JP2001033453A - Measuring method for ligand - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a trace component in a sample much more accurately as compared with conventional methods in a measuring method for a ligand, wherein a complex which is formed by reacting a labeling receptor with the ligand is separated by a separation means and the labeling receptor which does not participate in the reaction with the ligand and which passes the separation means is measured. SOLUTION: This measuring method for a ligand is provided with a process (a) wherein a complex is formed in such a way that particles which comprise a receptor specific with reference to the ligand and/or which comprise a receptor specific with reference to the ligand on the surface, particles which comprise a receptor specific to the ligand and/or which comprise a labeling receptor specific to the ligand and/or which comprise a labeling receptor specific to the ligand on the surface and a sample which contains the ligand are reacted. The measuring method is provided with a process (b) in which the formed complex is separated by a separation means. The measuring method is provided with a process (c) wherein particles which comprise the irradiated labeling receptor passing the separation means without participating in the reaction with the ligand and/or which comprise an unreacted labeling receptor are developed in a development phase. The measuring method is provided with a process (d) in which the amount of the developed unreacted labeling receptor is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring a ligand in a biological sample, and particularly to a method for measuring pregnancy, judging the presence or absence of fecal occult blood, and testing for bacteria and viruses. The present invention relates to a method for measuring a ligand contained in a sample using a chromatography medium.

[0002]

2. Description of the Related Art Conventionally, in the field of medical diagnostics, various analytical methods have been developed as methods for measuring trace substances contained in liquid samples. In particular, an immunoassay utilizing an antigen-antibody reaction is widely used as a high-sensitivity measurement method, and in the clinical field, trace substances are analyzed using an apparatus utilizing this measurement principle (JP-A-47-18597). 53-
No. 47518).

[0003] Furthermore, the necessity of simple measurement utilizing this immune reaction is increasing in clinics and general homes, and the development of a simple measuring device for the purpose is also progressing. Many devices or immunoassay methods have been proposed (Japanese Translation of PCT Application No. Sho 62-50103).
No. 4, JP-A-63-118657, JP-A-64-118657.
463).

In this immunoassay method, generally, an antigen in a sample developed using a chromatography medium is converted into an enzyme,
A so-called sandwich-type immune complex of an antibody-antigen-labeled antibody complex between a first antibody labeled with a labeling substance such as a fluorescent substance or a luminescent substance, and a second antibody immobilized on a chromatography medium. Is formed, and the amount of the substance to be measured is measured based on the color development, fluorescence, and luminescence derived from the immobilized marker (JP-A-5-15).
0881, 5-150882 and the like).

However, in these methods,
When a monoclonal antibody whose recognition site of the target substance is usually only one in the target substance is used as the marker-labeled antibody, a so-called sandwich-type immune complex consisting of an immobilized antibody, the target substance and the marker-labeled antibody is used. The number of marker particles (colloidal gold, colored latex, etc.) in the medium is at least one for each substance to be measured, so that there is a problem that sufficient sensitivity cannot always be obtained (Japanese Patent Laid-Open No. 10-1998). No. 68730).

In order to solve this problem, at least two types of monoclonal antibodies are used, and when a polyclonal antibody is used, sensitivity is improved by using one or more types of monoclonal antibodies separately. Was. However, in order to select such a combination, an enzyme immunoassay is generally performed, but if a good combination is not found, there is a problem that an antibody must be prepared again. .

[0007] In order to eliminate the complicated means for selecting an antibody combination, an antigen present in a liquid and an antibody specific to the antigen are immunologically bound and reacted with a labeled antibody. After forming an immune complex, a filter is provided for separating the immune complex and unreacted matter into physical sizes, absorbs the solution containing the unreacted matter, holds the immune complex, and performs measurement. An immunoassay device for detecting or quantifying the presence or absence of a sample by performing the measurement has been proposed (Japanese Patent Application Laid-Open No. 9-274039).

[0008]

However, the above-mentioned immunoassays and immunoassays only measure qualitatively or semi-quantitatively the immune complex, and separate the immunocomplex by filtration means or the like. There is no suggestion about a method for measuring an unreacted labeling substance that has passed through a filtration means. Accordingly, the present invention provides a novel ligand for measuring the unreacted labeled receptor which has passed through the separation means without being involved in the reaction with the ligand, by separating the complex formed by reacting the labeled receptor and the ligand by the separation means. It is intended to provide a measuring method.

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a complex of a ligand and a labeled receptor is captured by a separating means, and that the unreacted label which has passed through the separating means is unreacted. The present inventors have found that measuring the amount of a receptor or the amount of an unreacted labeled ligand makes it possible to more precisely measure a trace component in a sample, and arrived at the present invention.

The present invention is characterized by a method for measuring a ligand, which comprises the following steps. (a) a particle having the receptor specific to the ligand and / or the receptor specific to the ligand on the surface, and a labeled receptor specific to the ligand and / or the ligand specific to the surface Reacting a particle having the labeled receptor specific to the sample with the ligand to form a complex, (b) separating the formed complex by separation means, (c) Developing the unreacted labeled receptor which has passed through the separation means without participating in the reaction with the ligand, or particles having the unreacted labeled receptor in a developing phase, (d) developing the unreacted label Measuring the amount of receptor.

