JP3376955B2 - Complex separation method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to trace components in biological samples such as serum, blood, plasma, urine, etc., biological samples such as lymphocytes, blood cells, and various cells and their specific binding. 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 this separation method.

[0002]

BACKGROUND OF THE INVENTION Certain substances, such as antigens and antibodies, proteases and their proteinaceous protease inhibitors, sugar chains and lectins, enzymes and their corresponding substrates and coenzymes, physiologically active substances such as hormones, and their receptors and transports. It is known that proteins, a pair of polynucleotide chains of double-stranded DNA, and the like exert strong interactions with each other to form a strong complex.

As a method for measuring a trace component in a sample utilizing such interaction, the inventors of the present invention have previously developed JP-A-2-28557, JP-A-3-206964 and JP-A-3- There is a method described in Japanese Patent No. 221865. JP-A-2-285
The method described in Japanese Patent Publication No. 7 will be described as follows in the case of utilizing the interaction between an antigen and an antibody, for example. (1) In the measurement of a substance to be measured in a biological sample, an antibody prepared by using the substance to be measured as an antigen is labeled with a substance that can be detected by some method (hereinafter, abbreviated as a detected substance) ( (Labeled antibody) is mixed with the sample and reacted, and then the complex of the substance to be measured (antigen) and the labeled antibody and the free labeled antibody are separated by high performance liquid chromatography (HPLC). A measuring method, which comprises measuring the amount of a substance to be measured in a sample by measuring the amount of a substance to be detected in the body. (2) In the measurement of a substance to be measured in a sample derived from a living body, the substance to be measured to which a substance to be detected is bound and an antibody prepared using the substance to be measured as an antigen are mixed with a sample and reacted, and then detected. A complex of a substance to be measured (antigen) labeled with a substance and an antibody,
The substance to be measured (antigen) bound to the free type detection substance is separated by HPLC, and the amount of the substance to be detected in the complex or the substance to be measured (antigen) bound to the free type detection substance
A measuring method, which comprises measuring the amount of a substance to be measured in a sample by measuring the amount of a substance to be detected.

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

The invention disclosed in Japanese Patent Laid-Open No. 3-221865 discloses "two or more substances to be measured having the same action" such as isozymes and hormones having different sugar chain structures, or steroid hormones and humans. This is a measurement method in which “two or more measurement target substances having a similar structure but different actions” such as chorionic gonadotropin are the measurement target substances. This method is applied to placental villi-derived hCG and choriocarcinoma-derived hC.
The following is a description of an example in which G is the substance to be measured. That is, in a sample containing the above two types of hCG, anti-hCG-having binding ability to any hCG-
β-chain monoclonal antibody labeled with a detection substance,
Placental villus-derived hC capable of binding only to choriocarcinoma-derived hCG
After mixing and reacting with a lectin that does not bind to G,
Anti-hC labeled with placental villus-derived hCG and a detection substance
Complex with G-β chain monoclonal antibody, complex with anti-hCG-β chain monoclonal antibody and lectin labeled with choriocarcinoma-derived hCG and detection substance, and anti-hCG-labeled with free detection substance Separation from β-chain monoclonal antibody by HPLC, detection product in each complex
This is a measuring method characterized by measuring the amounts of two types of hCG in a sample by measuring the amount of quality.

As is clear from the above description, each of the measuring methods described in the above publications has a substance to be measured (or a substance to be measured labeled with a detection substance) and a substance capable of binding to the substance (hereinafter, binding ability). Abbreviated as "substance.") Separation of a complex (or a complex labeled with a detection substance) resulting from an interaction with a free binding substance (or a free substance to be measured labeled with a detection substance) HPLC
According to the method, the quantitative determination of trace components is carried out by conventional EIA (enzyme immunoassay), RIA (radioimmunoassay) or FIA (fluorescent immunoassay), etc. It is considered to be a method that is expected to be developed in the future because it can be performed easily and in a very short period of time with extremely high precision as compared with the method described in (1).

Further, in the above-mentioned publication, when forming a complex, two or more kinds of binding substances (specifically, those having a property of binding to different sites on a substance to be measured, respectively)
More than one kind of binding ability substance), further using two or more types of binding ability substance labeled with a detection substance, and the use of such two or more types of binding ability substance The molecular weight of the complex increases,
Since the range of fluctuation of the isoelectric point of the complex becomes large, etc., it becomes easier to separate the complex and the binding substance, and the measurement accuracy can be improved, and each binding substance is labeled with a detection substance. It is disclosed that the measurement sensitivity can be increased by making such preparations.

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

Therefore, the HPL in the above method is used.
In the separation operation using C, the complex (or the complex labeled with the detection substance) and the free binding substance (or the measurement labeled with the detection substance) can be measured by simply using two types of binding substances. (The target substance) is not sufficiently separated, or the elution position of the complex (or the complex labeled with the detection substance) overlaps with the elution position of biological components in serum or urine depending on the measurement 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-described situation, and it is a complex of a complex resulting from the interaction between a substance to be measured and a binding substance and a free binding substance and the like. When separating a coexisting substance that may affect the detection of a complex using HPLC, a method capable of more effectively separating the complex and a free binding-capacity substance and the like, and a method for separating the same are used. It is an object of the present invention to provide a method for measuring a trace amount of the component.

[0011]

The present invention is directed to a substance to be measured and a substance capable of binding to the substance to be measured (hereinafter,
It is abbreviated as a binding substance A ₁ . ) To a complex further modified with a substance capable of changing the hydrophobicity or ionicity of the complex (hereinafter abbreviated as a separation improving substance) and having a binding ability to the complex (hereinafter, (Abbreviated as modified binding ability substance A), and HP of the complex and free binding ability substance A ₁ (or free measurement target substance)
Separation by LC is carried out by using the hydrophobic or io
It is an invention of a method for separating the complex, which is characterized in that it is carried out based on the reactivity .

[0012] The present invention also, in the method of measuring analyte using the reaction between the analyte and the affinity substance A _1, resulting a result of the reaction, the analyte and the affinity substance A ₁ Further, a modified binding ability substance A is further bound to the complex of ( ₁ ), and the complex and the free binding ability substance A ₁ (or the free measurement target substance) are combined with the hydrophobicity or ion of the separation enhancing substance.
The amount of the complex or the free binding ability substance A ₁ (or the free measurement target substance) is measured after separation by HPLC based on the property , and the measurement target substance amount is determined based on the result. It is an invention of a measuring method.

Further, according to the present invention, a biological sample containing a substance to be measured, a substance to be measured labeled with a detection substance (hereinafter abbreviated as a labeled substance to be measured) and a binding substance A.
₁ is reacted with the modified binding ability substance A to form a complex of the measurement target substance, the binding ability substance A ₁ and the modified binding ability substance A, and a complex of the labeled measurement substance, the binding ability substance A ₁ and the modified binding ability substance A. After forming a body (hereinafter abbreviated as a labeled complex), the labeled complex and the labeled measurement substance are separated from each other by using a hydrophobicity- improving substance.
Or by measuring the amount of the detection substance contained in the separated labeled complex by high performance liquid chromatography according to the ionicity , and then determining the amount of the substance to be measured. It is an invention.

That is, the present inventors have found that the complex resulting from the interaction between the substance to be measured and the binding substance A ₁ and the free binding substance A ₁ (or the free substance to be measured. The substance may be labeled with a detection substance.) When the separation from a coexisting substance that may affect the detection of the complex, such as As a result of intensive studies in search of a method capable of more clearly separating A ₁ etc., a more suitable hydrophobic property for the complex was obtained .
Alternatively, an ionic separation-improving substance is bound, and based on the hydrophobicity or ionicity of the separation-improving substance, the complex and the complex such as a free binding ability substance A ₁ (or a free substance to be measured) When a separation operation with a coexisting substance that may affect the detection of the complex is performed, it is possible to freely adjust the elution position of the complex by appropriately selecting and using the separation enhancing substance. In other words, they have found that the complex and the free binding substance A ₁ etc. can be more clearly separated by binding an appropriate separation enhancing substance to the complex, and completed the present invention.

