JPS63238462A - Analysis of specific bonding - Google Patents

Abstract

PURPOSE:To reduce a time required for an analysis to enable the quick analysis to be performed by conducting the analysis utilizing a specific bonding reaction represented by an antigen-antibody reaction. CONSTITUTION:A material to be analyzed is measured or detected by utilizing a bonding reaction between the material to be analyzed and a specific bonding material having a specific affinity with the material to be analyzed. For this end, the specific bonding material labeled with a colored material and the material to be analyzed are reacted on each other. Then, the complex of the specific bonding material labeled with the colored material and the material to be analyzed is separated from a reactive liquid by separating means that does not pass the complex but passes the specific bonding material labeled with the colored material in a free condition and the material to be analyzed. Thus, the quantities of the colored material resulting from the separated complex/the specific bonding material labeled with the colored material in a free state are measured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an analysis method that utilizes a specific binding reaction, typified by the antigen-antibody reaction, which is widely used in the medical and biochemical fields.

[Prior art] Antigen (including hapten)-antibody, avidin-biotin,
Carbohydrates - Lectin, FCfi position of immunoglobulin - Protein A, substances that show different affinities such as specific DNA and complementary DNA, etc. Bares of similar substances are used for the analysis and separation and purification of various substances. . Especially antigen-antibody reactions and DNA
Hybridization is a reaction that is widely applied to analytical methods in the medical field and other fields.

What the main analytical methods using these reactions have in common is that
The substance involved in the reaction is labeled by some means, and the amount of label derived from the substance that participated (or did not participate) in the reaction is finally measured.

Labels that are currently in practical use include radioactive isotopes, enzymes (Japanese Patent Publication No. 47-13994), coenzymes (Japanese Patent Publications No. 60-52378 and No. 61-16938), and non-biological catalysts (Japanese Patent Publications No. 60-52378 and No. 61-16938). Kaisho 6l-249399),
These include fluorescent substances and luminescent substances.

Immunoturbidimetry, which does not particularly require a labeling substance, and a method that optically or visually tracks the reaction between an immunologically active substance sensitized to a particle carrier and its partner using particle carrier aggregation as a parameter, etc. For example, when an enzyme is used as a labeling substance, in addition to the step of carrying out the binding reaction in the specific binding analysis method, enzyme 2! This method requires a step to carry out an enzyme reaction using quality reagents, which wastes analysis time, and an increase in the number of reaction steps and types of reagents is not desirable as it can lead to analysis errors. The problems are also common to systems using labeling substances other than enzymes.

[Structure of the Invention] The present invention provides: 1) a specific binding analysis method for measuring or detecting an analyte using a binding reaction with a specific binding substance having a specific affinity for one analyte; a) React a specific binding substance labeled with a colored substance and an analyte.

b) Separation from the reaction solution using a separation means that does not allow the complex of the specific binding substance labeled with a colored substance and the analyte substance to pass through, but allows the free form of the specific binding substance labeled with a colored substance and the analyte substance to pass through. Separate the complex.

C) Measuring the amount of a colored substance derived from the separated complex and/or a specific binding substance labeled with a free colored substance.

and 2) a specific binding analysis method that measures or detects an analyte using a binding reaction with a specific binding substance that has a specific affinity for one analyte, comprising a) a fixed amount of an analyte; b) The reaction solution obtained in a) is labeled with a colored substance that has the same affinity for the specific binding substance as the analyte, and a certain amount of label-analyte is added to the reaction solution obtained in a). C) The complex is removed by separation means that does not pass the complex of the label-analyte and the specific binding substance but allows the free specific binding substance, analyte, and label-analyte to pass through. d) measuring the amount of a colored substance derived from the separated complex and/or the label-analyte in a free state. and 3) a specific binding analysis method for measuring or detecting an analyte using a binding reaction with a specific binding substance that has a specific affinity for the analyte, comprising: a) an analyte;
Mix a certain amount of label-analyte labeled with a colored substance that has the same affinity for the specific binding substance as the analyte, and b) add a certain amount of specific binding to the mixture obtained in a). C) Label - does not pass the complex of the analyte and the specific binding substance, but the specific binding substance of the free IE, the analyte substance,
and the label-analyte is separated from the reaction solution by a separation means, and d) measuring the amount of colored substance originating from the separated complex and/or the label-analyte in a free state. This is a specific binding analysis method characterized by the following.

