₃ They are not particularly limited. Specifically, for example, lectins such as an antibody having a binding ability to a substance to be measured, for example, concanavalin A, lentil lectin, kidney bean lectin, datura lectin, wheat germ lectin (however, binding to the binding substance A ₃ site different.) or the like, or for example, an antibody capable of binding to the binding affinity substance a ₃ or detection agents, e.g. concanavalin a, lentil lectin, Phaseolus vulgaris lectin, Datura stramonium lectin, wheat germ lectin lectin such as such Are preferred.

The method for binding a binding ability substance B ′ or C ′ and a separation enhancing substance according to the present invention includes a method of binding a specific reactive group of the binding ability substance B ′ or C ′ and a specific reactive group of the separation enhancing substance, A method in which a specific reactive group of the binding ability substance B 'or C' is replaced with a separation improving substance, a substance capable of binding to the binding ability substance B 'or C' (eg, an antibody, a lectin, an antigen, an inhibitor, DNA, etc.) Via the binding ability substance B 'or C'
And a method of binding the separation improving substance. More specifically, for example, 1) binding of an antibody to a labeling substance known per se, which is generally performed in a per se known enzyme immunoassay (EIA), radioimmunoassay (RIA), or fluorescence immunoassay (FIA). Methods (eg, Medical Experiment Account, Volume 8, Supervised by Yuichi Yamamura, First Edition, Nakayama Shoten,
1971; Illustrative fluorescent antibody, Akira Kawao, 1st edition, Soft Science Co., Ltd., 1983; Enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical Shoin, 1982, etc.) 2) Modification and binding methods of substances known per se (for example, chemical modification of proteins <upper><lower>, Ikuzo Uraya, Kensuke Shimura, Michinori Nakamura, edited by Masaru Funatsu, 1st edition, Gakkai Shuppan Co., Ltd.) , 198
1; Polyethylene glycol-modified protein, Yuji Inada et al., Biochemistry, Vol. 62, No. 11, P1351-1362, The Biochemical Society of Japan, 1990; DNA PROBES, George HK and Mark
MM STOCKTON PRESS, 1989, etc.) without exception, and these methods may be used.

The separation method of the present invention can be easily implemented, for example, as follows. That is, when the substances to be measured are two or more substances having the same action and the same detectable chemical property, first, the sample containing the substance to be measured and the binding ability substance A ₂ and a modified affinity substance B1, added to appropriate buffer if necessary, mixed and reacted, the particular analyte and the affinity substance a ₂ and modified affinity complex and the substance B1 bound after formation, be separated by the other coexisting substances such as affinity substance a ₂ free and the complex was a column packed with selected fillers depending on the nature of the separation-improving substance attached HPLC By
It can be easily implemented. Further, when the measurement target substance is two or more measurement target substances having the same action or two or more substances having a similar structure but different actions, first, a sample containing the measurement target substance, Binding substance A ₃
And the modified binding substance C, if necessary, are mixed in an appropriate buffer, mixed and reacted, and the substance to be measured and the binding substance A ₃
And a complex (complex B) in which a specific substance to be measured, a binding ability substance A ₃ and a modified binding ability substance C are bound, and then the complex is formed. The separation can be easily carried out by separating A from the complex B by HPLC equipped with a column packed with a filler selected according to the properties of the separation improving substance.

Although the separation method of the present invention can be effectively used for separating (or purifying) a specific trace component in a sample, it is particularly advantageous to use the separation method of the present invention when measuring the trace component. It is effective.

[0022]

[0023]

An example in which the present invention is used for measuring a trace component will be described below. (1) Measuring method in which the substance to be measured has the same action and two or more substances having the same detectable chemical property (measuring method (1)) First, a biological sample containing the substance to be measured And a substance that specifically binds to at least one of the substances to be measured but does not bind to at least one of them (that is, binding ability substance A ₂ ); Having a property of binding to the complex of the binding ability substance A ₂ (ie, the modified binding ability substance B)
1) and the addition to a suitable buffer if necessary, and reacted by mixing, after forming a complex with a particular analyte with the affinity substance A ₂ and modified affinity substance B1, the The complex and the free substance to be measured are separated by HPLC equipped with a column packed with a filler selected according to the properties of the separation improving substance. Next, if the amount of the measurement target substance contained in the separated complex and / or the amount of the free measurement target substance is determined by a measurement method according to the property of the measurement target substance, any of the measurement target substances in the sample is determined. Is required. At a above reaction, use concentration of affinity substance A ₂ in forming the complex may be appropriately set depending on setting a calibration limit or measuring sensitivity of the measuring object to what extent is not particularly limited However, usually, in the reaction solution, a concentration that can bind to all the substances to be measured corresponding to the set calibration limit concentration is at least a concentration, preferably at least twice the concentration, more preferably at least 5 times the concentration in the reaction solution. Is desirable. Further, the modified binding substance B1 for forming the complex
May be appropriately set depending on the calibration limit of the substance to be measured, and is not particularly limited. However, said use concentration is that the affinity substance A ₂ all must be set to more than the concentration capable of binding to a complex all produced by reacting with a specific analyte present in the reaction solution needless to say No.

(2) A measurement method in which the substance to be measured is two or more substances having the same action or two or more substances having a similar structure but different actions.
(2)) First, a biological sample containing the substance to be measured,
A substance that has a binding ability to all of the substances to be measured and is labeled with a detection substance (that is, a binding substance A ₃ ) and a substance that has a binding ability to a specific substance to be measured and is bound to the substance to be measured a ₃ may alter the properties of a complex between the substance (separation improving substance) by the modified material (i.e., modified affinity substance C) and was added to the appropriate buffer if necessary, and mixed reaction A complex of the substance to be measured and the binding ability substance A ₃ (that is, complex A) and a complex of the specific substance to be measured and the binding ability substance A ₃ and the modified binding ability substance C (that is, complex B) after forming a complex a, the complex B and free affinity substance a _3, separated by HPLC equipped with a column packed with selected fillers depending on the nature of the separation-improving substance. Next, if the amount of the detection substance contained in the separated complex A or / and the amount of the detection substance contained in the complex B is determined by a measuring method according to the property possessed by the detection substance, Any amount of the substance to be measured is determined.

When the binding ability substance A ₃ itself is a substance that can be measured (detected) by any method, the above-mentioned binding ability substance A ₃ not labeled with the detection substance is used.
The reaction of (2) was performed, and the binding ability substance A _{3 in the} obtained complex was obtained.
The amount also by determined by a measuring method suitable for properties of the affinity substance A ₃ of similarly can be determined analyte content of the sample.

[0027]

[0028]

The substance to be measured that can be measured by the measurement method (1) using the present invention can be measured (detected) by any method and has a strong mutual interaction with at least one of the substances to be measured. Act to form a strong complex, but at least one of them
The substance is not particularly limited as long as it has a substance having a property that it does not bind to one body. Examples thereof include biological fluids such as serum, blood, plasma, and urine, and biological organisms such as lymphocytes, blood cells, and various cells. Enzymes and the like contained in the sample are listed as typical examples. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GT
P), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase (PK), and tyrosine kinase. No.

As the binding substance A _{2 to the} substance to be measured according to the measuring method (1) utilizing the present invention, the substance A ₂ interacts strongly with at least one of the substances to be measured to form a strong complex. Is not particularly limited as long as it is a substance that does not bind to at least one of the substances to be measured. Examples thereof include an antibody against a specific partial structure of a substance having an antigenicity (including a hapten) or an antigen determining site, Lectins such as concanavalin A, lentil lectin, kidney bean lectin, kidney bean lectin, Hilo chawan lectin, juvenile lectin, peanut lectin, wheat germ lectin, etc., which have a binding ability to a sugar chain having a specific structure, such as amylase, creatine kinase ( CK), glutamate oxaloacetate transaminase (GO ) Inhibitors and the like for the enzyme, such as.