The separation-improving substance used in the separation method of the present invention may be a substance capable of changing properties such as hydrophobicity and isoelectric point of the complex of the substance to be measured and the binding substance A _1. Specific examples thereof include, but are not limited to, proteins such as α-chymotrypsinogen, β-galactosidase, lysozyme, cytochrome C, trypsin inhibitor, for example, phenylalanine,
Peptides containing amino acids such as proline, arginine, lysine, aspartic acid, glutamic acid, for example, halogen atoms such as bromine, chlorine, iodine, synthetic polymers such as polyethylene glycol, such as polyglutamic acid, polyaspartic acid, polylysine, polyarginine, Polyamino acids such as polyphenylalanine and polytyrosine, alkyl chains having 3 to 10 carbon atoms, such as fatty acids such as palmitic acid, oleic acid and stearic acid, such as N- (ε-maleimidocaproyloxy) succinimide [N- (ε- maleimidoca
proyloxy) succinimide, hereinafter abbreviated as EMCS. ], N-succinimidyl-6-maleimidohexanoate (N-Succinimidyl-6-maleimidohexanoate), bismaleimidohexane (Bismaleimidohexane, hereinafter BMH)
Is abbreviated. ), 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 ability substance A ₁ such as octylamine, and having hydrophobicity or ionicity.

A substance having a binding ability with respect to the complex of the substance to be measured and the binding substance A ₁ which is used for preparing the modified binding substance A according to the present invention (hereinafter referred to as binding substance A ′) Abbreviated), which does not inhibit the complex formation reaction between the substance to be measured and the binding substance A ₁ and the reaction for detecting the detection substance (or the binding substance A ₁ ) in the complex, If the substance to be measured has a binding ability to the binding substance A ₁ or a complex thereof, or if the substance to be measured or the binding substance A ₁ has a detection substance bound to it, There is no particular limitation as long as it is a substance having binding ability. Specifically, for example, an antibody having an ability to bind to a substance to be measured, for example, a lectin such as concanavalin A, lentil lectin, kidney bean lectin, dasula lectin, wheat germ lectin (provided that the substance capable of binding to the binding ability A ₁ Or the like, or an antibody having a binding ability to the binding substance A ₁ or the detection substance, such as an antibody, for example, lectins such as concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, wheat germ lectin, etc. Are preferred.

The binding method of the binding ability substance A'and the separation enhancing substance according to the present invention is a method of binding a specific reactive group of the binding ability substance A'and a specific reactive group of the separation enhancing substance, Method for substituting a specific reactive group with a separation-enhancing substance, a substance capable of binding to a binding-capacity substance A '(eg, antibody, lectin, antigen, inhibitor, DNA, etc.) and a separation-enhancing substance And the like. More specifically, for example, 1) enzyme immunoassay (EIA), radioimmunoassay (RIA) known per se.
Alternatively, a method for binding a labeling substance known per se to an antibody, which is generally used in fluorescence immunoassay (FIA) and the like (for example, Medical Experiment Account, Volume 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao, 1st edition,
Soft Science Co., Ltd., 1983; Enzyme-linked immunosorbent assay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical Institute, 1982,
Etc.), 2) Modification and binding method of substances known per se (for example, chemical modification of protein <up><down>, Ikuzo Uraya, Kensuke Shimura, Michinori Nakamura, Masaru Funatsu, 1st edition, Academic Society of Japan Publishing Center, 1981; Polyethylene glycol modified protein,
Yuji Inada et al., Biochemistry, Volume 62, No. 11, P1351-1362,
(Company) Japan Biochemical Society, 1990; DNA PROBES, George HK
and Mark MM STOCKTON PRESS, 1989, etc.) are mentioned without exception, and these methods may be used.

The separation method of the present invention can be easily carried out as follows, for example. That is, the sample containing the substance to be measured, the binding ability substance A ₁ and the modified binding ability substance A are added to and mixed in an appropriate buffer solution, if necessary, and allowed to react with each other. a substance a _1, after forming the modified affinity substance a is bound complexes, and other coexisting substances such as affinity substance a ₁ of the free and the complex, sparse separation improving substance
It can be easily carried out by separation by HPLC equipped with a column packed with a packing material selected depending on the aqueous or ionic property . Needless to say, in the above separation method, the substance to be measured labeled with the substance to be measured may coexist.

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

An example in which the present invention is applied to the measurement of trace components will be shown below. (1) Method for measuring amount of substance to be measured using non-competitive reaction (Measurement method (1)) First, a biological sample containing the substance to be measured, a binding ability substance A ₁ labeled with a detection substance, and a modified bond If necessary, the active substance A is added to an appropriate buffer, mixed and reacted to form a complex in which the substance to be measured, the binding ability substance A ₁ and the modified binding ability substance A are bound. Then, the complex and the free binding substance A ₁ are treated with a hydrophobic or
Are separated by HPLC equipped with a column packed with a packing material selected according to ionicity . Then, the amount of the detection substance contained in the separated complex is determined by a measuring method according to the property of the detection substance. Separately, measurement is performed by a similar method using a sample whose concentration of the substance to be measured is known, and a calibration curve showing the relationship between the amount of the substance to be measured and the amount of the substance to be detected in the complex is prepared, and this is used to The amount of the substance to be measured in the sample can be obtained by obtaining the amount of the substance to be measured corresponding to the amount of the substance to be detected in the body. In the above reaction, the concentration of the binding substance A ₁ used when forming the complex is
Although it varies depending on how much the calibration limit of the measurement target substance is set, in the reaction solution, it is usually higher than the concentration capable of binding to all the measurement target substances corresponding to the set calibration limit concentration, preferably It is desirable that 2 times or more concentration thereof, more preferably 5 times or more concentration thereof is present in the reaction solution. Although the concentration of the modified binding substance A to be reacted with the complex varies depending on how much the calibration limit of the substance to be measured is set, it is usually in the reaction solution.
It is desirable that a concentration equal to or higher than that of all the substances to be measured corresponding to the set calibration limit concentration, or higher, preferably 2 times or higher, more preferably 5 times or higher, is present in the reaction solution. When the binding ability substance A ₁ itself is a substance that can be measured (detected) by some method, the above reaction is carried out using the binding ability substance A ₁ which is not labeled with the detection substance, and the obtained complex is obtained. The amount of the substance to be measured in the sample can be similarly obtained by obtaining the amount of the binding substance A _{1 in} the body by a measuring method according to the property of the binding substance A ₁ .

(2) Method of Measuring Amount of Target Substance Using Competitive Reaction (Measuring Method (2)) First, a sample of biological origin containing a target substance of measurement, a target substance of measurement labeled with a detection substance (hereinafter, labeled measurement Abbreviated as a substance)), the binding ability substance A ₁ and the modified binding ability substance A are further added to and mixed in an appropriate buffer solution, if necessary, and reacted to obtain the measurement target substance and the binding ability substance A ₁ . Modified binding substance A
And a labeled measurement substance, a binding ability substance A ₁ and a modified binding ability substance A (hereinafter abbreviated as a labeling complex) are formed, and then, the labeling complex and the labeling measurement substance are formed. And are separated by HPLC equipped with a column packed with a packing material selected according to the hydrophobicity or ionicity of the separation enhancing substance. Then, the amount of the detection substance contained in the separated labeled complex is determined by a measuring method according to the property of the detection substance. Separately, measurement is performed by a similar method using a sample having a known concentration of the substance to be measured, and a calibration curve representing the relationship between the amount of the substance to be measured and the amount of the substance to be detected in the labeled complex is prepared, and using this, When the amount of the substance to be measured corresponding to the amount of the substance to be detected in the labeled complex is obtained, the amount of the substance to be measured in the sample can be obtained. In the above reaction, the use concentration of the binding ability substance A _{1 and} the use concentration of the labeled measurement substance in forming the labeled complex are appropriately determined depending on the calibration limit of the substance to be measured and the measurement sensitivity. It may be set, and is not particularly limited. However, it goes without saying that the concentration of the labeled measurement substance to be used must be set to a concentration at which it can bind to all the binding substances A ₁ present in the reaction solution. The concentration of the modified binding ability substance A used when forming the labeled complex may be appropriately set depending on how much the calibration limit of the measurement target substance is set, and is not particularly limited. However, it goes without saying that the use concentration must be set to a concentration higher than the concentration at which all the binding substances A ₁ present in the reaction solution can bind to all the complexes formed by the reaction with the labeled measurement substance.