The analyte substance and the specific binding substance in the present invention each include one of the pairs that exhibit specific binding properties as described later. For example, when an antigen is the analyte substance, the specific binding substance is On the other hand, when an antibody is used as an analyte, the specific binding substance is an antigen (
In some cases, it is also possible to use an antibody or protein A that recognizes the antibody.

In addition, the label-analyte may be one that exhibits the same affinity for the specific binding substance as the analyte, and does not necessarily need to be labeled with the same substance as the analyte. When a haptenic compound such as a steroid hormone is the substance to be analyzed, a so-called polyhapten, in which a large number of the steroid hormone or its derivatives having the same antigenicity are bound to an appropriate polymeric substance, can be labeled and used. . In this case, a colored substance may be used as the polymer substance.

Pairs of substances showing specific binding properties that can be applied to the present invention include antigen (including hapten)-antibody, avidin-biotin, sugar-lectin, Fc region of immunoglobulin-protein A, specific DNA and its complement. Examples include Nari NA. Therefore, any of the substances in the above pair can be analyzed using the specific binding analysis method according to the present invention.

More specifically, antigens include various hormones,
Haptens such as antigens derived from microorganisms and viruses, antigens derived from cancer cells, and drugs are used as antibodies, such as polyclonal antibodies and
Monoclonal antibodies, multiple types of monoclonal antibodies, and mixtures of these have sugar chains in molecules such as sugar chain antigens, which are attracting attention as markers of various tumors, in addition to literally pure sugar molecules. Also includes things.

Examples of the 41-color labeling substances include 1 dye, gold colloid, and colored latex. When using physical separation means as the separation means, the sizes of these labeled substances are such that they are compatible with the labeled specific binding substance (or label-analyte) and analysis so that separation can be performed quickly and accurately. It is recommended to set the difference between the size of the complex with the target substance (or specific binding substance) and the size when they are in the free state fEj to be as large as possible8. When using a single separation stage in which a second specific binding substance that has a specific affinity for the substance is immobilized, the desired substance can be reliably captured without being greatly affected by the size of a single molecule. Because there are billions to do,
Unlike when using physical separation means, a wider range of substances can be selected as labeling substances.

Labeling methods using colored substances include chemical bonding of dyes, colored latex, etc. with the desired substance, or physical adsorption of the desired substance to colored latex, gold colloid, etc. This method involves introducing a functional group such as a carboxyl group into a molecule of a labeling substance (or a molecule of a desired substance) and reacting this with a 7-mino group in a molecule of a desired substance (or a molecule of a labeling substance); A method of intervening a spacer such as a maleimide compound or aminocaproic acid between the desired substance and the desired substance to bind the two can be applied.

In addition, in addition to the above-mentioned labeling methods, labeling by staining can also be used when labeling substances consisting of sugars or proteins (eg, antigens, antibodies, protein A, sugars, etc.). That is, proteins such as antibodies can be stained with tetrabromophenol blue, Coomassie brilliant blue, pyrogallol red, etc. without impairing their specific affinity. To give a specific example of staining that does not impair specific affinity, if we take an antibody (immunoglobulin G) as an example of a substance to be labeled, the portions of the immunoglobulin G molecule other than the antigen-binding site, such as the C-terminus, H chain, etc. What is necessary is to stain the sugar chain moieties present in the protein, as well as the SH groups obtained by treatment with pepsin, etc.