Further, the present invention is characterized by a method for measuring a ligand, which comprises the following steps. (a) a receptor specific to a ligand and / or particles having the receptor specific to the ligand on the surface, a labeled ligand and / or particles having a labeled ligand, and a sample containing the ligand Reacting to form a complex, (b) separating the formed complex by separation means, (c) unreacted labeled ligand that has passed through the separation means without participating in the reaction with the receptor, Or a step of developing particles having a labeled ligand in a developing phase, and (d) a step of measuring the amount of the unreacted labeled ligand that has been developed.

Hereinafter, the present invention will be described in more detail.

In the present invention, the measurable ligand can be appropriately selected from known ligands as long as it has a specific binding action to the receptor. Specific examples of the ligand / receptor are not limited to antigens / antibodies, but include enzymes / substrates, single-stranded DNA / complementary strands of DNA, biotin / avidin, and the like. In particular, when used in an immunoassay, the ligand corresponds to an antigen (or an antibody), and the receptor corresponds to an antibody (or an antigen). The antibody is not particularly limited as long as it has a specific binding action to the antigen. In addition to commonly used polyclonal antibodies and monoclonal antibodies, polymerized antibodies, half-molecular antibodies (ha
Engineered antibodies such as lf IgG), Facb, F (ab ') 2, Fab, Fab', and Fv antibody fragments can also be suitably used. Here, the polymerized antibody refers to a covalent polymer of the antibody or the protein and the antibody, and the half-molecular antibody refers to, for example, an antibody molecule fragmented with a reducing agent. Preferred examples of such an immunological assay include a sandwich type reaction and a competitive type reaction.

The sample specimen applicable to the present invention includes any form of solution or colloid solution which is a fluid sample, and is preferably a biological fluid sample, for example,
Examples include blood, serum, plasma, urine, fecal suspension, cerebrospinal fluid, saliva and the like. Examples of ligands contained in these sample specimens include proteins such as albumin, globulin, fecal hemoglobin, and human chorionic gonadotropin (hC).
G), thyroid stimulating hormone (TSH), hormones such as insulin, thyroxine and estrogen, oncofetal proteins
(CEA), cancer markers such as alpha fetoprotein (AFP) basic fetoprotein (BFP), viruses such as human immunodeficiency virus (HIV), human adult T-cell leukemia virus (ATLV), hepatitis B virus (HBV), Interleukin-1 (IL-1), IL-2, IL
Cytokines such as -6, theophylline, phenytoin,
Various growth factors such as drugs such as valproic acid, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF), C-reactive protein (CRP) And proteins related to inflammation, bacteria and cells, or antibodies against these.

The label, which serves as an indicator for the measurement of the analyte, is reacted on a chromatography medium to form a complex between the ligand and the labeled receptor, and the unreacted labeled receptor or label which did not participate in the reaction. As long as the ligand can be measured by the detection means, it can be appropriately selected from known labels and used. Specific examples include enzymes, chromophores, fluorophores, luminophores, radioisotopes, stained gels, metal colloids, colored latex, etc. preferable.

Examples of such enzymes include peroxidase, glucose oxidase, cholesterol oxidase, glycerol phosphate oxidase, sarcosine oxidase, choline oxidase, acyl CoA oxidase, amino acid oxidase, uricase, alkaline phosphatase, acid phosphatase, β-galactosidase, and β-galactosidase. -D-glucosidase.

In the present invention, the means for separating the complex may be appropriately selected from known means for capturing the complex. For example, physical means such as a porous membrane such as a filter, a filter paper, a porous ceramic, and a fiber layer may be used. A typical separation means is mentioned, and a membrane filter is particularly preferable. The pore size of this separation means is such that the labeled receptor or the labeled receptor-bound particle is allowed to pass through, but the ligand is not allowed to pass through the complex of the labeled receptor or the labeled receptor-bound particle. Specifically, the pore size is in the range of 10 nm to 10 μm. It is preferred that

The most characteristic feature of the present invention is that the unreacted labeled receptor which has not been involved in the reaction with the ligand which has passed through the separation means, the particle having the same or the labeled ligand is developed in the developing phase. is there. By measuring the unreacted labeled receptor or unreacted labeled ligand, the amount of the ligand can be measured with high sensitivity that cannot be obtained by the conventional method. Examples of the measuring means include qualitative measurement and semi-quantitative measurement by visual inspection, and quantitative measurement by a detector such as a spectrophotometer and a reflectance measuring device. In particular,
By optically measuring the reflectance and the transmittance, it is possible to perform a more precise quantitative measurement of a trace component in a sample.

The developing phase used in the present invention functions as a medium for transferring unreacted labeled receptor or unreacted labeled ligand by the action of capillary action. The material is not particularly limited.

Examples of such materials include filter paper,
Examples thereof include a cellulose film, a glass fiber film, a nitrocellulose film, and a cellulose acetate film. These may be used alone or in combination. Among these materials, filter paper and cellulose acetate membrane are particularly preferable from the viewpoint that the physical adsorbability and development speed of antibodies and the like can be easily controlled. Although the size of the chromatography medium is not particularly limited, for use, for example, the width is 2 to 10 m.
m, a length of 5 to 150 mm, and a thickness of 0.02 to 1.5 mm are preferable.