[0022] The measurement affinity substance A ₁ itself by some method (detected) in the case of substances include the use of binding substances A ₁ which is not labeled with a detectable substance (1)
Or performs the reaction of (2), also by finding the obtained measuring method the amount of affinity substance A ₁ in the complex according to the nature of the affinity substance A _1, similarly analyte in a sample The quantity can be calculated.

Examples of the substance to be measured which can be measured by the method of the present invention include i) a substance capable of forming a strong complex by interacting strongly with the substance to be measured and forming a strong complex. Those that can be measured (detected) by a method, or that can be labeled with some detectable substance,
Or ii) the substance to be measured itself can be labeled with some kind of detection substance, and there is a substance capable of forming a strong labeled complex by interacting strongly with the substance to be measured. Examples of proteins, peptides, nucleic acids contained in biological samples such as serum, blood, plasma, urine, etc., biological samples such as lymphocytes, blood cells, various cells, etc. , Sugar chains,
Typical examples include lipids, hormones and drugs. More specifically, for example, α-fetoprotein (A
FP), CA19-9, prostate specific antigen (PSA), carcinoembryonic antigen (CEA), cancer markers such as substances having special sugar chains produced by cancer cells, for example, immunoglobulin A
(IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), β ₂ -microglobulin, albumin, serum proteins such as ferritin, such as C-peptide,
Peptides such as angiotensin I, eg amylase,
Enzymes such as alkaline phosphatase and γ-glutamyl transferase (γ-GTP), for example, rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, antiviral antibodies against clinically noticed viruses, pathogens such as viruses Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) or single-stranded polynucleotides that compose these nucleic acids, and antigenic substances derived from pathogens such as viruses, such as cedar and other allergens such as pollen and indoor dust of plants. Reactive antibodies such as lipids such as lipoproteins, proteases such as trypsin, plasmin, serine proteases such as insulin, human chorionic gonadotropin (hCG), thyroxine (T ₄ ), triiodothyronine (T ₃ ), prolactin, Thyroid stimulation e Examples thereof include hormones such as lumon (TSH), and drugs such as digoxin, phenytoin, morphine and nicotine.

The substance A ₁ capable of binding to the substance to be measured according to the method of the present invention is a substance which strongly interacts with the substance to be measured and forms a strong complex, and if necessary, itself. Those that can be measured (detected) by some method, or that can be labeled with some detectable substance that can be measured (detected) (this is only applicable if the substance to be measured itself can be labeled with some detectable substance) If it is not present), examples thereof include, but are not limited to, for example, an antibody against a substance having an antigenicity (including a hapten), an antigen against the antibody, a concanavalin A having an ability to bind to a sugar chain having a specific structure, Lectins such as lentil lectin, kidney bean lectin, datura lectin, and wheat germ lectin, for example, α ₁ -An against trypsin Macroglobulin, alpha ₂ relative to the serine protease - - Chitoripushin, alpha ₂ for plasmin inhibitors, for specific enzymes such as macroglobulin include complementary polynucleotide strand or the like 1-stranded polynucleotide is a measurement substance.

Examples of the detection substance used in the measuring method utilizing the separation method of the present invention include alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, which are used in enzyme immunoassay (EIA). Enzymes such as glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase,
Used in eg radioimmunoassay (RIA)
Radioisotopes such as ^99m Tc, ¹³¹ I, ¹²⁵ I, ¹⁴ C and ³ H, for example, fluorescein, dansyl, fluorescamine, coumarin 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, such as phenol, naphthol, anthracene, or their derivatives, which absorb in the ultraviolet, such as
4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl- α- (3,5-di-t-butyl-4-oxo
-2,5-cyclohexadiene-1-ylidene) -p-tolyloxyl such as a substance having a property as a spin labeling agent typified by a compound having an oxyl group,
Needless to say, it is not limited to these.

As a method of labeling the binding substance A ₁ with the above-described detection substance, a labeling method known per se (eg, medicinal chemistry) commonly used in EIA, RIA, FIA or the like known per se. Experimental course, Volume 8, edited by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao, 1st edition, Soft Science Co., 1983; Enzyme immunoassay, Eiji Ishikawa, Kawai Tadashi, Kiyoshi Miyai ed., 2nd edition, medical school, 1982, etc.) are all mentioned without exception, and the procedure may be carried out in accordance with these. Further, it goes without saying that a conventional method utilizing the reaction of avidin (or streptavidin) and biotin may be used as the labeling method.

Examples of the binding substance A _{1 according to} the present invention which can be measured (detected) by any method itself include, for example, an enzyme, a fluorescent substance, a luminescent substance or a substance having absorption in the ultraviolet region. As such, there may be mentioned those which themselves have the properties as the above-mentioned detection substances.

The separation-improving substance according to the present invention is characterized by the properties of the substance to be measured and the binding substance A ₁ (or / and the binding substance A ′) (eg pH stability, hydrophobicity, solubility in aqueous solution, isoelectric point). Etc.) and it is sufficient to select and use them appropriately.

Further, the type of the packing material of the column used in HPLC may be appropriately selected depending on the hydrophobicity or ionicity of the separation improving substance.

The combination of the filler and the separation improving substance will be described in more detail below.

[0031]

[0032] When using a packing material for hydrophobic chromatography Use the difference in hydrophobicity to separate the target substance from other coexisting substances.
Since it is a filler that has the property of releasing,
For example, α-chymotrypsinogen, β-galacto
Proteins with high hydrophobicity such as sidase, such as phenyl
A peptide containing highly hydrophobic amino acids such as alanine and proline.
Peptide, eg polyglutamic acid, polyasparagine
Acid, polylysine, polyarginine, polyphenylalani
And polyamino acids such as polytyrosine, and C3-10
Rutile chain, eg halogen atom such as bromine, chlorine, iodine,
For example, octylamine, EMCS, BMH, etc.
High chemicals such as palmitic acid, oleic acid, suture
Set the hydrophobicity of the complex such as fatty acid such as theearic acid appropriately.
Preferred examples are substances that can be used. For separation
When a peptide is used as the substance, it has a high hydrophobicity.
Peptides containing mino acids are preferred, depending on their chain length selection.
The hydrophobicity may be adjusted. Also, only hydrophobic amino acids
Peptides and polyamino acids composed of
If the number is 15 or more, the solubility in water will decrease, so
15 is preferable. When the separation improving substance is a halogen atom,
The modified binding substance A directly halogenates the binding substance A ′.
Can be easily obtained by changing the amount of halogen introduced.
The hydrophobicity can be adjusted accordingly. Highly hydrophobic chemistry
In addition to the above substances, the substance has a long alkyl chain.
Substances, which can be selected by appropriately selecting the alkyl chain length.
The hydrophobicity can be adjusted. In addition, it is not very hydrophobic
However, since the separation-enhancing substance has a high solubility in water,
When the binding reaction between the active substance A ′ and the separation enhancing substance is performed, an organic solvent is used.
It becomes necessary to use the modified binding ability substance obtained by denaturation or activity.
Or the modified binding substance is not soluble in water.
Separation-improved products may cause problems such as melting.
It is hard to say that the quality is preferable. Also, hydrophobic chroma
Examples of fillers for tography include butyl-NPR (East
Saw product name), Butyl MCI gel (Mitsubishi Kasei product)
Name), Phenyl MCI gel (trade name of Mitsubishi Kasei Co., Ltd.), etc.
To be