[0031]

The substance to be measured, which can be measured by the measuring method (2) using the present invention, is a substance which binds to all of the substances to be measured, and has a property which can be detected by any method or is itself detected. There is a substance that can be labeled with a substance, and further, it has a strong interaction (affinity or affinity) with at least one of the substances to be measured to form a strong complex. If there is a substance that does not bind to one,
Examples include, but are not limited to, enzymes, biologically active substances, cancer-related antigens, and the like contained in biological fluids such as serum, blood, plasma, and urine, and samples derived from living organisms such as lymphocytes, blood cells, and various cells. Typical examples include substances having a sugar chain. More specifically, for example, amylase,
Alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (L
DH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase, etc., such as steroid hormones, human chorionic gonadotropin (hCG), prolactin, thyroid stimulating hormone (TSH) ), Luteinizing hormone (LH)
Physiologically active substances such as prostate specific antigen (PSA),
α ₂ -macroglobulin, carcinoembryonic antigen (CEA), α-
Preferable examples include cancer-related antigens such as fetoprotein.

[0033] As affinity substance A ₃ for the measurement target material according to the measurement method using the present invention (2) is combined with all the analyte and itself has a property detectable by some method There is no particular limitation as long as it is a substance that is present or labeled with a detection substance. According to affinity substance A _3, as a specific example of a substance capable of binding to the measurement substance all, antibodies to a particular substructure or antigenic determinant sites of example substances having antigenicity (including haptens.) And lectins such as concanavalin A, lentil lectin, kidney bean lectin, dung bean lectin, Japanese locust lectin, chickpea lectin, peanut lectin, wheat germ lectin, or amylase, creatine kinase (CK), inhibitors, and the like for enzymes such as glutamic oxaloacetic transaminase (GOT), these those labeled detection agent may preferably be mentioned as specific examples of the affinity substance a _3.

[0034]

The detection substance used in the measurement method using the separation method of the present invention includes, for example, alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, and the like used in enzyme immunoassay (EIA). Enzymes such as glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase;
Used for example in radioimmunoassay (RIA)
Radioisotopes such as ^99m Tc, ¹³¹ I, ¹²⁵ I, ¹⁴ C, and ³ H, such as fluorescein, dansyl, fluorescamine, coumarin, and the like used in fluorescence immunoassay (FIA).
Fluorescent substances such as naphthylamine or derivatives thereof such as luciferin, isoluminol, luminol, bis
Luminescent substances such as (2,4,6-trifluorophenyl) oxalate, for example, substances having an ultraviolet absorption such as phenol, naphthol, anthracene or derivatives thereof, 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-cyclohexadiene-1-ylidene) -p-tolyloxyl and the like having a property as a spin labeling agent represented by a compound having an oxyl group, and the like.
It goes without saying that the present invention is not limited to these.

As a method for labeling the binding substance A ₃ with a detection substance as described above, EIA and RIA known per se are used.
Or a labeling method known per se generally used in FIA and the like (for example, Medical Chemistry Laboratory Course, Volume 8, supervised by Yuichi Yamamura, First Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody, Akira Kawao , 1st edition, Soft Science Co., Ltd., 1983; Enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical Shoin, 1982, etc., all without exception. Just do it. In addition, it goes without saying that a conventional method using a reaction between avidin (or streptavidin) and biotin may be used as the labeling method.

Examples of the binding substance A _{3 which} can be measured (detected) by any method according to the present invention include:
For example, substances having the properties of the above-described detection substance itself, such as an enzyme, a fluorescent substance, a luminescent substance, or a substance having absorption in the ultraviolet region, may be used.

The separation-improving substance according to the present invention may be a substance to be measured or a property of the binding substance A ₂ or A ₃ (or / and the binding substance B ′ or C ′) (for example, pH stability, hydrophobicity, In consideration of the solubility, isoelectric point, etc.), it is sufficient to select and use as appropriate.

It is sufficient that the type of the packing material of the column used in the HPLC is appropriately selected according to the properties of the separation improving substance.

Hereinafter, the combination of the filler and the separation improving substance will be described in more detail.

When a filler for gel filtration is used Since it is a filler having a property of separating a target substance from other coexisting substances by utilizing a difference in molecular weight, a separation improving substance such as protein or polyethylene glycol is used. Preferred are high-molecular substances such as synthetic polymers and polyamino acids. That is, the measurement target substance, the affinity substance A ₂ or A ₃ in the complex, by further modified affinity substance B1 or C bond, can change the molecular weight of said complex for the purpose Becomes The molecular weight of the modified binding substance B1 or C is at least 1.2 times the molecular weight of the complex, preferably
1.5 times or more, more preferably 2 times or more is preferable because high resolution can be obtained. The separation method based on the above principle is particularly effective when the sample containing the substance to be measured contains various substances that affect the analysis, such as serum, and their molecular weights have a wide range. That is, in order to avoid the influence of the coexisting substance in such a sample, the molecular weight of the complex should be larger than the maximum value of the molecular weight of the substance affecting the analysis. It is sufficient to select and use a compound having a molecular weight that satisfies the above. If the molecular weight of the complex is adjusted by the number of binding substances A ₂ or A ₃ to be bound as in the conventional method, (the target molecular weight / the molecular weight of the binding substance A ₂ or A ₃ ) since it is necessary to bond the material a ₂ or a ₃ to the target substance, the method, has a problem that the only available when the substance measured with the number of binding sites required It is clear that Further, in the present invention, when a separation-improving substance having a molecular weight larger than the molecular weight cut off of the packing material for gel filtration is bound, the complex containing the substance to be measured elutes in the void portion of the column. The analysis can be done in the shortest time.
In this case, it goes without saying that the separation improving substance may not be a substance having a single molecular weight, that is, a substance having a molecular weight larger than the molecular weight cut off of the packing material for gel filtration. As a filler for gel filtration, for example, YM
C-Pack Diol-200 (trade name of YMC), YMC-
Pack Diol-300 (trade name of YMC Co., Ltd.), TSK gel (trade name of Tosoh Corporation) and the like.

When a packing material for hydrophobic chromatography is used Since the packing material has a property of separating a target substance from other coexisting substances by utilizing a difference in hydrophobicity, examples of the separation improving substance include α-chymotrypsinogen. , Β
A protein having high hydrophobicity such as galactosidase, for example, a peptide containing a highly hydrophobic amino acid such as phenylalanine, proline, etc., for example, a polyamino acid such as polyglutamic acid, polyaspartic acid, polylysine, polyarginine, polyphenylalanine, polytyrosine; 3
~ 10 alkyl chains, e.g., halogen atoms such as bromine, chlorine, iodine, etc .; complexes of highly hydrophobic chemicals, e.g. octylamine, EMCS, BMH, e.g. fatty acids such as palmitic acid, oleic acid, stearic acid, etc. A substance whose hydrophobicity can be appropriately set is preferably exemplified. still,
When a peptide is used as the separation improving substance, a peptide containing a highly hydrophobic amino acid is preferable, and the hydrophobicity may be adjusted by selecting the chain length. In the case of peptides and polyamino acids composed of only hydrophobic amino acids, when the number of amino acids is 15 or more, the solubility in water is reduced. When the separation improving substance is a halogen atom, the modified binding substance B1 or C can be easily obtained by directly halogenating the binding substance B ′ or C ′,
The hydrophobicity can be appropriately adjusted by changing the amount of halogen introduced. Examples of the chemical substance having high hydrophobicity include substances having a long alkyl chain other than those described above. The hydrophobicity can be adjusted by appropriately selecting the alkyl chain length. Since the separation enhancing substance having too high hydrophobicity has low solubility in water, it is necessary to use an organic solvent at the time of the binding reaction between the binding enhancing substance B ′ or C ′ and the separation enhancing substance, resulting in the resulting modified binding ability. Since the denaturation of the substance or the decrease in the activity may occur, or the modified binding substance may become insoluble in water, it may not be preferable as a separation improving substance. Examples of the packing material for hydrophobic chromatography include butyl-NPR (trade name of Tosoh Corporation), butyl MCI gel (trade name of Mitsubishi Kasei Co., Ltd.), and phenyl MCI gel (trade name of Mitsubishi Kasei Co., Ltd.) Is mentioned.