The insoluble carrier particles used in the present invention can be appropriately selected from those conventionally used as carrier particles for an immune reaction. Specific examples thereof include inorganic substance powder and organic polymer substance powder. Examples of the inorganic substance include, but are not particularly limited to, metals such as gold, titanium, iron, and nickel, metal oxides such as alumina and titania, and silica. The organic polymer is not particularly limited,
Styrene polymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate copolymer, vinyl acetate-acrylate Copolymers and the like can be mentioned, and latex particles in which these polymer powders are uniformly suspended in water are particularly preferable.

The sensitization of these insoluble carrier particles with an antigen or antibody can be carried out according to a known method. Specific examples thereof include, for example, a chemical bonding method using a so-called coupling agent such as glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluoronitrobenzene, carbodiimides, quinones, chromium chloride, and tannic acid, and an antigen. Alternatively, a physical adsorption method and the like in which an antibody and a carrier are brought into contact with each other in a water-soluble solvent (for example, water, physiological saline, various buffer solutions, and the like) may be used.

In the present invention, although not particularly required, a surfactant such as polyethylene glycol, polyoxyethylene octyl phenyl ether, or polyvinyl alcohol is added in order to adjust the flow rate of the capillary phenomenon in the reaction layer or the spreading layer. It is also possible. The surfactant to be added is not particularly limited as long as it changes the viscosity that does not adversely affect the reaction of the entire system, and may be appropriately selected from known surfactants.

[0024]

According to the present invention, a complex formed by reacting a labeled receptor with a ligand is separated by separation means,
This is a novel ligand measurement method that measures unreacted labeled receptor that has passed through the separation means without being involved in the reaction with the ligand, and enables more accurate measurement of trace components in a sample than conventional methods. .

[0025]

EXAMPLES The present invention will be described below based on test examples and examples, but the present invention is not limited to these examples.

Test Example 1 Preparation of mouse anti-human albumin monoclonal antibody-bound latex suspension Mouse anti-human albumin monoclonal antibody (homemade,
Clone 1) 1 ml of 600 μg / ml and 1 ml of 20 mg / ml latex emulsion {Ceradyne Co., Ltd., 0.3 μm in diameter} were mixed with 0.02 M
After mixing in 5 ml of a PBS pH 7.2 solution and incubating at 37 ° C. for 60 minutes, the mixture was centrifuged at 15,000 rpm for 20 minutes. To the resulting precipitate was added 10 mg / ml skim milk solution,
After incubation at 3 ° C. for 3 hours, an antibody-bound latex suspension was obtained.

Test Example 2 Preparation of Peroxidase-Labeled Mouse Anti-Human Albumin Monoclonal Antibody-Bound Latex Suspension Enzyme-linked immunosorbent assay Toyobo Co., Ltd.} and a mouse anti-human albumin monoclonal antibody (homemade, clone 2) to prepare a peroxidase-labeled mouse anti-human albumin monoclonal antibody. Created peroxidase-labeled mouse anti-human albumin monoclonal antibody 600μ
g / ml 1ml and 20mg / ml latex suspension
1 ml of 0.3 μm in diameter and 5 ml of 0.02 M PBS pH 7.2 solution
After incubating at 37 ° C for 3 hours.
Centrifuged at 5,000 rpm for 20 minutes. 10 mg to the obtained precipitate
After adding 3 ml of skim milk solution and incubating at 25 ° C. for 3 hours, a suspension of peroxidase-labeled mouse anti-human albumin monoclonal antibody-bound latex was obtained.

Test Example 3 Preparation of latex suspension-containing sheet 1 ml of 40 mg / ml mouse anti-human albumin monoclonal antibody-bound latex suspension obtained in Test Example 1 and 40 mg / ml peroxidase-labeled mouse anti-human albumin monoclonal antibody obtained in Test Example 2 Antibody-bound latex suspension 1 m
l, and 0.1 M containing 2 ml of 100 mg / ml glucose solution
10 ml of a phosphate buffer (pH 7.2) was prepared. A polyethylene sheet {Asahi Kasei Co., Ltd. thickness 1 mm, water content 40
μl / cm ² }, and lyophilized to obtain a latex suspension-containing sheet.

Test Example 4 Preparation of Latex Suspension and Enzyme-Linked Antibody-Fixed Sheet 1 mg of 40 mg / ml mouse anti-human albumin monoclonal antibody-bound latex suspension obtained in Test Example 1, 5 mg / ml peroxidase-labeled mouse obtained in Test Example 2 0.1 M phosphate buffer (pH 7.2) containing 0.5 ml of anti-human albumin monoclonal antibody solution and 2 ml of 100 mg / ml glucose solution
10 ml were prepared. A polyethylene sheet was added to this solution.
(Thickness: 1 mm, water absorption: 40 μl / cm ² ), and lyophilized to obtain an enzyme-linked antibody-immobilized sheet.

Test Example 5 Preparation of Peroxidase Detection Pad Glucose Oxidase {Roche Co., Ltd. 200 u / m
g｝ 2.5 mg and 2,7-diaminofluorene hydrochloride {Sigma Co., Ltd.
0.1M MES buffer (pH 6.0) with 0.27 mg
00 ml was prepared. Add white filter paper {Toyo Filter Paper Co., Ltd. N
o.1, water absorption of 10 μl / cm ² } was immersed, air-dried at 45 ° C. for 2 hours, and further dried in a vacuum desiccator for 3 hours to obtain a peroxidase detection pad.