In the present invention, in addition to the direct labeling method described above, the following indirect labeling method may be used. For example, when using anti-C as the substance to be analyzed and antibody as the specific binding substance. For example, in addition to directly labeling antibodies with colored substances, e! It is possible to identify the

(2) The antibody is labeled with avidin or a derivative thereof, and then reacted with biotin or a derivative thereof labeled with a colored substance.

■Antibodies are labeled with lectin and reacted with sugars labeled with colored substances.

(2) Immunoglobulin G is used as an antibody, and protein A labeled with a colored substance is reacted with it.

(2) Use the antibody as it is and react it with an antibody that recognizes this antibody and is labeled with a colored substance.

(2) Label an antibody with an antigen, and react with an antibody that recognizes the antigen and is labeled with a colored substance.

Of course, these labeling methods can be applied not only to antibodies but also to labeling other substances. Furthermore, even if the relationship between avidin and biotin or sugar and lectin is reversed, these indirect labeling methods can still be used.Furthermore, when it comes to labeling immunoglobe G, it is possible to use these indirect labeling methods even if the relationship between avidin and biotin or sugar and lectin is reversed. , tJ! present in the molecule. A labeled lectin can also be bound to the J chain portion.

One of the separation means suitable for the present invention includes physical separation means such as porous membranes such as membrane filters, filter paper, porous ceramics, and fiber layers. The pore size of these physical separation means allows for accurate and efficient separation of the complex ultimately formed between the labeled substance and its corresponding substance and the other substance present in its free state. In the specific binding analysis method of the present invention, a complex formed by binding substances other than the labeled substance may be generated, but since this complex is not labeled, Even if separation disequilibrium occurs, it will not affect the analytical results.

Other separation means include those in which a second specific binding substance for the analyte or label-analyte is immobilized. Antigen, antibody, lectin, sugar, protein A
Accurate and rapid separation can be achieved by capturing the desired substance with a substance fixed on cellulose or its derivatives. In addition, one separation means can be divided into several sections to prevent interference between different types of samples and reagents when performing multiple analyzes simultaneously.

It is advisable to take measures such as partitioning with an impermeable material or providing a notch as shown in FIG. 1, for example.

If a material with excellent liquid absorption properties such as a glass filter or a polymeric absorber such as a polyvinyl formal l compound is used in conjunction with a separation means such as a biped, rapid separation is expected as it absorbs unnecessary liquid. can. As the polymer absorbent material, various materials that have been put into practical use in disposable diapers, feminine hygiene products, etc. can be used.

The present invention will be explained in more detail below based on Examples.

[Example], l) Analysis using colloidal gold as a colored substance and anti-[(hCG) as one target substance to be analyzed/Colloidal gold labeled anti-hCG antibody Anti-CE antibody to colloidal gold (particle size 1-40u set) is the usual method [
The Journal of Histochemistry and
Cytochemistry (J, Histocha■.

(:ytochem,) 26.1074 (1978)]
A reddish-purple colloidal gold-labeled anti-hCG antibody was obtained.

・Creating a filter Circular filter paper with a diameter of 2 c” (manufactured by Atoppanczuk, NO.
.゛・Test 1 Urine containing 200 and 1000 slU/ml of LCG,
In addition, urine not containing hCG was used as a sample.

Trial 4200JJI and colloidal gold labeled anti-hCG antibody 20
0 Jll was mixed, left at room temperature for several minutes, and then added dropwise to the above Filter-L. When separation by the filter did not occur quickly, a syringe was connected to the filter to apply pressure.

No colloidal gold color was observed on the filter with urine that did not contain hCG, but when urine containing hCG was
Urine containing III/ml has a pale reddish-purple color, but 1000■
A strong reddish-purple color was observed in the urine containing III/ml.

2) Analysis using colored latex as a colored substance and CEA as an analysis target substance Colored latex labeled anti-CEA antibody Colored latex (manufactured by Japan Synthetic Rubber ■, particle size 0.1-1.
The 6 sensitization conditions were as follows.
Anti-CEA mouse monoclonal antibody (1%) 50J+ was added to 0.1 ml of the colored latex suspension (10% W/V), left at room temperature for 4 hours, and then washed with physiological saline.