When a filler for ion-exchange chromatography is used. The target substance and other coexisting substances are separated by utilizing the difference in ionicity. Therefore, examples of the separation improving substance include basic proteins such as lysozyme and cytochrome C, and the like. For example, acidic proteins such as trypsin inhibitor, for example, residues containing basic amino acid residues such as arginine and lysine, and peptides containing acidic amino acid residues such as aspartic acid and glutamic acid, and polyamino acids containing 50 or more amino acid residues as described above Preferred examples thereof include fatty acids such as palmitic acid, oleic acid, and stearic acid. In addition, in the case of ion exchange chromatography, in general, it is better to elute the target substance once it is adsorbed on the column, since high resolution and high specificity can be obtained. It is preferable to use a packing material for cation exchange chromatography, and to use a packing material for anion exchange chromatography when an anionic separation improving substance is used. When the separation improving substance is a peptide or polyamino acid composed of only basic amino acid residues (or acidic amino acid residues), the elution time of the complex can be freely adjusted by adjusting the number of amino acid residues. When a peptide or polyamino acid composed of usually 5 or more, preferably 50 or more, more preferably 100 or more residues is used, the elution position of the complex can be completely separated from that of the biological component in serum or urine. So desirable. When the peptide or polyamino acid is a synthetic peptide or synthetic polyamino acid, the length of the peptide (or polyamino acid) is proportional to the ionicity. By adjusting and using it as a separation improving substance, the elution position of the complex can be easily adjusted. In addition, even when there are a plurality of serum components affecting the measurement, if the ionicity of the complex is made larger than the ionicity of these serum components by using a separation improving substance, the stepwise gradient can be used. The effect that the time required for analysis can be shortened is produced. Packing materials for ion-exchange chromatography generally have a high exchange capacity (absolute adsorption capacity for ionic substances), so when analyzing samples with a large absolute amount of ionic coexisting substances such as biological samples such as serum. Even so, all the complexes to which the separation improving substance is bonded can be adsorbed, so that the complexes can be eluted at a position where the influence of the coexisting substance can be almost avoided. In addition, since the separation improving substance that can be used in the present method has high solubility in water, the water solubility of the complex to which the separation improving substance is bound is higher than that before the binding. There is almost no possibility that the object to be measured will be denatured or deactivated during the formation reaction of the complex. As a packing material for ion exchange chromatography, for example, DEAE-MCI gel (Mitsubishi Chemical
(Trade name), QAE MCI gel (Mitsubishi Kasei Corporation), Wakobeads DEAE gel (Wako Pure Chemical Industries, Ltd.), etc., a filler for anion exchange chromatography, or, for example, SP MCI
Gel (Mitsubishi Chemical Corporation), CM MCI Gel (Mitsubishi Chemical Corporation)
(Trade name), Wako Beads CM Gel (Wako Pure Chemical Industries, Ltd.)
Fillers for cation exchange chromatography such as (trade name).

The present invention can be carried out by HPLC using any of the above-mentioned chromatographys.
Among them, the use of ion exchange chromatography is most preferred for the present invention. The reasons are as follows, for example. For example, in order to carry out the separation method of the present invention using gel filtration chromatography, it is necessary to use a column of an appropriate length. There is a disadvantage that the separation time is longer than in the case of using the lithography. Therefore, it is preferable to use ion exchange chromatography when a reduction in the separation time is required. In addition, gel filtration chromatography requires very high molecular weights (molecular
There is also a drawback that it is not suitable for separation of substances to be measured having a thickness of 1000 Å or more. In the case of hydrophobic chromatography, when the substance to be measured is a physiologically active substance having a high-dimensional structure such as a protein, the high-dimensional structure is destroyed by the organic solvent used in the separation operation, and the measurement in the complex is performed. There may be a problem that the activity of the target substance is lost. For these reasons, it is not preferable to use a substance having high hydrophobicity as the separation improving substance. Further, there is a problem that the higher the hydrophobicity of the binding ability substance B ′ or C ′ to which the hydrophobic substance is bound as the separation improving substance, the lower the water solubility of the complex and the more the precipitate is easily precipitated, so that the separation becomes difficult. On the other hand, ion exchange chromatography is based on subtle differences in ionicity,
The substance to be measured can be separated more effectively. Furthermore, according to ion exchange chromatography, a separation improving substance having various ionic properties can be arbitrarily selected, so that a substance to be measured can be separated under an optimum pH condition. Furthermore, since the separation enhancer used in ion exchange chromatography has high water solubility itself, even if the separation enhancer is bound to the target substance, there is almost no possibility that the target substance will precipitate. Thus, the separation operation can be performed in a stable state.

When a measurement target substance is measured using the measurement method of the present invention, the peak of the target measurement target substance can be moved to a position that is not affected by components such as serum and urine. In addition, by appropriately selecting and using the modified binding substance B1 or C according to the substance to be measured, the elution positions of the complexes containing various substances to be measured can be matched, so that the specific analysis conditions There is also an effect that various kinds of substances to be measured can be measured using HPLC.

In the measurement method of the present invention, the detection contained in the complex separated by HPLC (including both those bound by the modified binding substance B1 or C and those not bound). substance (or affinity substance a ₃ or analyte) measurement of the amount of the detectable substance (or affinity substance a ₃ or analyte) has, depending on the nature that can be detected by some method Each is performed according to a predetermined method. For example, if the property is enzymatic activity, the standard method of EIA, for example, "Enzyme immunoassay, protein nucleic acid enzyme extra volume No. 31, Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji,
Edited by Eiji Ishikawa, 51-63, Kyoritsu Shuppan Co., Ltd., September 1987
The measurement may be carried out according to the method described in "10 days issued", etc., and when the detected substance is a radioactive substance, immersion is performed according to the type and intensity of the radiation emitted by the radioactive substance according to the RIA standard method. The measurement may be performed by appropriately selecting and using measuring instruments such as a type GM counter, a liquid scintillation counter, a well-type scintillation counter, and a HPLC counter (for example, Medical Chemistry Experiment Course, Vol. 8, supervised by Yuichi Yamamura, 1st edition). Edition, Nakayama Shoten, 1971, etc.). When the property is fluorescent, a standard FIA method using a measuring instrument such as a fluorometer, for example, "Illustration Fluorescent Antibody, Akira Kawao, First Edition, Soft Science Corp., 1983", etc. The measurement may be carried out according to the method described in, and when the property is luminescent, a conventional method using a measuring instrument such as a photon counter, for example, "enzyme immunoassay, protein, nucleic acid, enzyme, extra volume"
No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 2
52 to 263, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 ”or the like. Further, when the property is a property having absorption in the ultraviolet, measurement may be performed by a conventional method using a measuring instrument such as a spectrophotometer, and when the detection substance is a substance having a spin property, electron spin resonance is used. A conventional method using an apparatus, for example, "Enzyme Immunoassay, Protein Nucleic Acid Enzyme Supplement No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 264-271, Kyoritsu Shuppan Co., Ltd., September 1987 The measurement may be performed according to the method described in “10th issue”.