Test Example 6 Preparation of developing phase White filter paper cut to 5 mm in width and 20 mm in length (No.
1, a water absorption of 10 μl / cm ² } was attached to a position 15 mm from one end of a vinyl chloride support 5 having a width of 5 mm and a length of 50 mm with a double-sided tape to obtain a white filter paper (development phase) 3 for development.

Embodiment 1 Quantification of Human Albumin On one end of the developed phase 3 obtained in Test Example 6, the peroxidase detection pad (enzyme detection coloring pad) 4 cut in 5 mm on a side obtained in Test Example 5 was overlapped. A 7 mm square filtration membrane filter 2 {manufactured by Toyo Roshi Kaisha Co., Ltd., cellulose acetate membrane, pore size 0.45 μm} was overlaid on the other end, and further obtained in Test Example 3 so as not to directly touch the developing phase 3 below. Latex suspension containing sheet (antigen-antibody reaction pad) 1 (width 5 mm length 2
0 mm), and 2 from one end of the latex suspension-containing sheet 1
Except for the 5 mm square part located at a distance of mm, the whole was covered with a cover film and each part was fixed to prepare a test piece.
(refer graph1).

A phosphate buffer solution containing 0, 6.25, 12.5, 25, and 50 μg / ml of human albumin {manufactured by Sigma Co., Ltd.} was added to one part of the latex suspension-containing pad of the prepared test piece.
00 μl was dropped, and the peroxidase detection pad 4 which turned blue four minutes later was measured with a color difference meter {trade name of Tokyo Denshoku Co., Ltd .: color analyzer TC-1800M}. 600n measured
The K / S value calculated by the following formula based on the reflectance (R value) of m was plotted on the ordinate and the concentration of human albumin was plotted on the abscissa to create a standard quantitative curve (see FIG. 2). As shown in FIG. 2, a curve of an inverse sigmoid in which the K / S value decreases depending on the concentration of human albumin was drawn, and it was found that the method was quantitative. Calculation formula for calculating K / S K / S = (1-a) 2 / 2a, a = (reflectance at 600 nm) / 100

Example 2 Quantification of Human Albumin The latex suspension and the enzyme-labeled antibody-fixed sheet obtained in Test Example 4 were used as an antibody-fixed sheet, and a membrane filter (manufactured by Toyo Roshi Kaisha Co., Ltd. (cellulose acetate membrane, pore size 0.22 μm)) was used. A test piece was prepared in exactly the same manner as in Example 1 except that the test piece was used. Human albumin {manufactured by Sigma Co., Ltd.} was added to the latex suspension and the enzyme-conjugated antibody-immobilized sheet portion of the prepared test piece, respectively, at 0 and 6.2.
100 μl of phosphate buffer containing 5, 12.5, 25, and 50 μg / ml was added dropwise, and the peroxidase detection pad that turned blue after 4 minutes was measured with a color difference meter {trade name of Tokyo Denshoku Co., Ltd .: Color Analyzer TC -1800M}. 600nm measured
The K / S value calculated by the formula of Example 1 based on the reflectance of Example 1 was plotted on the ordinate and the human albumin concentration was plotted on the abscissa to create a standard quantitative curve (see FIG. 3). As shown in FIG. 3, a curve of an inverse sigmoid in which the K / S value becomes smaller depending on the concentration of human albumin was drawn, and it was found that the method was quantitative.

Test Example 7 Preparation of Latex Suspension Containing Mouse Anti-basic Fetoprotein Monoclonal Antibody Mouse Antibasic Fetoprotein Monoclonal Antibody
{Manufactured by Nippon Kayaku Co., Ltd.} Clone No. K1 300 μg / ml, and Clone No. 5C6 300 μg / ml 20 mg / ml latex emulsion
{Ceradyne Co., Ltd., 0.3 μm in diameter} 1 ml and 0.02M PBS p
The mixture was mixed in 5 ml of H7.2 solution, incubated at 37 ° C. for 60 minutes, and then centrifuged at 15,000 rpm for 20 minutes. 10 mg / ml bovine albumin solution was added to the obtained precipitate, and the mixture was added at 25 ° C.
After incubation for 3 hours, an antibody-bound latex suspension was obtained.

Test Example 8. Preparation of Peroxidase-Labeled Mouse Antibasic Fetoprotein Monoclonal Antibody-Bound Latex Suspension Peroxidase and mouse antibasic fetoprotein monoclonal antibody {Nippon Kayaku Co., Ltd. And clone No. 5C2} to obtain a mouse anti-basic fetoprotein monoclonal antibody labeled with peroxidase. The obtained peroxidase-labeled mouse anti-basic fetoprotein monoclonal antibody 600 μg / ml
1 ml and 20 mg / ml latex suspension {Ceradyne Co., Ltd.
0.3 μm} was mixed in 5 ml of 0.02M PBS pH 7.2 solution and incubated at 37 ° C. for 3 hours, after which 15,000 rp
and centrifuged for 20 minutes. A 10 mg / ml bovine albumin solution was added to the obtained precipitate, and the mixture was incubated at 25 ° C. for 3 hours to obtain a suspension of peroxidase-labeled mouse anti-basic fetoprotein monoclonal antibody-bound latex.