[0033] When using a packing material for ion exchange chromatography Targets and other coexisting substances can be used by utilizing the difference in ionicity.
As the separation-enhancing substance, for example, lysozyme
, Basic proteins such as cytochrome C, eg birds
Acidic proteins such as pusin inhibitors, eg Argi
Residues of basic amino acids such as nin and lysine, or asparagus
PE containing acidic amino acid residues such as formic acid and glutamic acid
Peptide, a poly containing 50 or more amino acid residues as described above.
Amino acids such as palmitic acid, oleic acid, steari
Preferable examples include fatty acids such as acid. Ion exchange
In exchange chromatography, the measurement target is generally
Higher resolution is obtained by adsorbing once on the column and then eluting.
Cationic separation-enhancing substance due to its high specificity
For cation exchange chromatography if used
When using a filler, an anionic separation-enhancing substance
You can use packing materials for anion exchange chromatography.
And are preferred. The separation enhancing substance is a basic amino acid residue (or
Is a peptide or polypeptide composed only of acidic amino acid residues)
In the case of amino acids, by adjusting the number of amino acid residues
The elution time of the complex can be adjusted freely, but
The number is usually 5 or more, preferably 50 or more, and more preferably
Peptide or polyamino acid consisting of 100 or more
If used, the elution position of the complex will be
It is desirable because it can be completely separated from it. Also, the above
Cide or polyamino acid is a synthetic peptide or synthetic polyamino acid
For acids, the length of the peptide (or polyamino acid) and the
Since the nucleophilicity is proportional, the peptide (or
Is a polyamino acid) and the length of
Can be easily adjusted by adjusting the elution position of the complex
can do. In addition, serum components that affect measurement
Even when there are multiple
To be more ionic than those of these serum components.
Then use a stepwise gradient
Can reduce the time required for analysis
The effect occurs. Packing for ion exchange chromatography
Agents generally have high exchange capacity (absolute adsorption capacity for ionic substances)
Therefore, ionic coexisting substances like biological samples such as serum
Even when performing analysis on a sample with a large absolute amount of
Can absorb all complexes with enhancer
Therefore, the influence of the coexisting substance on the complex can be almost avoided.
It is possible to elute in position. Also, this method is
Separation enhancing substances that can be used have high solubility in water
So the water solubility of the complex with which it is bound is better than that before binding.
In this method, the separation-enhancing substance is bound to be formed.
The denaturation and deactivation of the measurement target during the formation reaction of the combined complex
It is unlikely to happen. Ion exchange chromatography
Examples of fillers for use include DEAE-MCI gel (Mitsubishi Kasei
(Trade name), QAE MCI gel (trade name of Mitsubishi Kasei), Wa
Kobe's DEAE gel (trade name of Wako Pure Chemical Industries, Ltd.)
Packing material for Nion exchange chromatographies, or for example SP MCI
Gel (trade name of Mitsubishi Kasei), CM MCI gel (Mitsubishi Kasei)
(Trade name), Wako beads CM gel (Wako Pure Chemical Industries, Ltd.)
Packing material for cation exchange chromatography such as (trade name)
Etc.

The present invention can be carried out by HPLC using any of the above-mentioned chromatographies. Among them, the use of ion exchange chromatography is most preferable for the present invention. The reason is 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 having an appropriate length. Therefore, when gel filtration chromatography is used, ion exchange chromatography is used. There is a drawback in that the separation time becomes longer than that when using graphy. Therefore, it is preferable to utilize ion exchange chromatography when shortening the separation time is required. In addition, gel filtration chromatography has a very high molecular weight (molecular size of about 1000
It also has the drawback that it is not suitable for the separation of substances to be measured that have a thickness of Å or more). In the case of hydrophobic chromatography, when the substance to be measured is a physiologically active substance with a high-dimensional structure such as a protein, the high-dimensional structure is destroyed by the organic solvent used during 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 this reason, it is not preferable to use a substance having high hydrophobicity as the separation improving substance. Furthermore, the higher the hydrophobicity of the binding ability substance B, C or D to which the hydrophobic substance is bound as the separation improving substance is,
There is also a problem that separation of the complex becomes difficult because the water solubility of the complex decreases and precipitation easily occurs. On the other hand, ion exchange chromatography can more effectively separate the substance to be measured based on the subtle difference in ionicity. Furthermore,
According to the ion exchange chromatography, the separation enhancing substance having various ionic properties can be arbitrarily selected, so that the substance to be measured can be separated under the optimum pH condition. Furthermore, since the separation-improving substance used in ion exchange chromatography has a high water solubility itself, even if the separation-improving substance is bound to the measurement target substance, there is almost no risk of precipitation of the measurement target substance. , It becomes possible to execute the separation operation in a stable state.

When the substance to be measured is measured using the measuring method utilizing the separation method of the present invention, the peak of the target substance to be measured is moved to a position not affected by components such as serum and urine. Can be made. Not only that, by appropriately selecting and using the modified binding substance A according to the substance to be measured, the elution positions of the complex containing the various substances to be measured can be made to coincide with each other.
There is also an effect that various kinds of substances to be measured can be measured using the PLC.

In the measuring method using the separation method of the present invention, the complex separated by HPLC (including both the one with the modified binding ability substance A bound and the one without it). The detection substance contained (or the binding substance A)
₁ or the substance to be measured)
The binding ability substance A ₁ or the substance to be measured) is carried out according to a predetermined method depending on the property that can be detected by some method. For example, when the property is enzymatic activity, a conventional method of EIA, for example, “enzyme immunoassay, protein / nucleic acid / enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pp. 51-63, Kyoritsu Publishing Co., Ltd., September 1987
It may be measured in accordance with the method described in "Issued 10th of a month", etc., and when the substance to be detected is a radioactive substance, the liquid is determined according to the type and intensity of the radiation emitted by the radioactive substance according to the usual method of RIA. Measurement may be performed by appropriately selecting and using a measuring instrument such as an immersion type GM counter, a liquid scintillation counter, a well type scintillation counter, an HPLC counter (for example, medical chemistry experiment course, Volume 8, supervised by Yuichi Yamamura, No. 1). 1st edition, Nakayama Shoten, 1971 etc.). When the property is fluorescent, a conventional FIA method using a measuring instrument such as a fluorometer, for example, “illustrated fluorescent antibody, written by Kawao Aki,
First Edition, Soft Science Co., Ltd., 1983 ”, etc. may be used for the measurement, and when the property is luminescent, a conventional method using a measuring device such as a photon counter, for example,“ Enzyme Immunoassay, Protein Nucleic Acid Enzyme Separate Volume N
Edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji and Eiji Ishikawa, o.31, 25
2 to 263, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 ”and the like. Further, when the property has a property of absorbing in the ultraviolet, the measurement may be carried out by a conventional method using a measuring instrument such as a spectrophotometer,
When the substance to be detected has a spin property, a conventional method using an electron spin resonance apparatus, for example, "enzyme immunoassay,
Protein Nucleic Acid Enzyme Separate Volume No.31, Tsunehiro Kitagawa, Toshio Minamihara
Edited by Akio Tsuji and Eiji Ishikawa, pages 264-271, Kyoritsu Shuppan Co., Ltd.,
The measurement may be performed in accordance with the method described in “September 10, 1987”.

In the present invention, the reaction conditions for forming a complex by reacting the substance to be measured with the binding substance A ₁ which is labeled or not, the binding ability of the substance to be measured and the labeled substance to be measured. The reaction conditions for forming a labeled complex by reacting with the substance A ₁ or for the binding reaction between the complex and the modified binding substance A are complex (or labeled complex). ), Or the binding reaction between the complex and the modified binding substance A, is not particularly limited as long as it is a condition known in the art, such as EIA, RIA, FIA, affinity chromatography and the like. The method may be carried out according to the reaction conditions for forming a complex or the like. For example, when a buffer solution is used in the reaction, the buffer agent and other reagents to be used may be appropriately selected from those used in the methods known per se. The pH during the reaction is the complex (or labeled complex).
Is not particularly limited as long as it does not hinder the formation reaction of the above or the binding reaction between the complex and the modified binding substance A, but the range is usually from 2 to 10, preferably from 5 to 9. . The temperature during the reaction is not particularly limited as long as it does not hinder the complex (or labeled complex) forming reaction or the binding reaction between the complex and the modified binding substance A. The range of 0 to 50 ° C, preferably 20 to 40 ° C is preferably mentioned. The reaction time is such that complexes (or labeled complexes) are formed, and these complexes and modified binding ability substance A
Since the time required for binding with the substance varies depending on the properties of the substance to be measured, the binding substance A ₁ and the modified binding substance A, it may be appropriately reacted for several seconds to several hours depending on each property.

In the separation method of the present invention, a complex (or a labeled complex) (including both those bound with the modified binding ability substance A and those not bound; the same applies hereinafter) and free. HPL used when separating the binding substance A ₁
As C, the apparatus itself can be used without any particular problem as long as it is one that is usually used in the field of analysis and a constant flow rate can be obtained.