In the present invention, the reaction conditions for forming a complex by reacting the substance to be measured with the binding ability substance A ₂ or A ₃ , or these complexes and the modified binding ability substance B 1 or C
The reaction conditions for the binding reaction with are not particularly limited as long as they do not prevent the complex formation reaction or the binding reaction between these complexes and the modified binding substance B1 or C. For example, the reaction may be performed according to reaction conditions for forming a complex or the like by a method known per se such as EIA, RIA, FIA, and affinity chromatography. For example, when a buffer is used during the reaction, a buffer and other reagents to be used may be appropriately selected from those used in a method known per se. The pH during the reaction is not particularly limited as long as it does not hinder the complex formation reaction or the binding reaction between these complexes and the modified binding substance B1 or C. ~
10, preferably in the range of 5-9. The temperature during the reaction is not particularly limited as long as it does not hinder the complex formation reaction or the binding reaction between these complexes and the modified binding substance B1 or C. And preferably in the range of 20 to 40 ° C. The reaction time, the complex (or the labeled complex) is formed, and the time required for binding of the modified affinity substance B1 or C to these complexes, bind to analyte affinity substance A ₂ or A ₃ And the properties of the modified binding substance B1 or C, and the reaction may be appropriately carried out for several seconds to several hours depending on each property.

In the separation method of the present invention, a complex (including a substance bound to the modified binding substance B1 or C and a substance not bound to the modified binding substance B1 or C. The same applies hereinafter) or a free binding substance A _{As a} HPLC used for separating ₂ or A ₃ or a free substance to be measured, there is no particular problem as long as the apparatus itself is one generally used in the field of analysis and a constant flow rate can be obtained. It can be used without.

As a solvent (eluent) used for separating the complex and the free binding substance A ₂ or A ₃ by HPLC, the formed complex or the like is again the substance to be measured and the binding substance A ₂ or A ₃ (and the modified binding substance B1 or C) without being decomposed into the binding substance A ₃ (or the substance to be measured) contained in the complex detecting substances affinity substance a ₃ holds has, there may be mentioned, without being limited particularly unless such that occupies lose the property that can be detected by some method, typically for example EIA, RIA, Those used as a buffer in a method known per se, such as FIA and affinity chromatography, are preferably used. Specific examples include phosphates, acetates, citrates, buffers of Good, and buffers such as tris (hydroxymethyl) aminomethane, and salts such as sodium chloride, potassium chloride, and ammonium sulfate. Polar organic solvents such as methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran and the like, and a surfactant or the like are used as a complex (or labeled complex), a free binding substance A ₂ or A ₃ , or a free measurement target substance. Prepared by appropriately selecting, adding and mixing according to the properties,
A buffer having a pH of 2 to 10 is preferably used.

In the measurement method using the separation method of the present invention, the measurement method after separation by HPLC is as follows.
For example, the effluent from an HPLC column as described in “Latest Liquid Chromatography, Shoji Hara and Akio Tsuji, First Edition, pp. 92-104, Nanzando, published February 1, 1978” is used. directly guided to the detection unit, the detection substance contained in the complex in the effluent (or affinity substance a ₃ or analyte) method for directly measuring the amount of is more preferable because rapidly perform the measurement. In this case, if the property of the binding ability substance A ₃ (or the substance to be measured) or the detection substance held by the binding ability substance A ₃ can be detected by any method, for example, enzyme activity For example, it goes without saying that it is necessary to provide a so-called post-column method reaction section between the HPLC column and the detection section, in which a reagent for enzyme activity measurement is added and reacted with the effluent. Detectable substance (or affinity substance A ₃ or analyte) reagents for measuring the enzyme activity which is used in the reaction section when said properties is an enzyme activity of a conventional method, for example, "Enzyme Immunoassay , Proteins, Nucleic Acids and Enzymes Separate Volume No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji and Eiji Ishikawa, pp. 51-63, Kyoritsu Shuppan Co., Ltd., September 1, 1987
One prepared according to the method described in “0-day issue” or the like may be used, or a reagent of a commercially available clinical test kit may be appropriately selected and used. The detection substance (or affinity substance A ₃ or analyte) also said properties of In cases other than enzyme activity, adding a predetermined reagent for the purpose of increasing the detection sensitivity, in order to react, HPLC It is optional to provide an appropriate reaction section between the column and the detection section.

When a plurality of components having different components are used as eluents for HPLC in the separation method of the present invention, the operation method is a concentration gradient method (linear gradient method).
The stepwise method can be performed easily, the operation can be simplified, the actual analysis time can be shortened, and the target peak can be sharpened. There are advantages such as
More desirable. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0052]

【Example】

Embodiment 1 FIG. Measurement of human chorionic gonadotropin (hCG) and α-fetoprotein (AFP) (when β-galactosidase is used as a separation-improving substance and a filler for gel filtration is used) (eluent) monosodium phosphate 3.9 g, 81 g of disodium phosphate (12-hydrate) and 44 g of sodium chloride were dissolved in ion-exchanged water, and the pH was adjusted to 7.5. (Substrate solution) Dissolve 1.66 g of 3- (p-hydroxyphenyl) propionic acid in 80 ml of eluent, and add 1N to adjust pH to 7.5.
After addition of NaOH, the total volume was made up to 100 ml. With this solution
A 30% hydrogen peroxide solution was diluted to prepare a 20 mM solution of H ₂ O ₂ , which was used as a substrate solution. (Antibody solution 1) An anti-hCG-α chain monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was treated by a conventional method to obtain Fab ′, and horseradish peroxidase (POD) was added thereto by a conventional method.
POD-labeled anti-hCG-α chain-Fab ′ obtained by labeling
The antibody solution 1 was added to a phosphate buffer (pH 7.5, containing 150 mM sodium chloride) so as to have a protein concentration of 3 nM. (Antibody solution 2) An anti-hCG-β chain monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) is processed by a conventional method to obtain Fab ′, which is then treated with β-galactosidase (β-Gal, Oriental Yeast Co., Ltd.) Sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate [Sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexa]
ne-1-carboxylate (Sulfo-SMCC), manufactured by Pierce Co.] and β-Gal-bound anti-hCG-β obtained by a conventional method.
The chain-Fab 'was added to a 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) so as to have a protein concentration of 50 nM to prepare an antibody solution 2. (Antibody solution 3) A POD-labeled anti-AFP-Fab ′ obtained by treating an anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) in accordance with the method for preparing antibody solution 1 was treated with a 50 mM phosphate buffer ( Antibody Solution 3 was added to a protein solution of pH 7.5, containing 150 mM sodium chloride so as to have a protein concentration of 3 nM. (Antibody solution 4) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) confirmed to have a different recognition site from the monoclonal antibody used in antibody solution 3 was treated according to the method for preparing antibody solution 2. Obtained β-Gal binding anti-AFP-Fab ′
Was added to a 50 mM phosphate buffer solution (pH 7.5, containing 150 mM sodium chloride) so as to have a protein concentration of 50 nM to prepare an antibody solution 4. (HCG sample solution) A commercially available hCG (derived from placental villi, manufactured by Sigma) was added to a 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) to a concentration of 250 mIU / ml, and the hCG sample solution was added. And (AFP sample solution) AFP (manufactured by Wako Pure Chemical Industries, Ltd.) purified from human placenta was used in 50 mM phosphate buffer (pH 7.5, 150 mM).
(Containing sodium chloride) to give an AFP sample solution at a concentration of 1 nM. (Conditions for Using HPLC) An outline of the system is shown in FIG. -Column: 0.8φx30cm. Filler: YMC Pack Diol-200 (trade name of YMC Co., Ltd.). Flow rate: eluent: 1.0 ml / min, substrate liquid: 0.1 ml / min.・ Reaction part: 0.025φx1000cm (Insulation at 55 ° C) ・ Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. (Measurement procedure) Samples 1 to 5 having the composition shown in Table 1 below were prepared,
After each sample was left at 30 ° C. for 30 minutes, 50 μl of each sample was
Analyzed by PLC.