Test Example 9 Preparation of Latex Suspension-Containing Sheet 1 mg of the 20 mg / ml mouse anti-basic fetoprotein monoclonal antibody-bound latex suspension obtained in Test Example 7 and 20 mg / ml of the latex suspension obtained in Test Example 8 Oxidase-labeled mouse anti-basic fetoprotein monoclonal antibody-bound latex suspension 1 ml, 200 mg / ml polyethylene glycol (# 6000)
0.1 ml containing 2 ml, and 2 ml of 100 mg / ml glucose solution
10 ml of M phosphate buffer (pH 7.2) was prepared. A polyethylene sheet (thickness: 1 mm, water absorption: 40 μl / cm ² ) was immersed in the prepared solution, and then lyophilized to obtain a latex suspension-containing sheet.

Test Example 10 Preparation of Expanded Phase White filter paper cut to a width of 5 mm and a length of 20 mm (No. 1, manufactured by Toyo Roshi Kaisha, Ltd.)
A water-absorbing amount of 10 μl / cm ² } was attached to a position 30 mm from one end of a vinyl chloride support 5 having a width of 5 mm and a length of 60 mm with a double-sided tape to obtain a developed phase 3.

Example 3 Determination of Basic Fetoprotein On one end of the developed phase 3 obtained in Test Example 10, the pad 4 for detecting peroxidase cut in 5 mm on a side obtained in Test Example 5 was placed. A 7 mm-square membrane filter 2 (manufactured by Toyo Roshi Kaisha Co., Ltd., cellulose acetate membrane, pore size 0.45 μm) is overlaid on the end of the other developing phase 3, and the width 5 mm long so as not to directly touch the lower developing phase 3. One end of a developing phase 3 (white filter paper) having a thickness of 20 mm {No.1, water absorption 10 μl / cm ² } (manufactured by Toyo Roshi Kaisha, Ltd.) was superposed on the filter 2 (to overlap with a side of 5 mm square). Furthermore, the latex suspension-containing sheet 1 (width 5 mm, length 20 mm) obtained in Test Example 9 was overlapped with the other end of the developing phase 3 (so that each side overlaps a 5 mm square), and the latex suspension-containing sheet 1 Except for the portion of 5 mm square on one side located at a distance of 2 mm from one end, the whole was covered with a cover film and each part was fixed to prepare a test piece
(See FIG. 4).

A phosphate buffer solution containing 0, 2, 10, 50, and 250 ng / ml of basic fetoprotein (made in-house) was added to the latex suspension-containing sheet portion of the prepared test piece.
μl was dropped, and the peroxidase detection pad which turned blue after 10 minutes was measured using a color difference meter {trade name, manufactured by Tokyo Denshoku Co., Ltd .: color analyzer TC-1800M}. The K / S value calculated by the equation of Example 1 based on the measured reflectance at 600 nm was plotted on the ordinate, and the basic fetoprotein concentration was plotted on the abscissa, to create a standard quantitative curve (see FIG. 5). As shown in FIG. 5, depending on the concentration of basic fetoprotein, K / S
The value plots a curve of inverse sigmoid with decreasing values, indicating that the method is quantitative.

Test Example 11. Introduction of maleimide group into antibody Mouse anti-human albumin monoclonal antibody (manufactured in-house)
5 mg was dissolved in 360 μl of 0.1 M phosphate buffer pH 7.2. N-solution dissolved in 10 mg / ml of dimethylformamide was added to this solution.
(8-maleimidocapryloxy) sulfosuccinimide sodium salt {trade name: Sulfo-HMCS} manufactured by Dojin Chemical Co., Ltd.
μl was added and reacted at 30 ° C. for 30 to 60 minutes. After the reaction, PD-
Gel filtration using 10 {trade name of Pharmacia Co., Ltd.}
The protein portion was recovered. Then, maleimide group introduction
The collected IgG was concentrated to about 3.5 mg / ml by centrifugal concentration.

Test Example 12. Introduction of a thiol group into an antibody Mouse anti-human albumin monoclonal antibody (made in-house)
10 mg was dissolved in 1.0 ml of 0.1 M phosphate buffer pH 7.2. To this solution was added 10 mg of N-succininimidyl S-acetylthioacetate per 1 ml of dimethylformamide {Pierce Co., Ltd.
39 μl of a solution prepared by dissolving the product was added. After reacting at 30 ° C. for 30 to 90 minutes, 200 μl of 1 M tris (hydroxymethyl) aminomethane hydrochloride buffer pH 8.0, 100 μl of 1 M hydroxylamine hydrochloride solution, and 0.1 M ethylenediaminetetraacetic acid-0.1 M tris ( (Hydroxymethyl) aminomethane solution (50 μl) was sequentially added and reacted at 30 ° C. for 10 to 30 minutes. After the reaction, gel filtration was performed using PD-10 {trade name of Pharmacia Co., Ltd.} using a 0.1 M phosphate buffer containing 0.01 M ethylenediaminetetraacetic acid as an eluent to recover a protein portion. Further, the recovered solution of the SH group-introduced antibody was concentrated to about 3.5 mg / ml by centrifugal concentration.