Complex (or labeled complex) by HPLC
As the solvent (eluent) used when separating the free binding ability substance A ₁ and the formed complex (or labeled complex), the measurement target substance and the binding ability substance A ₁ (and the modified substance) are again used. affinity substance a) and without as decomposed into, affinity substance a ₁ to and included in the complex (or the labeled complex)
(Or substance to be measured) or a binding substance A ₁
(Or labeled measurement substance) is not particularly limited as long as it does not lose the property of the detection substance held by (or the labeled measurement substance) that can be detected by any method, but usually, for example, EIA, Those used as buffers in methods known per se such as RIA, FIA and affinity chromatography are preferably used. Specific examples include, for example, phosphates, acetates, citrates, Good's buffers, buffers such as tris (hydroxymethyl) aminomethane, salts such as sodium chloride, potassium chloride, ammonium sulfate, etc. Polar organic solvents such as methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran, and surfactants are appropriately selected and added according to the properties of the complex (or labeled complex) or the free binding substance A ₁ . A buffer having a pH of 2 to 10 prepared by mixing is preferably used.

Further, in the measuring method using the separating method of the present invention, the measuring method after separation by HPLC is as follows.
For example, the effluent from the HPLC column as described in "Latest Liquid Chromatography, Shoji Hara and Akio Tsuji, First Edition, pages 92 to 104, Nanzandou, published February 1, 1978", etc. The method that leads to the detection unit as it is and directly measures the amount of the detection substance (or the binding ability substance A ₁ or the measurement target substance) contained in the complex (or the labeled complex) in the effluent is faster in the measurement. It is more preferable because it can be performed. In this case, the binding substance A ₁ (or the measurement target substance) or the detection substance held by the binding substance A ₁ (or the labeled measurement substance) has a property that can be detected by some method. Needless to say, for example, in the case of enzyme activity, it is necessary to provide a so-called post-column method reaction section between the HPLC column and the detection section to add a reagent for enzyme activity measurement and react with the effluent. . When the property of the substance to be detected (or the substance capable of binding A ₁ or the substance to be measured) is enzymatic activity, the reagent for measuring the enzyme activity used in the reaction part is a conventional method, for example, “enzyme immunoassay method”. , Protein Nucleic Acid Enzyme Separate Volume No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 51-63, Kyoritsu Shuppan Co., Ltd., published September 10, 1987. What was prepared by this may be used, and the reagent of the commercially available clinical test kit may be appropriately selected and used. In addition, even when the property of the detection substance (or the binding ability substance A ₁ or the measurement target substance) is other than the enzyme activity, in order to increase the detection sensitivity, a predetermined reagent is added and reacted with HPLC. It is optional to provide a suitable reaction part between the column and the detection part.

When a plurality of different eluents for HPLC are used 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 actual analysis time can be shortened, and the target peak can be sharpened. Since 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.

[0042]

EXAMPLES Experimental Example 1. 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, After 81 g of disodium phosphate (12-hydrate) and 44 g of sodium chloride were dissolved in ion-exchanged water to adjust the pH to 7.5, the total amount was 5 liters to give an eluent. (Substrate liquid) 3- (p-hydroxyphenyl) propionic acid in 80 ml of eluent
Dissolve 1.66g and adjust to pH 7.5 with 1N NaOH
After adding, the total amount was 100 ml. A 30% hydrogen peroxide solution was diluted with this solution to prepare a 20 mM H ₂ O ₂ solution, 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 ', which was labeled with horseradish peroxidase (POD) by a conventional method. Antibody liquid 1 was prepared by adding POD-labeled anti-hCG-α chain-Fab 'to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a protein concentration of 3 nM. (Antibody solution 2) An anti-hCG-β chain monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was processed into Fab 'by a conventional method, and this was combined with β-
Galactosidase (β-Gal, Oriental Yeast Co., Ltd.
Co., Ltd.) and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate [Sulfosuc
cinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carbo
xylate (Sulfo-SMCC), manufactured by Pierce Co., Ltd.] was used to bind β-Gal-bonded anti-hCG-β chain-Fab ′.
Was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a protein concentration of 50 nM to give antibody solution 2. (Antibody solution 3) Anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.)
Was treated according to the method for preparing antibody solution 1 to obtain POD-labeled anti-AFP-Fab ′, which was treated with 50 mM phosphate buffer (pH 7.5, 150).
Antibody solution 3 was prepared by adding 3 mM of sodium chloride to mM protein concentration. (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. The obtained β-Gal-conjugated anti-AFP-Fab ′ was added to 50 nM in 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride).
The antibody solution 4 was added to give the protein concentration of. (HCG sample solution) Commercially available hCG (derived from placenta villi, manufactured by Sigma) was added to 25 mM in 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride).
The hCG sample solution was prepared by adding the solution to a concentration of 0 mIU / ml. (AFP sample solution) AFP purified from human placenta (manufactured by Wako Pure Chemical Industries, Ltd.)
Was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a concentration of 1 nM to prepare an AFP sample solution. (Use condition of HPLC) The outline of the system is shown in FIG.・ Column: 0.8φx30cm. Filler: YMC pack Diol-200 (YMC product name). Flow rate: eluent: 1.0 ml / min, substrate solution: 0.1 ml / min. -Reaction part: 0.025φ x 1000 cm (incubated at 55 ° C) -Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. (Measurement operation) Samples 1 to 5 having the compositions shown in Table 1 below were prepared, and each sample was heated to 30 ° C.
After standing for 30 minutes at 50 μl of each sample was analyzed by HPLC.

[Table 1] Table 1 * 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) (Results) From the analysis results of Sample 1 and Sample 2, POD-labeled anti-hCG-
α-chain-Fab 'was 10.5 minutes later, and POD-labeled anti-hCG-α-chain-
After 9.5 minutes, the complex of Fab 'and hCG (Complex-1)
POD-labeled anti-hCG-α chain-Fab 'and β-Gal bound anti-h
The complex of CG-β chain-Fab 'and hCG (complex-2) is
It was found that each elutes after 6.8 minutes. From the analysis results of Samples 3 and 4, POD-labeled anti-AFP-Fab ′ 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-AFP-Fa
It was found that the complex of b'and β-Gal binding anti-AFP-Fab 'and AFP (complex-4) was eluted after 6.8 minutes. In the case of Sample 2 and Sample 4, Complex-1 and Complex-3 were 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. Identical (both are 6.8
It will be understood that the elution occurs after a minute), 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. Further, in the case of the specimen 5, the complex-2 is 6.
After 8 minutes, the complex-3 was detected after 9.1 minutes by POD-labeled anti-hCG.
-Α chain-Fab 'and POD-labeled anti-AFP-Fab' eluted after 10.5 minutes. As is clear from this result, different measurement targets (hCG
And AFP) can be measured at the same time.

Example 1 AFP measurement (when β-Gal is used as a separation improving substance and a packing material for hydrophobic chromatography is used) (eluent A) 50 mM phosphate buffer (pH 7.5) containing 1.7 M ammonium sulfate is used as eluent A And (Eluent B) 50 mM phosphate buffer (pH 7.5) was used as eluent B. (Substrate solution) Same as Experiment 1. (Antibody solution 1) The same as the antibody solution 3 of Experiment 1. (Antibody solution 2) The same as the antibody solution 4 of Experiment 1. (Sample) Experiment Same as the AFP sample solution of Example 1. (Use condition of HPLC) The outline of the system is shown in FIG.・ Column: 0.46φ x 2.5 cm. -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: 0.025φ x 1000 cm (incubated 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 the eluent A and the eluent B is shown in FIG. (Measurement operation) Experiment 1 having the same composition as that of the specimen 3 of Experiment 1 and Experiment 1
Sample 2 with the same composition as Sample 4 and each sample at 30 ℃
After standing for 30 minutes at 50 μl of each sample was analyzed by HPLC. (Result) From the analysis result by HPLC, POD-labeled anti-AFP-Fa
b ′ was 4.9 minutes later, the complex of POD-labeled anti-AFP-Fab ′ and AFP was 6.7 minutes later, and POD-labeled anti-AFP-Fab ′ and β were conjugated.
The complex between -Gal-conjugated anti-AFP-Fab 'and AFP is 9.7.
After a minute, it was found that each elutes. From the above results, POD-labeled anti-A can be obtained by using the modified binding substance.
It can be seen that it becomes possible to more clearly separate FP-Fab 'and the complex.