[Table 1] (Results) From the analysis results of the sample 1 and the sample 2, the POD-labeled anti-hCG-α chain-Fab '
The complex of -α chain-Fab 'and hCG (complex-1) was 9.5
Minutes later, POD-labeled anti-hCG-α chain-Fab ′ and β-Gal
It was found that each of the complexes of the bound anti-hCG-β chain-Fab ′ and hCG (complex-2) eluted after 6.8 minutes. From the analysis results of Samples 3 and 4, POD-labeled anti-AFP-Fa
b ′ was 10.5 minutes later, the complex of POD-labeled anti-AFP-Fab ′ and AFP (complex-3) was 9.1 minutes later, and POD-labeled anti-A
It was found that the complex of FP-Fab ′ and β-Gal-bound anti-AFP-Fab ′ and AFP (complex-4) eluted after 6.8 minutes, respectively. In the case of Sample 2 and Sample 4, complex-1,
Complex-3 was not detected. From the above results, it is possible to more clearly separate the POD-labeled antibody and the complex by using the modified binding substance, and the elution time of the complex-2 and the complex-4 It is the same (all eluted after 6.8 minutes), in other words, the use of the modified binding substance makes it possible to perform analysis using HPLC under the same conditions even when the measurement target is different. I understand. In the case of sample 5, the complex-
2 after 6.8 minutes, complex-3 after 9.1 minutes, POD-labeled anti-hCG-α chain-Fab 'and POD-labeled anti-AFP-Fab'
It eluted after 0.5 minutes. As is apparent from the results, it is possible to simultaneously measure different analytes (hCG and AFP) by appropriately using the modified binding substance.

Embodiment 2 FIG. AFP measurement (when β-Gal is used as a separation-improving substance and a packing material for hydrophobic chromatography is used) (Eluent A) A 50 mM phosphate buffer (pH 7.5) containing 1.7 M ammonium sulfate is used as an eluent A And (Eluent B) 50 mM phosphate buffer (pH 7.5)
And (Substrate solution) Same as in Example 1. (Antibody solution 1) Same as antibody solution 3 of Example 1. (Antibody solution 2) Same as antibody solution 4 of Example 1. (Sample) Same as the AFP sample solution of Example 1. (Conditions for Using HPLC) An outline of the system is shown in FIG. -Column: 0.46φx2.5cm. Filler: butyl-NPR (trade name of Tosoh Corporation).・ Flow rate: eluent A + B; 1.0 ml / min, substrate solution: 0.1 ml / mi
n.・ Reaction part: 0.025φx1000cm (Insulation at 55 ° C) ・ Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. Gradient: The gradient of eluent A and eluent B is shown in FIG. 3 (Measurement procedure) Sample 1 having the same composition as sample 3 of Example 1 and Sample 2 having the same composition as sample 4 of Example 1. age,
After each sample was left at 30 ° C. for 30 minutes, 50 μl of each sample was
Analyzed by PLC. (Results) From the analysis results by HPLC, POD-labeled anti-A
FP-Fab 'was 4.9 minutes later, and the complex of POD-labeled anti-AFP-Fab' and AFP was 6.7 minutes later, POD-labeled anti-AFP-F
It was found that the complexes of ab ', β-Gal-bound anti-AFP-Fab' and AFP eluted after 9.7 minutes, respectively. The above results show that the use of the modified binding substance makes it possible to more clearly separate the POD-labeled anti-AFP-Fab 'and the complex.

Embodiment 3 FIG. AFP measurement (when iodine is used as a separation improving substance and a packing material for hydrophobic chromatography is used) (Antibody solution 1) Same as antibody solution 3 in Example 1 (Antibody solution 2) Antibody solution 4 in Example 1 The anti-AFP monoclonal antibody used for the preparation was purified by the chloramine T method known per se (Biochemical Experiment Course 16 "Hormone", edited by The Biochemical Society of Japan, published by Tokyo Kagaku Dojin, pages 117-180 and 230-231). , 1977.). Each of the iodinated anti-AFP monoclonal antibodies obtained with the iodination reaction time of 5 seconds, 30 seconds or 2 minutes was subjected to a protein concentration of 50 nM in 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride). Antibody solution 2-1, antibody solution 2-2, and antibody solution 2-3 were added as needed. (Sample) Same as the AFP sample solution of Example 1 (use conditions of HPLC) Same as Example 2. (Measurement procedure) 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride), antibody solution 2-1, antibody solution 2-2 or antibody solution 2-3 were added to 30 μl of antibody solution 1 and 30 μl of AFP sample solution.
After mixing 30 μl and leaving the mixture at 30 ° C. for 30 minutes, 50 μl of the obtained mixture was analyzed by HPLC. (Results) From the analysis results by HPLC, POD-labeled anti-A
It was found that FP-Fab 'eluted after 4.9 minutes and the complex A of POD-labeled anti-AFP-Fab' and AFP eluted after 6.7 minutes. In addition, the complex of POD-labeled anti-AFP-Fab 'and iodinated anti-AFP-Fab' and AFP
-1 was used after 8.2 minutes, antibody solution 2-2 was used after 9.5 minutes, and antibody solution 2-3 was used after
It was found that each eluted after 9.9 minutes. From the above results, it can be seen that the elution position of the complex of POD-labeled anti-AFP-Fab 'and AFP can be freely adjusted by using an antibody having a different binding amount of iodine (modified binding substance).

Embodiment 4 FIG. AFP measurement [When octylamine, phenylalanine tetramer (hereinafter abbreviated as Phe4), BMH or EMCS is used as a separation improving substance, and a packing material for hydrophobic chromatography is used] (Antibody solution 1) Same as Antibody Solution 3 (Antibody Solution 2) 0.1 M phosphate buffer [pH 7.0, containing 50% dimethylformamide (DMF). Octylamine and Sulfo-SMCC (manufactured by Pierce) dissolved in an equimolar amount by a conventional method, and an anti-AFP monoclonal antibody having a recognition site different from that of the antibody used in the antibody solution 1 is added to the reaction solution. Fab '(anti-AFP-Fab') [0.1
M phosphate buffer (pH 7.0)], 1/100 times the molar amount of octylamine, and the suspension was incubated at 30 ° C for 30 minutes. The reaction solution was purified by an ordinary method to obtain octylamine-conjugated anti-AFP-Fab ′, which was added to a 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride).
The antibody solution 2 was added by adding so as to have a protein concentration of 50 nM. (Antibody solution 3) Using the same reagent as used for preparing antibody solution 2 except that Phe4 peptide was used in place of octylamine, the same operation was carried out to obtain Phe4-bound anti-AFP
-Fab 'was prepared and mixed with 50 mM phosphate buffer (pH 7.5, 15
(0 mM sodium chloride) to give an antibody solution 3 at a protein concentration of 50 nM. (Antibody solution 4) The same anti-AFP-Fab ′ used in preparing the antibody solution 2 was reacted with BMH by a conventional method, and N-acetyl-L-cysteine (AC) was added to this reaction solution with BMH. 100
A double molar amount was added, and the mixture was further incubated at 30 ° C. for 30 minutes. The reaction solution was purified by a conventional method to obtain AC-BMH-conjugated anti-AFP-Fab ′, which was added to a 50 mM phosphate buffer (pH 7.
5, containing 150 mM sodium chloride) to give a protein concentration of 50 nM. (Antibody solution 5) The same anti-AFP-Fab ′ used in preparing the antibody solution 2 was reacted with EMCS by a conventional method, and glycine was added to this reaction solution in a molar amount 1000 times the EMCS.
Incubated at 30 ° C. for 30 minutes. The reaction solution was purified by a conventional method, and glycine-EMCS-conjugated anti-AFP-Fa
b ′ was obtained and added to a 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) so as to have a protein concentration of 50 nM to prepare an antibody solution 5. (Sample) Same as the AFP sample solution of Example 1. (Conditions for HPLC) Same as in Example 2. (Measurement method) A solution of 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride), antibody solution 2, antibody solution 3, antibody solution 4 or antibody solution 5 was added to 30 μl of antibody solution 1 and 30 μl of AFP sample solution.
After mixing 30 μl and leaving at 30 ° C. for 30 minutes, 50 μl of the mixture
Was analyzed by HPLC. (Results) As a result of analysis by HPLC, POD-labeled anti-AF
After 4.9 minutes, P-Fab 'and POD-labeled anti-AFP-Fab'
The complexes with FP were found to elute after 6.7 minutes, respectively. Further, the complex of POD-labeled anti-AFP-Fab 'and AFP and each modified anti-AFP-Fab' (modified binding ability substance) was 7.5 minutes after using octylamine-conjugated anti-AFP-Fab '. After 8.5 minutes when using Phe4-conjugated anti-AFP-Fab ', and 7.2 minutes after using AC-BMH-conjugated anti-AFP-Fab', anti-AF with glycine-EMCS was used.
When P-Fab 'was used, it was found that each eluted after 7.1 minutes. From the above results, it can be seen that the elution position of the target complex can be freely adjusted by changing the type of the separation improving substance.