Test Example 13. Preparation of polymerized antibody 3.6 mg of maleimide group-introduced mouse anti-human albumin monoclonal antibody obtained in Test Example 11 and 3.6 mg obtained in Test Example 12
Was mixed with a thiol group-introduced mouse anti-human albumin monoclonal antibody (reaction ratio: 1: 1), and incubated at 30 ° C. for 30 to 60 minutes to obtain a gel-like reaction product. After freeze-drying and weighing the obtained reaction product, 0.1M HEP
The gel was hydrated again by adding an ES-NaOH buffer solution (pH 7.5), and the gel was crushed by sonication for 10 minutes while cooling on ice to prepare a suspension of the polymerized antibody. ELS-800 (OTSUKA ELECT
RONICS) to measure the particle size of the polymerized antibody.
The average particle size was 0.2463 μm.

Test Example 14 Preparation of Peroxidase-Labeled Polymerized Antibody A homopolymerized antibody of a mouse anti-human albumin monoclonal antibody (made in-house) was prepared in exactly the same manner as in Test Examples 11 to 13. Furthermore, utilizing the residual thiol group of the homogeneously polymerized antibody, the antibody was chemically bonded to peroxidase to prepare a peroxidase-labeled polymerized antibody.

Test Example 15 Preparation of Maleimide Group-Introduced Peroxidase 8 mg of peroxidase {manufactured by Toyobo Co., Ltd.} was dissolved in 0.6 ml of 0.1 M phosphate buffer pH 7.2. Add 200 μl of DMF to this solution.
10 mg of N- (6-maleimidocaproyloxy) dissolved in l
Succinimide (trade name: EMCS) manufactured by Dojin Chemical Co., Ltd. 60 μl
And incubated at 30 ° C. for 60-90 minutes.
After the reaction, the eluate was 0.1 M phosphate buffer (pH 7.2), and PD-1
The mixture was subjected to gel filtration using 0 {manufactured by Pharmacia Co., Ltd.} to obtain a maleimide group-introduced peroxidase portion.

Test Example 16 Reaction of homopolymerized antibody with maleimide group-introduced peroxidase Mouse anti-human albumin monoclonal antibody (homemade)
A homogeneously polymerized antibody of 3.5 mg / ml solution 0.4 ml and 1.2 mg / ml maleimide group-introduced peroxidase 0.4 ml were mixed at 30 ° C.
Incubated for 0-90 minutes. After the reaction, gel filtration was performed using TSK-GEL G4000SWXL (manufactured by Tosoh Corporation) using a 0.1 M phosphate buffer (pH 6.5) to remove unreacted peroxidase, and to prepare a peroxidase-labeled polymerized antibody. Got the part.

Test Example 17 Preparation of Immobilized Sheet of Polymerized Antibody and Peroxidase-Labeled Polymerized Antibody 1 ml of a 10 mg / ml mouse anti-human albumin monoclonal antibody (made in-house) polymerized antibody solution obtained in Test Example 13 1 ml of a polymerized antibody solution of 10 mg / ml peroxidase-labeled mouse anti-human albumin monoclonal antibody (made in-house) obtained in Test Example 14, 200 mg / ml polyethylene glycol (# 6000)
0.1 M containing 2 ml, and 2 ml of 100 mg / ml glucose solution
10 ml of a phosphate buffer (pH 7.2) was prepared. A polyethylene sheet (thickness: 1 mm, water absorption: 40 μl / cm ² ) was immersed in the prepared solution, and then lyophilized to obtain an antibody-fixed sheet.

Test Example 18 Preparation of Expanded Phase A white filter paper {manufactured by Toyo Roshi Kaisha, Ltd., water absorption 10 μl / cm ² } cut to a width of 5 mm and a length of 30 mm was applied to a polyvinyl chloride support 5 having a width of 5 mm and a length of 80 mm. Attach 30mm from one end with double-sided tape to deploy
Got three.

Example 4 Quantification of Human Albumin One side obtained in Test Example 5 was placed at a position 15 mm away from one end (position 25 mm from the end of the support) of the developed phase 3 obtained by the method of Test Example 18. A 5 mm square peroxidase detection pad 4 is stacked on the other end, and a 5 mm square absorbent membrane passing liquid absorbing pad 6 on the other end {Product name: 7 chromatograph, water absorption 40 manufactured by Whatman Co., Ltd.
μl / cm ² }. Further, a 7 mm square membrane filter 2 (manufactured by Toyo Roshi Kabushiki Kaisha, cellulose acetate membrane, pore size 0.
45 μm}, and do not directly touch the developed phase 3 at the bottom.
Developing phase 3 with width 5mm and length 20mm {Toyo Roshi Kaisha, Ltd., water absorption 10μ
l / cm ² } was overlapped on the filter 2 (so as to overlap at a side of 5 mm square). Furthermore, on the other end of the developing phase 3, a fixed sheet (width 5 mm, length 20 mm) of the polymerized antibody obtained in Test Example 17 and the peroxidase-labeled polymerized antibody was overlapped (to overlap at a side of 5 mm square), and latex was used. Except for a 5 mm square part located at a distance of 2 mm from one end of the suspension-containing sheet 1, the whole was covered with a cover film and each part was fixed to prepare a test piece.
(See FIG. 6).