Example 2 Measurement of AFP (iodine is used as a separation improving substance,
(When using a packing material for hydrophobic chromatography) (Antibody solution 1) The same as the antibody solution 3 of Experiment 1 (Antibody solution 2) The anti-AFP monoclonal antibody used to prepare the antibody solution 4 of Experiment 1 was used. , The chloramine T method known per se (Biochemistry Experiment Course 16 "Hormones", edited by The Japan Biochemical Society, published by Tokyo Kagaku Dojin, pages 117-180 and 230-231.
Page, 1977. ). Each iodinated anti-AF obtained by setting the iodination reaction time to 5 seconds, 30 seconds or 2 minutes
P monoclonal antibody was added to 50 mM phosphate buffer (pH
7.5 and 150 mM sodium chloride was added) so as to have a protein concentration of 50 nM to obtain antibody solution 2-1, antibody solution 2-2 and antibody solution 2-3. (Sample) Experiment Same as the AFP sample solution of Example 1 (conditions for using HPLC) Same as Example 1 . (Measurement procedure) Antibody solution 1 30 μl and AFP sample solution 30 μl, 50 mM phosphate buffer solution (pH 7.5, containing 150 mM sodium chloride), antibody solution 2
-1, 30 μl of antibody solution 2-2 or antibody solution 2-3 were mixed and left at 30 ° C. for 30 minutes, then 50 μl of the resulting mixture
Was analyzed by HPLC. (Result) From the analysis result by HPLC, POD-labeled anti-AFP-Fa
It was found that b ′ was eluted after 4.9 minutes, and the complex A of POD-labeled anti-AFP-Fab ′ and AFP was eluted after 6.7 minutes. In addition, POD-labeled anti-AFP-Fab 'and iodinated anti-AF
Regarding the complex of P-Fab ′ and AFP, the antibody solution 2-3 was used after 8.2 minutes when the antibody solution 2-1 was used and after 9.5 minutes when the antibody solution 2-2 was used. In some cases, it was found that each elutes after 9.9 minutes. From the above results, it is understood that the elution position of the complex of POD-labeled anti-AFP-Fab ′ and AFP can be freely adjusted by using an antibody (modified binding ability substance) having a different iodine binding amount.

Example 3 Measurement of AFP [octylamine, phenylalanine tetramer (hereinafter abbreviated as Phe4), BM
When H or EMCS is used as a separation improving substance and a packing material for hydrophobic chromatography is used] (Antibody solution 1) Same as the antibody solution 3 of Experimental Example 1 (Antibody solution 2) 0.1M phosphate buffer [pH 7.0 , 50% dimethylformamide (DMF) included. ] Dissolved in octylamine and Sulf
After reacting an equimolar amount of o-SMCC (manufactured by Pierce) by a conventional method, this reaction solution was treated with an anti-AFP monoclonal antibody having a recognition site different from that of the antibody used in antibody solution 1 by a conventional method. The obtained Fab '(anti-AFP-Fab') [dissolved in 0.1M phosphate buffer (pH 7.0)] was added at a molar ratio of 1/100 times that of octylamine, and this suspension was incubated at 30 ° C for 30 minutes. . This reaction solution was purified by a conventional method to obtain octylamine-conjugated anti-AFP-Fab ', which was added to 50 mM phosphate buffer (p
Antibody solution 2 was added to H7.5, containing 150 mM sodium chloride so as to have a protein concentration of 50 nM. (Antibody solution 3) Except that Phe4 peptide was used instead of octylamine,
Using the same reagents used to prepare antibody solution 2,
Phe4-binding anti-AFP-Fab 'was prepared in the same manner, and this was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a protein concentration of 50 nM to prepare an antibody solution. It was set to 3. (Antibody solution 4) The same anti-AFP-Fab 'used to prepare antibody solution 2
And BMH were reacted by a conventional method, N-acetyl-L-cysteine (AC) was added to this reaction solution in 100 times the molar amount of BMH, and the mixture was further incubated at 30 ° C. for 30 minutes. This reaction solution was purified by a conventional method to obtain AC-BMH-conjugated anti-AFP-F.
Ab 'was obtained, and this was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a protein concentration of 50 nM to obtain antibody solution 4. (Antibody solution 5) The same anti-AFP-Fab 'used to prepare antibody solution 2
And EMCS were reacted by a conventional method, glycine was added to this reaction solution in a 1000-fold molar amount of EMCS, and the mixture was further incubated at 30 ° C. for 30 minutes. This reaction solution was purified by a conventional method to obtain glycine-EMCS-bound anti-AFP-Fab ', which was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a protein concentration of 50 nM. To obtain antibody solution 5. (Sample) Experiment Same as the AFP sample solution of Example 1. (Use condition of HPLC) Same as in Example 1 . (Measurement method) To 30 μl of antibody solution 1 and 30 μl of AFP sample solution, 30 μl 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. Mix and leave at 30 ° C for 30 minutes, then use 50 μl of the mixture by HPLC.
Was analyzed by. (Result) As a result of analysis by HPLC, POD-labeled anti-AFP-Fab '
Was found to elute after 4.9 minutes, and the complex of POD-labeled anti-AFP-Fab ′ and AFP after 6.7 minutes, respectively. Also,
POD-labeled anti-AFP-Fab ', AFP and each modified anti-AFP-
Regarding the complex with Fab ′ (modified binding ability substance), 7.5 minutes after using octylamine-conjugated anti-AFP-Fab ′, 8.5 minutes after using Phe4-conjugated anti-AFP-Fab ′, When using AC-BMH-conjugated anti-AFP-Fab '
After 7.2 minutes, when glycine-EMCS-conjugated anti-AFP-Fab 'was used, it was found to elute after 7.1 minutes, respectively.
From the above results, it is understood that the elution position of the target complex can be freely adjusted by changing the type of the separation enhancing substance.

Example 4 Measurement of AFP (when polyaspartic acid was used as a separation improving substance and a packing material for anion exchange chromatography was used) (Eluent A) 10 mM phosphate buffer (pH 7.5) was used as eluent A. (Eluent B) 50 mM phosphate buffer (pH 7.
5) was used as an eluent B. (Substrate solution) 2.5 mM of 4-methylumbelliferylgalactopyranoside was dissolved in a 50 mM phosphate buffer solution (pH 7.5) containing 20% DMF and 150 mM sodium chloride to prepare a substrate solution. (Antibody solution 1) Anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.)
Is processed into Fab 'by a conventional method, and β-G
β-Gal labeled anti-AFP-Fab ′ obtained by labeling al
Antibody solution 1 was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a protein concentration of 4 nM. (Antibody solution 2) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) whose recognition site was confirmed to be different from the monoclonal antibody used in antibody solution 1 was processed into Fab 'by a conventional method, This and polyaspartic acid (average molecular weight: 6165,
13000 or 28800, manufactured by Sigma Co., Ltd.) by a conventional method using Sulfo-SMCC (manufactured by Pierce), and then purified by a conventional method to obtain various polyaspartic acid-binding anti-AFP-
I got Fab '. Add these to 50 mM phosphate buffer (pH 7.5, 150
Antibody solution 2 was prepared by adding each of them to a protein concentration of 50 nM. (Sample) Experiment Same as the AFP sample solution of Example 1. (Use condition of HPLC) The outline of the system is shown in FIG.・ Column: 0.46φx3.0cm. -Filler: DEAE-MCI gel (trade name of Mitsubishi Kasei Co., Ltd.).・ Flow rate: Eluent A + B; 1.0 ml / min, substrate solution; 0.1 ml / mi
n. -Reaction part: 0.025φ x 1000 cm (45 ° C heat retention). -Detection: Fluorescence was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm. -Gradient: The gradient of the eluent A and the eluent B is shown in FIG. (Measurement method) Antibody solution 1 (30 μl), AFP sample solution (30 μl), and 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) or antibody solution 2 containing anti-AFP-Fab ′ bound with a predetermined polyaspartic acid 30 μl of the above was mixed and left at 30 ° C. for 30 minutes, and then 50 μl of the mixed solution was analyzed by HPLC. (Results) As a result of analysis by HPLC, β-Gal labeled anti-AFP-
Fab ′ was labeled with β-Gal-labeled anti-AFP-Fab ′ 3.33 minutes later.
The complex with FP was found to elute after 4.2 minutes. Regarding the complex of β-Gal-labeled anti-AFP-Fab ′ and AFP with polyaspartic acid-binding anti-AFP-Fab ′ (modified binding ability substance), when the average molecular weight of the bound polyaspartic acid is 6165, Was found to elute after 4.99 minutes, in the case of 13000 after 7.90 minutes, and in the case of 28800 after 8.85 minutes. From the above results, it is understood that the elution position of the complex can be freely adjusted by changing the property of the separation enhancing substance, that is, the molecular weight of polyaspartic acid.