Embodiment 5 FIG. AFP measurement (when polyaspartic acid is used as a separation improving substance and a packing material for anion exchange chromatography is used) (Eluent A) 10 mM phosphate buffer (pH 7.5) is used as eluent A
And (Eluent B) Eluent B was 50 mM phosphate buffer (pH 7.5) containing 1 M sodium chloride. (Substrate solution) 5 containing 20% DMF and 150 mM sodium chloride
2.5 mM 4-methylumbelliferyl galactopyranoside was dissolved in 0 mM phosphate buffer (pH 7.5) to obtain a substrate solution. (Antibody solution 1) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was treated by a conventional method to obtain Fab ′, and β-Gal-labeled anti-AF obtained by labeling this with β-Gal by a conventional method.
P-Fab 'was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) to a protein concentration of 4 nM to prepare antibody solution 1. (Antibody solution 2) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) whose recognition site is confirmed to be different from that of the monoclonal antibody used in the antibody solution 1 is processed by a conventional method to obtain Fab ′, This and Sulfo-polyaspartic acid (average molecular weight: 6165, 13000 or 28800, manufactured by Sigma)
Binding was carried out by a conventional method using SMCC (manufactured by Pierce), followed by purification by a conventional method to obtain various polyaspartic acid-conjugated anti-AFP-Fab's. These were each added to a 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) so as to have a protein concentration of 50 nM to prepare an antibody solution 2. (Sample) The same system as the AFP sample liquid of Example 1 (use conditions of HPLC) is schematically shown in FIG. -Column: 0.46φx3.0cm. -Filler: DEAE-MCI gel (Mitsubishi Kasei Corporation).・ Flow rate: eluent A + B; 1.0 ml / min, substrate solution: 0.1 ml / mi
n.・ Reaction part: 0.025φ × 1000cm (Insulation at 45 ° C.) ・ Detection: Fluorescence was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm. Gradient: The gradient between eluent A and eluent B is shown in FIG. 4. (Measurement method) Antibody solution 1 (30 μl), AFP sample solution (30 μl), 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) or specified Anti-AF conjugated with polyaspartic acid
After 30 μl of the antibody solution 2 containing P-Fab ′ was mixed and left at 30 ° C. for 30 minutes, 50 μl of the mixture was analyzed by HPLC. (Results) As a result of analysis by HPLC, β-Gal-labeled anti-AFP-Fab ′
The complex of Fab 'and AFP was found to elute after 4.2 minutes. In addition, the complex of β-Gal-labeled anti-AFP-Fab ′ and AFP and polyaspartic acid-conjugated anti-AFP-Fab ′ (modified binding ability substance) is obtained when the average molecular weight of the bound polyaspartic acid is 6165. Is 4.99 minutes, 13000
It was found that eluted after 7.90 minutes in the case of, and after 8.85 minutes in the case of 28800. From the above results, it can be seen that the elution position of the complex can be freely adjusted by changing the properties of the separation improving substance, that is, the molecular weight of polyaspartic acid.

Embodiment 6 FIG. Examination of avoiding the influence of hemolysis in AFP measurement (when polyaspartic acid is used as a separation improving substance and a filler for anion exchange chromatography is used) (eluent A) Same as eluent A in Example 5. (Eluent B) Same as eluent B of Example 5. (Substrate solution) Same as the substrate solution of Example 1. (Antibody solution 1) Same as antibody solution 3 of Example 1. (Antibody solution 2) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) whose recognition site is confirmed to be different from that of the monoclonal antibody used in the antibody solution 1 is processed by a conventional method to obtain Fab ′, This was combined with polyaspartic acid (average molecular weight: 13000, manufactured by Sigma) by a conventional method using Sulfo-SMCC (manufactured by Pierce), and then purified by a conventional method to obtain an antibody to which polyaspartic acid was bound. AFP-
Fab 'was obtained. This was added to a 50 mM phosphate buffer (pH 7.5, 150 mM
Antibody solution 2 by adding 50 nM protein concentration to the solution (containing sodium chloride). (Hemolysis) 1000mg / d hemoglobin obtained from human red blood cells
Diluted with 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) so as to obtain 1 l, was defined as hemolyzed blood. (Sample) Same as the AFP sample solution of Example 1. (Conditions for Using HPLC) An outline of the system is shown in FIG. -Column: 0.46φx3.0cm. -Filler: Wako Beads DEAE gel (trade name of Wako Pure Chemical Industries, Ltd.).・ Flow rate: eluent A + B; 1.0 ml / min, substrate solution: 0.1 ml / mi
n.・ Reaction part: 0.025φx1000cm (Insulation at 55 ° C) ・ Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. Gradient: The gradient between eluent A and eluent B is shown in FIG. 4. (Measurement method) A 50 mM phosphate buffer (pH 7.5, 150 mM sodium chloride) was added to 30 μl of the antibody solution 1, 30 μl of the AFP sample solution and 10 μl of the lysed blood. 30 μl of Antibody Solution 2
After standing at 30 ° C. for 30 minutes, 50 μl of each mixture was analyzed by HPLC. (Results) The obtained elution pattern is shown in FIG. In FIG.
A shows the elution pattern obtained by analyzing the hemolyzed blood diluted 10-fold with a 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride), and B shows the antibody solution 1, AFP sample solution, and hemolysate. And C, the elution pattern obtained as a result of analyzing the mixture with the phosphate buffer and antibody buffer 1.
The elution patterns obtained as a result of analyzing a mixture of the P sample solution, the hemolyzed solution, and the antibody solution 2 are shown respectively. As is clear from FIG. 5A, the hemolytic component elutes as a plurality of peaks during 0.5 to 5.5 minutes. On the other hand, PO
D-labeled anti-AFP-Fab 'was 1.10 minutes later, POD-labeled anti-AF
After 3.25 minutes, the complex of P-Fab 'and AFP was labeled with POD-labeled anti-AFP-Fab' and polyaspartic acid-conjugated anti-AFP-Fa.
It can be seen that the complexes of b 'and AFP elute after 7.20 minutes, respectively. From the above results, it can be seen that by using the separation method of the present invention, the complex can be eluted at an elution position that is not affected by hemolysis at all, in other words, by using the separation method of the present invention, the measurement can be performed at the time of measurement. It can be seen that the measurement target substance can be measured without being affected by the hemolytic component in the sample to be measured, so that the measurement accuracy can be improved as compared with the conventional case.