Human albumin {manufactured by Sigma Co., Ltd.} was added to the fixed sheet portion of the prepared test piece with the polymerized antibody and the peroxidase-labeled polymerized antibody at 0, 6.25, 12.5, and 12.5, respectively.
150 μl of phosphate buffer containing 25, 50 μg / ml was added dropwise, and 5
After a minute, the peroxidase detection pad that turned blue afterwards was colored with a color difference meter {trade name of Tokyo Denshoku Co., Ltd .: Color Analyzer TC-
1800M). The K / S value calculated by the formula of Example 1 based on the measured reflectance at 600 nm was plotted on the ordinate, and the human albumin concentration was plotted on the abscissa to create a standard quantitative curve.
(See FIG. 7). As shown in FIG. 7, a curve of an inverse sigmoid in which the K / S value becomes smaller depending on the concentration of human albumin was drawn, and it was found that the present method was quantitative.

Example 5 Quantification of Human Albumin The immobilized sheet of the polymerized antibody and the peroxidase-labeled antibody obtained in Test Example 17 was used as the antibody immobilized sheet, and the membrane filter 2 was manufactured by Toyo Roshi Kaisha, Ltd., cellulose acetate membrane. A test piece was prepared in exactly the same manner as in Example 4, except that the pore size was 0.1 μm. Phosphate buffer solution 1 containing 0, 6.25, 12.5, 25, and 50 μg / ml of human albumin {manufactured by Sigma Co., Ltd.) on the immobilized sheet portion of the prepared test piece with the polymerized antibody and the peroxidase-labeled antibody, respectively.
50 μl was dropped, and the peroxidase detection pad which turned blue after 5 minutes was measured using a color difference meter {trade name of Tokyo Denshoku Co., Ltd .: color analyzer TC-1800M}. It was measured
The K / S value calculated by the formula of Example 1 based on the reflectance at 600 nm was plotted on the ordinate and the human albumin concentration was plotted on the abscissa to create a standard quantitative curve (see FIG. 8). As shown in FIG. 8, a curve of an inverse sigmoid in which the K / S value becomes smaller depending on the concentration of human albumin was drawn, and it was found that the present method has a quantitative property.

Test Example 19 Preparation of Peroxidase-Labeled Human Albumin Peroxidase-labeled human albumin was prepared in exactly the same manner as in Test Example 2 except that human albumin was used instead of the mouse anti-human albumin monoclonal antibody.

Test Example 20 Preparation of peroxidase-labeled human albumin immobilized sheet 1 ml of the 500 μg / ml peroxidase-labeled human albumin solution obtained in Test Example 19, and 100 mg / ml glucose solution 2
10 ml of a 0.1 M phosphate buffer (pH 7.2) containing ml was prepared. A polyethylene sheet (1 mm thick, water content 40 μl / cm ² ) was immersed in the prepared solution, and then lyophilized to obtain a peroxidase-labeled human albumin fixed sheet.

Test Example 21 Preparation of Latex Suspension-Containing Sheet 1 ml and 100 mg / m of the 40 mg / ml mouse anti-human albumin monoclonal antibody-bound latex suspension obtained in Test Example 1
1 M glucose solution containing 2 ml of 0.1 M phosphate buffer (pH
7.2) 10 ml was prepared. A polyethylene sheet (thickness 1 mm, water content 40 μl / cm ² ) was immersed in the prepared solution, and then lyophilized to obtain a latex suspension-containing sheet.

Test Example 22 Preparation of Expanded Phase White filter paper {manufactured by Toyo Roshi Kaisha Co., Ltd., water absorption 10 μl / cm ² } cut to a width of 5 mm and a length of 20 mm was applied to a polyvinyl chloride support 5 having a width of 5 mm and a length of 80 mm. Attach 45mm from one end with double-sided tape to deploy
Got three.

Example 6 Quantification of Human Albumin At one end (position 15 mm from the end of the support) of the developing phase 3 obtained by the method of Test Example 22, the peroxidase having a side of 5 mm square obtained in Test Example 5 was placed. The detection pads 4 were stacked. Further, a membrane filter 2 (manufactured by Toyo Roshi Kaisha Co., Ltd., cellulose acetate membrane, pore size 0.20 μm) having a side of 7 mm square is overlapped on one end of the developing phase 3 (position 45 mm from the end of the support). To avoid direct contact with 3, one end of a developing phase 3 (white filter paper) 5 mm wide and 20 mm long {manufactured by Toyo Roshi Kaisha, Ltd., water absorption 10 μl / cm ² } is placed on the filter (5 mm square on each side) like). The latex suspension fixing sheet 1 (width 5 mm, length 20 mm) obtained in Test Example 21 was overlaid on the other end of the developing phase 3 (to overlap with a side of 5 mm square). Furthermore, the peroxidase-labeled human albumin fixing pad 7 (width 5 mm, length 20 mm) obtained in Test Example 20 was placed at one end of the latex suspension-containing sheet 1 (to overlap 2 mm), and the peroxidase-labeled human Except for a 5 mm-square portion located at a distance of 2 mm from one end of the albumin fixing sheet 7, the whole was covered with a cover film and each part was fixed to prepare a test piece (see FIG. 9).