Example 5 Examination of avoiding influence of hemolysis in AFP measurement (when polyaspartic acid is used as a separation improving substance and a packing material for anion exchange chromatography is used) (eluent A) The same as eluent A of Example 4 . (Eluent B) The same as the eluent B of Example 4 . (Substrate solution) The same as the substrate solution of Experiment 1. (Antibody solution 1) The same as the antibody solution 3 of Experiment 1. (Antibody solution 2) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) whose recognition site was confirmed to be different from the monoclonal antibody used in antibody solution 1 was processed into Fab 'by a conventional method, This and polyaspartic acid (average molecular weight: 1300
0, manufactured by Sigma) was bound by a conventional method using Sulfo-SMCC (manufactured by Pierce), and then purified by a conventional method to obtain anti-AFP-Fab ′ conjugated with polyaspartic acid. This was added to 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) at a protein concentration of 50 nM to obtain antibody solution 2. (Hemolysis) Hemoglobin obtained from human erythrocytes was diluted with 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) to 1000 mg / dl and used as hemolysis. (Sample) Experiment Same as the AFP sample solution of Example 1. (Use condition of HPLC) The 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: 0.025φ x 1000cm (55 ℃ heat retention). -Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. -Gradient: The gradient of the eluent A and the eluent B is shown in FIG. (Measurement method) 30 μl of antibody solution 1, 30 μl of AFP sample solution, and 10 μl of hemolyzed blood were mixed with 30 μl of 50 mM phosphate buffer solution (pH 7.5, containing 150 mM sodium chloride) or antibody solution 2 at 30 ° C. for 30 minutes. After standing, 50 μl of each mixture was analyzed by HPLC. (Results) The obtained elution pattern is shown in FIG. In FIG. 5, A is the elution pattern obtained by analyzing the hemolyzed blood diluted 10 times with 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride), and B is the antibody solution 1, AFP sample. The elution pattern obtained as a result of the analysis of the mixed solution of the liquid, hemolyzed blood and a phosphate buffer was obtained as a result of the analysis of the mixed liquid of the antibody liquid 1, the AFP sample liquid, the hemolyzed blood and the antibody liquid 2. The respective dissolution patterns are shown. As is clear from FIG. 5A, it can be seen that the hemolytic component elutes as a plurality of peaks during 0.5 to 5.5 minutes. In contrast, the POD-labeled anti-AFP-Fab 'was 1.10 minutes later,
The complex of b'and AFP was 3.25 minutes later, and the POD-labeled anti-AF was detected.
P-Fab 'and polyaspartic acid binding anti-AFP-Fab' and A
It can be seen that the complex with FP elutes after 7.20 minutes, respectively.
From the above results, it is possible to elute the complex at the elution position that is not affected by hemolysis by using the separation method of the present invention, in other words, by using the separation method of the present invention, at the time of measurement. Since it is possible to measure the substance to be measured without being affected by the hemolytic component in the sample, it can be understood that the measurement accuracy can be improved as compared with the conventional case.

Example 6 Fractional measurement of hCG having different sugar chain structure (when polyaspartic acid is used as a separation improving substance and a packing material for anion exchange chromatography is used) (Antibody solution) The same as the antibody solution 1 of Experimental Example 1. (Lectin solution 1) Datura lectin (manufactured by Wako Pure Chemical Industries, Ltd.) is 50 mM tris (hydroxymethyl) aminomethane (hereinafter, Tris)
-HCl buffer (pH 7.5, 150 mM sodium chloride and 1 mM
Lectin solution 1 was obtained by adding calcium chloride (containing calcium chloride) to a protein concentration of 1 mg / ml. (Lectin solution 2) The same datura lectin and polyaspartic acid (average molecular weight 28800) that were used to prepare lectin solution 1 were added to Disuccinimidyl s
uberate, manufactured by Pierce Co., Ltd.) was used to prepare polyaspartic acid-binding datura lectin, which was added to 50 mM Tris-
A lectin solution 2 was prepared by adding a hydrochloric acid buffer solution (pH 7.5 containing 150 mM sodium chloride and 1 mM calcium chloride) to a protein concentration of 1 mg / ml. (HCG sample solution) Human choriocarcinoma-derived hCG (manufactured by Wako Pure Chemical Industries, Ltd.)
A hCG sample solution was prepared by adding 250 mM IU / ml to a mM Tris-hydrochloric acid buffer solution (containing pH 7.5 150 mM sodium chloride and 1 mM calcium chloride) so as to have a concentration of 250 mIU / ml. (Use condition of HPLC) The same as in Example 5 . (Measurement procedure) hCG sample solution 10 μl, antibody solution 20 μl, lectin solution 1
Alternatively, 70 μl of lectin solution 2 was mixed and allowed to stand at 30 ° C. for 30 minutes, and 50 μl of the resulting mixed solution was analyzed by HPLC. (Results) As a result of analysis by HPLC, POD-labeled anti-hCG-α chain-Fab ′ was 1.10 minutes later, and POD-labeled anti-hCG-α chain-Fab ′ was found.
After 3.50 minutes, the complex of hCG and hCG was detected by POD-labeled anti-hCG-
The complex of α-chain-Fab ', hCG and datura lectin is 3.82.
After a minute, the complex of POD-labeled anti-hCG-α chain-Fab ', hCG and polyaspartic acid-conjugated datura lectin was 7.9.
It was found that after a minute, each elutes. From the above results, it is possible to more clearly separate the lectin-binding complex and the lectin-non-binding complex by using the polyaspartic acid-binding datura lectin, in other words, the hCG amount having a specific sugar chain in all hCG is more accurate. It turns out that it becomes possible to measure well.

Example 7 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) is used as Eluent A did. (Eluent B) 20 mM Tris-HCl buffer containing 1 M sodium chloride (p
H8.0) was used as the eluent B. (Substrate solution) 1.66 g of 3- (p-hydroxyphenyl) propionic acid was dissolved in 80 ml of eluent A, and the pH was adjusted to 8.0 with 1N sodium hydroxide.
After that, the total amount of eluent A was adjusted to 100 ml. A 30% hydrogen peroxide solution was diluted with this solution to prepare a solution containing 20 mM H2O2, which was 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 Obtained POD
Labeled anti-T ₄ -Fab ′ with 20 mM Tris-HCl buffer (pH 8.0)
Antibody solution 1 added to the inside to give a protein concentration of 3 nM
And (Antibody solution 2) Anti-POD monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.)
Is treated with a conventional method to obtain Fab ', and this and polyglutamic acid (average molecular weight: 95100, manufactured by Sigma) are combined with Sulfo-SMC.
The polyglutamic acid-conjugated anti-POD-Fab ′ obtained by binding by a conventional method using C was added to 20 mM Tris-HCl buffer (pH
8.0) to give a protein concentration of 50 nM to give antibody solution 2. The anti-POD monoclonal antibody used this time has the property of binding to POD but not inhibiting its enzymatic activity. (T ₄ sample solution) Commercially available L-thyroxine (manufactured by Sigma) was added to 20 mM Tris-hydrochloric acid buffer solution (pH 8.0) at a concentration of 10 nM to prepare a T ₄ sample solution. (Use condition of HPLC) The 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: 0.025φ x 1000cm (55 ℃ heat retention). -Detection: Fluorescence was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 404 nm. -Gradient: The gradient of the eluent A and the eluent B is shown in FIG. (Measurement Operation) T4 sample solution 30 [mu] l antibody solution 1 30 [mu] l and 20mM Tris - HCl buffer (pH 8.0) a mixture of a 30 [mu] l and the sample 1, T ₄ sample solution 30 [mu] l antibody solution 1 30 [mu] l antibody solution 230μl
Sample 2 was a mixture of and. After leaving each sample to stand at 30 ° C. for 30 minutes, 50 μl of each sample was analyzed by HPLC. (Result) From the analysis result by HPLC, POD-labeled anti-T ₄ -Fab '
3.3 minutes later, the complex of POD-labeled anti-T ₄ -Fab 'and T ₄ was 2.3 minutes later, and the complex of POD-labeled anti-T ₄ -Fab' and polyglutamic acid-conjugated anti-POD-Fab 'was 8.9 minutes later. , POD
Labeled anti-T ₄ -Fab 'and polyglutamic acid-conjugated anti-POD-Fa
It was found that the complex of b ′ and T ₄ was eluted after 7.4 minutes. From the above results, polyglutamic acid-conjugated anti-PO
D-Fab 'by using, POD-labeled anti-T _4-Fab'
And POD-labeled anti-T _4-Fab 'and being able to vary the elution position of the complex with T _4, POD-labeled anti-T _4-Fab other words', a POD-labeled anti-T _4-Fab' and T ₄ It can be seen that it can be more clearly separated from the complex of.