Embodiment 7 FIG. Differentiation measurement of hCG having different sugar chain structures (when polyaspartic acid is used as a separation-improving substance and a filler for anion exchange chromatography is used) (Antibody solution) Same as antibody solution 1 in Example 1. (Lectin solution 1) Datsura lectin (manufactured by Wako Pure Chemical Industries, Ltd.) was treated with 50 mM Tris (hydroxymethyl) aminomethane (hereinafter, Tris) -hydrochloric acid buffer (pH 7.5, 150 mM)
1 mg in sodium chloride and 1 mM calcium chloride)
Lectin solution 1 was added by adding so as to have a protein concentration of / ml. (Lectin solution 2) The same method as used for preparing lectin solution 1 was used for the same method as in the use of datsura lectin and polyaspartic acid (average molecular weight: 28800) using disuccinimidyl suberate (Pierce). To make polyaspartic acid-binding datura lectin,
Lectin solution 2 was added to a 50 mM Tris-HCl buffer (pH 7.5, 150 mM sodium chloride, containing 1 mM calcium chloride) to a protein concentration of 1 mg / ml.
And (HCG sample solution) Human choriocarcinoma-derived hCG (manufactured by Wako Pure Chemical Industries, Ltd.) was treated with 50 mM Tris-HCl buffer (pH 7.5 150 m
250 M in sodium chloride and 1 mM calcium chloride)
The solution added to a concentration of mIU / ml was used as an hCG sample solution. (Conditions for HPLC) Same as in Example 6. (Measurement procedure) 10 μl of hCG sample solution, 20 μl of antibody solution, and 70 μl of lectin solution 1 or lectin solution 2 were mixed.
After leaving at 0 ° C. for 30 minutes, 50 μl of the resulting mixture was
Analyzed by LC. (Results) As a result of HPLC analysis, POD-labeled anti-hC
G-α chain-Fab ′ was 1.10 minutes later, POD-labeled anti-hCG-α
The complex of chain-Fab 'and hCG was 3.50 minutes later, the complex of POD-labeled anti-hCG-α chain-Fab' and hCG and datura lectin was 3.82 minutes later, and the complex of POD-labeled anti-hCG-α chain-Fab 'and h
It was found that the complexes of CG and polyaspartic acid-conjugated datura lectin eluted after 7.9 minutes, respectively. From the above results, it can be seen that the use of polyaspartic acid-binding datura lectin allows the lectin-binding complex and the lectin-non-binding complex to be more clearly separated, in other words, all hC
It turns out that the amount of hCG having a specific sugar chain in G can be measured with higher accuracy.

Embodiment 8 FIG. Measurement of thyroxine (T ₄ ) (when polyglutamic acid is used as a separation-improving substance and a packing material for anion exchange chromatography is used) (Eluent A) 20 mM Tris-HCl buffer (pH 8.0) was used as eluent A did. (Eluent B) Eluent B was 20 mM Tris-HCl buffer (pH 8.0) containing 1 M sodium chloride. (Substrate solution) 3- (p-hydroxyphenyl) in 80 ml of eluent A
After dissolving 1.66 g of propionic acid and adjusting the pH to 8.0 with 1N sodium hydroxide, the total volume was adjusted to 100 ml with eluent A. 30
% Hydrogen peroxide solution was diluted with this solution to prepare a solution containing 20 mM H ₂ O ₂ and used as a substrate solution. (Antibody solution 1) anti-T ₄ monochrome - what was monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was treated by a conventional method Fab ', by a conventional method horseradish peroxidasin - peptidase (POD) labeled The POD-labeled anti-T ₄ -Fab ′ thus obtained was added to a 20 mM Tris-HCl buffer (pH 8.0) to a protein concentration of 3 nM to prepare an antibody solution 1. (Antibody solution 2) An anti-POD monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was treated by a conventional method to obtain Fab ′, which was then mixed with polyglutamic acid (average molecular weight: 95100, manufactured by Sigma).
And polyglutamic acid-conjugated anti-POD-Fab 'obtained by binding Sulfo-SMCC using a conventional method was added to a 20 mM Tris-HCl buffer (pH 8.0) so as to have a protein concentration of 50 nM. Liquid 2. The anti-PO used this time
The D monoclonal antibody has the property of binding to POD but not inhibiting its enzymatic activity. (T ₄ sample solution) commercially available L- thyroxine (Sigma) 2
0mM Tris - was T ₄ sample solution was added to a concentration of 10nM in hydrochloric acid buffer solution (pH 8.0). (Conditions for Using HPLC) An outline of the system is shown in FIG. -Column: 0.46φx3.0cm. -Filler: Wako Beads DEAE gel (trade name of Wako Pure Chemical Industries, Ltd.).・ Flow rate: eluent A + B; 1.0 ml / min, substrate solution: 0.1 ml / mi
n.・ Reaction part: 0.025φx1000cm (Insulation at 55 ° C) ・ Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. Gradient: The gradient of eluent A and eluent B shown in FIG. 4 (Measurement Operation) T ₄ sample solution 30 [mu] l antibody solution 1 30 [mu] l and 20mM
Sample 1 was prepared by mixing 30 μl of a Tris-HCl buffer (pH 8.0), and Sample 2 was obtained by mixing 30 μl of a T ₄ sample solution, 30 μl of an antibody solution, and 230 μl of an antibody solution. After each sample was left at 30 ° C for 30 minutes, 50 μl of each sample was
Was analyzed by (Results) The POD-labeled anti-T
_4-Fab 'after 3.3 minutes, POD-labeled anti-T _4-Fab' complex with the T ₄ after 2.3 minutes, POD-labeled anti-T _4-Fab 'and polyglutamate conjugated anti POD-Fab' complexes of After 8.9 minutes,
POD-labeled anti-T ₄ -Fab ′ and polyglutamic acid-conjugated anti-PO
Complex with D-Fab 'and T ₄ was found to come to each eluted after 7.4 minutes. From the above results, by using polyglutamic acid-conjugated anti-POD-Fab ′, POD-labeled anti-T ₄
-Fab 'and POD-labeled anti-T _4- It is possible to change the elution position of the complex of -Fab' and T ₄ , in other words, P
OD label 'and, POD-labeled anti-T _4-Fab' anti-T _4-Fab and T ₄
It can be seen that the complex can be more clearly separated from the complex.

Embodiment 9 FIG. Measurement of thyroxine (T ₄ ) (when polyglutamic acid is used as a separation improving substance and a packing material for anion exchange chromatography is used) (Labeled antigen) Conventional method (NAKANE method: Nakane, PK and Kawao
i, A., J.Histochem.Cytochem., vol.22,1084-1091,197
POD-labeled T ₄ was prepared by 4), 20 mM Tris - was a solution obtained by dissolving to a concentration of 10nM HCl buffer (pH 8.0) and labeled antigen solution. (Antibody solution 1) anti-T ₄ monochrome - the monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) Fab which was obtained by treating in a conventional manner ', which 20-fold molar amount of N- ethylmaleimide added to 60 at 37 ° C
It was left to react for minutes. Then, N-ethylsuccinimide-modified Fab ′ obtained by purifying the same by a conventional method was added to a 20 mM Tris-HCl buffer (pH 8.0) so as to have a protein concentration of 2 nM to prepare an antibody solution 1. (Antibody solution 2) Same as antibody solution 2 of Example 8. (T4 sample solution) the same as T ₄ sample solution of Example 8. (Hemolysis) Same as the hemolysis in Example 6. (Conditions for HPLC) Same as in Example 8. (Measurement operation) Samples 1 to 3 having the composition shown in Table 2 below were prepared,
After each sample was left at 30 ° C. for 30 minutes, 50 μl of each sample was
Analyzed by PLC.