The prepared test piece was provided with human albumin on the peroxidase-labeled human albumin-fixed sheet 7.
{Sigma Co., Ltd.} respectively, 0, 12.5, 25, 50, 100 μg / m
The phosphoric acid buffer solution containing 150 μl was dropped, and the peroxidase detection pad which turned blue after 5 minutes was measured using a colorimeter {trade name of Tokyo Denshoku Co., Ltd .: color analyzer TC-1800M). . The K / S value calculated by the equation of Example 1 based on the measured reflectance at 600 nm was plotted on the ordinate, and the human albumin concentration was plotted on the abscissa to create a standard quantification curve (see FIG. 10). As shown in FIG. 10, a K / S value was plotted as a sigmoid curve in which the K / S value increased depending on the concentration of human albumin, indicating that the method was quantitative.

[Brief description of the drawings]

FIG. 1 is a schematic view of a test piece according to one embodiment of the present invention.

FIG. 2 is a graph plotting the K / S value calculated from the albumin concentration and the reflectance at 600 nm in Example 1 of the human albumin sample measurement using the test piece according to the present invention.

FIG. 3 is a graph plotting the K / S value calculated from the albumin concentration and the reflectance at 600 nm in Example 2 of the human albumin sample measurement using the test piece according to the present invention.

FIG. 4 is a schematic view of an example of a preferred embodiment of a test piece according to the present invention.

FIG. 5 is a graph showing a curve plotting a basic fetoprotein concentration and a K / S value calculated from a reflectance at 600 nm in Example 3 of basic fetoprotein sample measurement using a test piece according to the present invention. .

FIG. 6 is a schematic view of an example of a preferred embodiment of a test piece according to the present invention.

FIG. 7 is a graph showing a curve plotting the K / S value calculated from the albumin concentration and the reflectance at 600 nm in Example 4 of the human albumin sample measurement using the test piece according to the present invention.

FIG. 8 is a graph showing a curve plotting the K / S value calculated from the albumin concentration and the reflectance at 600 nm in Example 5 of the human albumin sample measurement using the test piece according to the present invention.

FIG. 9 is a schematic view of an example of a preferred embodiment of a test piece according to the present invention.

FIG. 10 is a graph showing a curve in which the K / S value calculated from the albumin concentration and the reflectance at 600 nm in Example 6 of the human albumin sample measurement using the test piece according to the present invention is plotted.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Pad for antigen-antibody reaction 2 Membrane filter 3 Developing phase 4 Enzyme detection coloring pad 5 Support 6 Membrane passage liquid absorption pad 7 Fixing sheet

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/545 G01N 33/545 A 33/553 33/553 // G01N 33/537 33/537 F term ( Reference) 4B033 NA22 NB12 NB27 NB32 NB45 NB62 NB63 NB65 NB68 ND03 NG09 NG10 NH06 4B063 QA01 QA18 QQ01 QQ03 QQ67 QQ70 QQ75 QQ79 QQ87 QR02 QR13 QR82 QR85 QS15 QS17 QX01

Claims (9)

[Claims] 1. A method for measuring a ligand, comprising the following steps: (a) a particle having the receptor specific to the ligand and / or the receptor specific to the ligand on the surface, and a labeled receptor specific to the ligand and / or the ligand specific to the surface Reacting a particle having the labeled receptor specific for the sample with a sample containing the ligand to form a complex, (b) separating the formed complex by a separation means, Developing the unreacted labeled receptor and / or particles having the unreacted labeled receptor that have passed through the separation means without participating in the reaction with the ligand in a developing phase; Measuring the amount of labeled receptor in the reaction. 2. A method for measuring a ligand, comprising the following steps: (a) a receptor specific to a ligand and / or particles having the receptor specific to the ligand on the surface, a labeled ligand and / or particles having a labeled ligand, and a sample containing the ligand (B) separating the formed complex by separation means, (c) unreacted label that has passed through the separation means without participating in the reaction with the receptor. A step of developing the ligand or the particles having the labeled ligand in the developing phase, and (d) a step of measuring the amount of the developed unreacted labeled ligand. 3. The method according to claim 1, wherein the separation means is a physical separation means such as a porous membrane such as a filter, a filter paper, a porous ceramic, a fiber layer and the like. 4. The ligand is an antigen (or an antibody), an enzyme, a single-stranded DNA or biotin, and the corresponding receptor is an antibody (or an antigen), a substrate, a complementary strand of the DNA,
3. The method according to claim 1, which is avidin. 5. The method according to claim 4, wherein the ligand is an antigen (or an antibody) and the receptor is an antibody (or an antigen) or a processed antibody. 6. The processed antibody is a polymerized antibody, half IgG, Facb,
The method according to claim 5, which is F (ab ') 2, Fab, Fab', Fv. 7. The method according to claim 1, wherein the label is an enzyme, chromophore, fluorophore, luminophore, radioisotope, stained gel, metal colloid, or colored latex. 8. The method according to claim 1, wherein the measurement is performed quantitatively by reflectance or transmittance. 9. The method according to claim 1, wherein the particles are insoluble carrier particles, preferably latex particles.