After washing, treat with fresh blood albumin solution (0.1%),
After further washing with physiological saline, glycine buffer (0.2M
) Float in 20s+l and add colored latex-labeled anti-CE.
A mouse monoclonal antibody was obtained.

・Preparation of filter 0.5 μg/++1 anti-CEA antibody was dropped into the center of a 2-c Peng immunoaffinity membrane (manufactured by Nippon Ball ■, trade name) in a diameter range of 5 to 10 mm, and left at room temperature for 10 hours. After fixation, add skim milk powder to phosphate buffer (
0.2M, pH 7.2) solution (0.5%) and washed several times to obtain a filter.

・Test: 0 colored latex-labeled anti-eEA mouse monoclonal antibody 1 using serum containing 5.20 and 40 ng/ml of CEA and serum without CEA as samples.
Mix 00JII and 400μI of the sample.

After leaving it for 3 minutes, it was dropped onto the antibody-immobilized portion of the filter and the color remaining on the filter was observed.

In serum without CEA, no coloration was observed, but in serum containing CEA, -L' of CEAC degree was observed.
Along with yl, the strong color tone of 7i color latex is 1Bl.
It was noticed.

3) Polynomial 11 using colloidal gold as a colored substance and H) IgG and H) I gM as target substances
Simultaneous analysis/colloidal gold-labeled anti-Human IgG goat antibody and colloidal gold-labeled anti-Human IgM goat antibody Example 1. A colloidal gold-labeled anti-human IgG goat antibody and a colloidal gold-labeled anti-human IgM goat antibody were obtained in the same manner as above.

The concentration of the mixture of colloidal gold-labeled anti-human IgG goat antibody and colloidal gold-labeled anti-human IgM goat antibody (the concentration of colloidal gold has an absorbance of 2.2 in the 540n layer), and the amount of antibody immobilized on the paper are as follows: Human IgG of 1500 mg/d1 or more and human IgM of 150 mg/d1 or more
(both values are considered normal values).

・Production of filter A filter paper having a shape with a notch as shown in Figure 1 (
Example 2. Anti-human IgG rabbit antibody and anti-human IgG rabbit antibody were added to opposing parts across the notch of Whatman (GF/D), each having a diameter of 1c+s. Immobilize in the same manner as.

Antibody-immobilized filter paper was obtained. In order to make this antibody-immobilized filter paper easier to handle, a plastic plate of the same type as the filter paper with a hole of 8 mm in diameter was made at the position corresponding to the antibody-immobilized portion, and a plastic plate of the same type as the filter paper was used. It was sandwiched between the absorbent batts used in the above to make a filter. An exploded perspective view of this filter is shown in FIG.

- Test serum i 00JII was dropped into each of the two holes of the test filter and allowed to completely penetrate. Next, colloidal gold-labeled anti-Human IgG goat antibody and colloidal gold-labeled anti-Human antibody)
A mixture of IgM goat antibodies was dropped into each of the two holes of the filter at 200 JII.

In the part where the anti-human IgG rabbit antibody was immobilized, human Ig
Depending on the grade. In addition, in the area where the anti-Human IgM rabbit antibody was immobilized, coloring due to colloidal gold that corresponded to the Human IgM concentration was observed.

[Effects of the Invention] According to IJI, specific binding analysis can be performed without using special reagents such as a substrate solution, as shown in the Examples and IJI. Therefore, the time required for analysis is shortened, and rapid analysis becomes possible.

Further, as in the example, semi-quantitative measurement is possible simply by reading the appearance of color with the naked eye, and analysis can be performed without using special equipment such as a colorimeter.

Compared to general specific binding analysis methods, such as immunological carrier agglutination reactions, which require skill to read the degree of aggregation of particle carriers, the specific binding analysis method of the present invention performs analysis using a simple operation of color determination. This has the advantage of being able to do the following. Of course, by reading the way colors appear with a color difference meter, etc.

Mechanization of the analysis is also possible.