[Table 2] (Results) From the analysis results by HPLC, the POD-labeled T ₄
After 2.6 minutes, POD-labeled T ₄ and N- ethyl succinimide of Fab 'complex of after 3.5 minutes, POD-labeled T ₄ and polyglutamic acid conjugated anti POD-Fab' complex of after 8.3 minutes, P
OD labeled T ₄ and N- ethyl succinimide of Fab 'and polyglutamate conjugated anti POD-Fab' complex with was found to come to each eluted after 9.1 minutes. On the other hand, it was found that the hemolytic component eluted as a plurality of peaks in 0.5 to 5.5 minutes as in Example 6. From the above results, it can be seen that by using the separation method of the present invention, the complex can be eluted at an elution position that is not affected by hemolysis at all, in other words, by using the separation method of the present invention, the measurement can be performed at the time of measurement. It can be seen that the measurement target substance can be measured without being affected by the hemolytic component in the sample to be measured, so that the measurement accuracy can be improved as compared with the conventional case.

[0061]

As described above, the present invention relates to a complex formed as a result of the interaction between a substance to be measured and a substance capable of binding thereto and a free binding substance (or a free substance to be measured).
In a measurement method in which separation from other substances such as is performed using high-performance liquid chromatography, the properties of the complex can be freely changed by further binding a separation-improving substance to the complex. In which the elution position of the complex can be freely adjusted in the present invention. A trace component in a biological sample such as serum was measured using the separation method of the present invention. In this case, a remarkable effect that high-precision measurement can be performed easily and in a very short time as compared with conventional measurement methods such as EIA and RIA is also exerted. Invention.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a high-performance liquid chromatography (HPLC) system used in Example 1.

FIG. 2 shows a schematic diagram of the HPLC system used in Examples 2, 3, 4, 5, 6, 7, 8, and 9.

FIG. 3 shows HPLC in Examples 2, 3 and 4.
The vertical axis represents the concentration of ammonium sulfate (M), and the horizontal axis represents time (minutes).

FIG. 4 shows HPLC gradient patterns in Examples 5, 6, 7, 8 and 9, wherein the vertical axis represents the concentration (%) of eluent B, and the horizontal axis represents time (%). Minutes) respectively.

FIG. 5 shows the elution pattern of the sample obtained by HPLC obtained in Example 6, wherein A shows that the lysed blood was diluted 10-fold with a 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride). B shows the elution pattern obtained as a result of analyzing the diluted solution, B shows the elution pattern obtained as a result of analyzing the mixture of antibody solution 1, AFP sample solution, hemolyzed solution and phosphate buffer, and C shows the antibody solution. 1 shows the elution patterns obtained as a result of analyzing a mixture of the AFP sample solution, the hemolyzed solution, and the antibody solution 2, respectively.

 ──────────────────────────────────────────────────続 き Continuing on the front page Judge Chieko Goto, Judge of the Joint Panel Judge Kiyozumi Yazawa Judge, Koichi Itsubo (56) References JP-A-3-221865 (JP, A) JP-A-61-110059 (JP, A)

Claims (11)

(57) [Claims] 1. A measurement kit in which the substances to be measured are two or more substances having the same action as each other and having the same detectable chemical properties, wherein (a) the substance to be measured is While at least one specific binding substance having does not bind to at least one property of these (hereinafter, abbreviated as affinity substance a _2.) and, (b) analyte and the affinity substance a substance having the property of binding to a complex of ₂ and the modified and analyte by separation-improving substance which is capable of changing the nature of the complex resulting bound to affinity substance a ₂ (hereinafter, modified affinity abbreviated as material B 1.) the comprising at kit. 2. A measuring kit in which the substance to be measured is two or more substances having the same action or two or more substances having a similar structure but different actions,
(A ′) a substance that has binding ability to all of the substances to be measured and is labeled with a detection substance (hereinafter, binding ability substance A ₃
Abbreviated. ) And (b ′) a substance modified with a separation improving substance ( hereinafter, referred to as a substance having a binding ability to a specific substance to be measured and capable of changing the property of a complex of the substance to be measured and the binding substance A ₃₎ Abbreviated as modified binding substance C.)
And a kit comprising: 3. The separation enhancing substance is a protein, a peptide,
Yes synthetic polymer compound, poly amino acid, a halogen atom, an alkyl chain, fatty acids, or the analyte and the binding ability complex or specific reactive group capable of binding to a substance capable of binding to the target substance with a substance A ₂ The kit according to claim 1, wherein the kit is a chemical substance having hydrophobicity or ionicity. 4. A biological sample containing two or more substances to be measured having the same action and the same detectable chemical property, and a binding ability substance A ₂ and a modified binding ability substance B 1 . After the reaction, a complex of the specific substance to be measured, the binding ability substance A ₂ and the modified binding ability substance B 1 is formed, and the complex is separated from the free substance to be measured, and then contained in the complex. A measurement method characterized by determining any amount of a measurement target substance in a sample by measuring the amount of a measurement target substance and / or the amount of a free measurement target substance. 5. A biological substance containing two or more substances to be measured having the same action or two or more substances to be measured having a similar structure but different functions, and a binding ability substance A.
₃ and the modified binding substance C to react with each other to form a complex (complex A) between the substance to be measured and the binding substance A ₃ and a specific substance to be measured, the binding substance A ₃ and the modified binding substance C After forming the complex (complex B) and separating the complex A and the complex B, the amount of the detection substance contained in the complex A or / and the amount of the detection substance contained in the complex B are determined. A measuring method characterized by determining any amount of a substance to be measured in a sample by measuring. 6. The method according to claim 4, wherein the separation is by liquid chromatography. 7. The method according to claim 4, wherein the packing of the column used in the liquid chromatography is a packing for ion exchange chromatography. 8. The separation enhancing substance is a protein, a peptide,
Yes synthetic polymer compound, poly amino acid, a halogen atom, an alkyl chain, fatty acids, or the analyte and the binding ability complex or specific reactive group capable of binding to a substance capable of binding to the target substance with a substance A ₂ The method according to claim 4, wherein the chemical substance is a hydrophobic or ionic chemical substance. 9. The method according to claim 6, wherein the separation enhancing substance is a protein, a synthetic high molecular compound or a polyamino acid, and the packing material of the column used in liquid chromatography is a packing material for gel filtration. 10. The separation enhancing substance is a protein, a peptide,
Polyamino acids, the alkyl chain, halogen atom, fatty acids, or the analyte and the affinity substance A ₂ complexes or specific measuring object having a reactive group capable of binding to a substance capable of binding to a substance and a hydrophobic Chemical substances that have
The method according to claim 6, wherein the packing material of the column used in the liquid chromatography is a packing material for hydrophobic chromatography. 11. The separation improving substance is a synthetic polymer compound, a protein, capable of binding to a substance capable of binding to the complex or specific analyte polyamino acid, or a substance to be measured and the affinity substance A ₂ reaction The method according to claim 6, which is a chemical substance having a group and ionicity, a fatty acid or a peptide, and wherein the packing material of a column used in liquid chromatography is a packing material for ion exchange chromatography.