Example 8 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 Kawaoi, A., J. Histoc
. hem.Cytochem, the POD-labeled T ₄ was prepared by vol.22,1084~1091,1974), 20mM Tris - HCl buffer (pH 8.
What was dissolved in 0) at a concentration of 10 nM was used as a labeled antigen solution. (Antibody solution 1) Fab ′ obtained by treating an anti-T ₄ monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) by a conventional method and 20 times the molar amount of N-ethylmaleimide were added to the Fab ′. And left to react at 37 ° C for 60 minutes. Then, this was purified by a conventional method to obtain N-
Ethylsuccinimidated Fab 'was added to 20 mM Tris-hydrochloric acid buffer (pH 8.0) at a protein concentration of 2 nM to prepare antibody solution 1. (Antibody solution 2) Same as the antibody solution 2 of Example 7 . (T ₄ sample solution) Same as the T ₄ sample solution of Example 7 . (Hemolysis) The same as the hemolysis of Example 5 . (Use condition of HPLC) Same as in Example 7 . (Measurement operation) Samples 1 to 3 having the compositions shown in Table 2 below were prepared, and each sample was stored at 30 ° C.
After standing for 30 minutes, 50 μl of each sample was analyzed by HPLC.

[Table 2] Table 1 * 1: 20 mM Tris-hydrochloric acid buffer (pH 8.0) * 2: 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride) (Results) From the analysis results by HPLC, POD-labeled T ₄ was detected after 2.6 minutes by 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, POD-labeled T ₄ and N- ethyl succinimide of It was found that the complex of Fab ′ and polyglutamic acid-conjugated anti-POD-Fab ′ was eluted after 9.1 minutes. On the other hand, it was found that the hemolyzed components were eluted as a plurality of peaks during 0.5 to 5.5 minutes as in Example 6. From the above results, it is possible to elute the complex at the elution position that is not affected by hemolysis by using the separation method of the present invention, in other words, by using the separation method of the present invention, at the time of measurement. Since it is possible to measure the substance to be measured without being affected by the hemolytic component in the sample, it can be understood that the measurement accuracy can be improved as compared with the conventional case.

[0051]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a complex and a free binding ability substance (or a free measurement subject substance) resulting from the interaction between the measurement subject substance and the binding ability substance thereto.
In the measuring method in which the separation from other substances such as, for example, is performed by high performance liquid chromatography, the complex or the complex is further bound with a separation-improving substance to make the complex hydrophobic or ionic.
The present invention provides a method having the effect of freely adjusting the elution position of the complex in high-performance liquid chromatography by freely changing the sex , and using the separation method of the present invention, serum, etc. When a trace amount of a component in a biological sample is measured, it has a remarkable effect that high-precision measurement can be performed easily and in an extremely short time as compared with the conventional measurement methods such as EIA and RIA. It is a great invention that contributes to the industry.

[Brief description of drawings]

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

FIG. 2 is a schematic diagram of Examples 1 , 2, 3, 4, 5, 6, 7, and 7.
9 is a schematic view of the HPLC system used in FIGS.

FIG. 3 shows HPLC in Examples 1, 2 and 3.
The ordinate represents the ammonium sulfate concentration (M), and the abscissa represents the time (minutes).

FIG. 4 shows HPLC gradient patterns in Examples 4, 5, 6, 7, and 8 , in which 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 by HPLC obtained in Example 5 , where A is a 10-fold increase in hemolyzed blood with 50 mM phosphate buffer (pH 7.5, containing 150 mM sodium chloride). The elution pattern obtained as a result of the analysis of the diluted product, B is the elution pattern obtained as a result of the analysis of the mixed solution of the antibody solution 1, the AFP sample solution, the hemolysate and the phosphate buffer solution, and C is the antibody solution. 1 shows the elution patterns obtained as a result of analysis of a mixed solution of AFP sample solution, hemolyzed blood, and antibody solution 2.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI // G01N 33/536 G01N 33/536 A (72) Inventor Shinji Satomura 6-1 Takada-cho, Amagasaki-shi, Hyogo Jun Wako (56) References JP 61-110059 (JP, A) JP 3-221865 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 30/00-30/96 G01N 33/531 G01N 33/538 G01N 33/536

Claims (8)

(57) [Claims] 1. A complex of a substance to be measured and a substance capable of binding to the substance to be measured (hereinafter abbreviated as binding substance A ₁ ), further comprising the hydrophobicity of the complex or
A substance modified with a substance capable of changing ionicity (hereinafter abbreviated as a separation-improving substance) and having a binding ability to the complex is bound to form a complex-binding substance A ₁ (or a free binding substance). Ku separation by high-performance liquid chromatography and analyte) Wakashi hydrophobic of the separation-improving substance
Is a separation method characterized by being performed based on ionicity . 2. The separation enhancing substance is a protein, a peptide,
A synthetic polymer compound, a polyamino acid, a halogen atom, an alkyl chain, a fatty acid, or a hydrophobic group or an ion having a reactive group capable of binding to a substance capable of binding to a complex of a substance to be measured and a substance capable of binding A _1. The separation method according to claim 1, which is a chemical substance having properties. 3. A separation group having a reactive group capable of binding to a protein, a peptide, a polyamino acid, an alkyl chain, a halogen atom, a fatty acid or a substance having a binding ability to a complex of a substance to be measured and a binding substance A _1. The separation method according to claim 1, which is a chemical substance having a hydrophobic property or an ionic property, and the packing material of a column used in high performance liquid chromatography is a packing material for hydrophobic chromatography. 4. The separation-improving substance has a reactive group capable of binding to a synthetic polymer compound, a protein, a polyamino acid, a substance capable of binding to a complex of a substance to be measured and a binding substance A ₁ , and is hydrophobic. Alternatively, the separation method according to claim 1, which is a chemical substance having an ionic property, a fatty acid or a peptide, and the packing material of the column used in high performance liquid chromatography is a packing material for ion exchange chromatography. 5. A method for measuring a substance to be measured using a reaction between a substance to be measured and a substance capable of binding A ₁ , wherein a complex of the substance to be measured and the substance capable of binding, which results from the reaction, is further included. , A substance that is modified with a separation-enhancing substance and has a binding ability to the complex (hereinafter, abbreviated as modified binding ability substance A), and the complex and the free binding ability substance A
₁ (or free analyte) and sparse of the separation-improving substance
After aqueous Wakashi Ku is separated by high performance liquid chromatography based on ionic, measuring the amount of complex or free affinity substance A ₁ (or free analyte), measured on the basis of the result A measuring method characterized by determining the amount of a substance. 6. The measuring method according to claim 5 , wherein the packing material for the column used in high performance liquid chromatography is a packing material for ion exchange chromatography. 7. A biologically-derived sample containing a measurement target substance, a measurement target substance labeled with a detection substance (hereinafter abbreviated as a labeled measurement substance), and a binding ability substance A ₁ are reacted with a modified binding ability substance A. , A complex of the substance to be measured, the binding substance A ₁ and the modified binding substance A, and a complex of the labeled measurement substance, the binding substance A ₁ and the modified binding substance A (hereinafter abbreviated as labeled complex) .) Is formed, the labeled complex and the labeled measurement substance are separated by high performance liquid chromatography according to the hydrophobicity or ionicity of the separation enhancing substance, and then,
A measuring method, characterized in that the amount of a substance to be measured is determined by measuring the amount of a detection substance contained in the separated labeled complex. 8. The measuring method according to claim 7 , wherein the column packing material used in high performance liquid chromatography is an ion exchange chromatography packing material.