Furthermore, in the present invention, since the colored substance used as a labeling substance is generally chemically more stable than conventional labeling substances such as enzymes, a reagent with a long life can be provided.

[Brief explanation of the drawing]

FIG. 1 is an exploded perspective view showing one embodiment of a separation means suitable for performing multinomial specific binding analysis according to the present invention. In the figure, 1... P paper, 2... Notch, 3... Antibody immobilization part, 4... Plastic plate, 5... Absorption bat. 6...hole

Claims (1)

[Claims] 1) A specific binding analysis method for measuring or detecting an analyte using a binding reaction with a specific binding substance that has a specific affinity for the analyte, comprising a) a colored A specific binding substance labeled with a substance and an analyte are reacted, and b) a complex of a specific binding substance labeled with a colored substance and an analyte does not pass through, but a specific binding substance labeled with a free colored substance is formed. separating the complex from the reaction solution by a separation means that allows the sexual substance and the analyte to pass through; c) a colored substance derived from the separated complex and/or a specific binding substance labeled with a free colored substance; A specific binding analysis method characterized by: measuring the amount of . 2) Specific binding according to claim 1, wherein the separation means is formed by immobilizing a second specific binding substance that has a specific affinity for the substance to be analyzed. Analysis method. 3) A specific binding analysis method that measures or detects an analyte using a binding reaction with a specific binding substance that has specific affinity for the analyte; b) A certain amount of labeled analyte labeled with a colored substance that has the same affinity for the specific binding substance as the analyte is added to the reaction solution obtained in a). c) separation means that does not pass the complex of the label-analyte and the specific binding substance but allows the free specific binding substance, the analyte, and the label-analyte to pass through; d) measuring the amount of a colored substance derived from the separated complex and/or the free label-analyte. 4) A specific binding analysis method that measures or detects an analyte using a binding reaction with a specific binding substance that has a specific affinity for the analyte, comprising: a) an analyte;
Mix a certain amount of label-analyte labeled with a colored substance that has the same affinity for the specific binding substance as the analyte, and b) add a certain amount of specific binding to the mixture obtained in a). and c) separation means that does not allow the complex of the label-analyte and the specific binding substance to pass through, but allows the free state of the specific binding substance, the analyte, and the label-analyte to pass through. A specific binding analysis method comprising: separating the complex from the reaction solution; and d) measuring the amount of a colored substance derived from the separated complex and/or the label-analyte in a free state. . 5) Claims 3 or 4, characterized in that the separation means is formed by immobilizing a second specific binding substance that has a specific affinity for the specific binding substance. Specific binding analysis method described in . 6) A patent characterized in that the substance to be analyzed is selected from the group consisting of antigens, antibodies, avidin, biotin, sugars, lectins, Fc regions of immunoglobulins, protein A, DNA, and derivatives thereof. Claim 1
4. The specific binding analysis method according to any one of Items 1, 4, and 5. 7) Specific binding substance or label labeled with a colored substance - The analyte in the analyte is an antigen, antibody, avidin, biotin, sugar, lectin, Fc region of immunoglobulin, protein A, DNA, or a derivative thereof. The specific binding analysis method according to any one of claims 1, 3, and 4, characterized in that the specific binding analysis method is selected from the group consisting of: 8) Claim 6 or 7, characterized in that the antibody is one or a combination of two or more selected from the group consisting of polyclonal antibodies, monoclonal antibodies, and multiple types of monoclonal antibodies. Specific binding analysis method described in . 9) The colored substance is a pigment, gold colloid, colored latex,
The specific binding analysis method according to any one of claims 1, 3, and 4, characterized in that the specific binding analysis method is selected from the group consisting of: and colored ribosomes. 10) The specific binding analysis method according to any one of claims 1, 3, and 4, wherein the separation means is a physical separation means. 11) The physical separation means is made of at least one material selected from the group consisting of porous membranes, filter paper, porous ceramics, porous resins, and fibers. Specific binding analysis method according to item 10.