JPS61272659A - Fluorescent immunoassay method - Google Patents

Abstract

PURPOSE:To make quick and exact immune analysis by bringing the antigen or antibody in a sample together with an antibody or antigen labeled with a fluorescent material into reaction with the antibody (antigen) fixed to a porous film then the water in the porous film with a specific org. solvent and measuring the fluorescent intensity of the fluorescent material in the film. CONSTITUTION:The antigen or antibody is deposited on the porous film such as cellulose acetate film and the antigen (antibody) immobilized film is obtd. The film is grasped by a glass ball joint and a tris.glycine buffer soln. is filled on the film. The sample liquid contg. the antibody (antigen) such as human IgG is injected thereto and the antibody (antigen) in the sample is migrated by an electrophoretic method, etc. so as to react with the antigen (antibody) in the film. The water in the film is replaced with the org. solvent such as ethanol having small dipole moment after the reaction. The quantitive determination of a slight amt. of the antigen (antibody) in the sample with high sensitivity is made possible by the replacement even if the fluorescent material such as fluorescein isothiocyanate having the small quantum yield for an excitation wavelength is used. The range for selecting the labeling material is thus considerably expanded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to an immunoassay method, and more particularly to a method for rapidly and accurately quantifying trace biological components using antigen-antibody reactions.

[Background of the invention]

BACKGROUND ART Methods that utilize antigen-antibody reactions to quantify trace amounts of hormones, enzymes, etc. present in blood and the like are widely applied in medical diagnosis and the like. Conventionally, this immunoassay method is 1251
Radioimmunoassay (RIA), which uses reagents in which antigens or antibodies are labeled with radioactive isotopes, has been widely used. On the other hand, immunoassay methods that do not use radioactive substances, ie, methods that use enzymes, bacteriophage, free radicals, chemiluminescent substances, fluorescent substances, etc. as labels, have also been studied and some have been put into practical use.

Among non-radioactive quantitative methods, the present invention relates to a fluorescence immunoassay method (FI) using a fluorescent substance as a label.
A). It is known that FIA is effective as a method for quantifying trace amounts of hormones and other substances. but,
The excitation light and fluorescence wavelengths of fluorescent substances conventionally used as labeling substances for FIA are close to the absorption wavelengths and fluorescence wavelengths of proteins and other blood components, so there is a limit to the improvement of S/N. The reality is that FIA's quantification limits are not as high as RIA's. For example, the maximum excitation wavelength of fluorescein isothiocyanate, which is a typical labeled fluorescent substance, is 492 nm and the maximum fluorescence wavelength is 518 nm.
On the other hand, proteins have wavelengths of 280 nm and 340 nm, respectively.
It has a maximum value at nm. Blood also contains non-protein components that emit fluorescence with a maximum wavelength of 450 to 470 nm. Even if these substances are present in trace amounts, if the detected component is in extremely trace amounts, they become significant interfering substances. Furthermore, if the difference between the excitation wavelength and fluorescence wavelength of the labeling substance, that is, the Stokes shift, is small, scattering of the incident excitation light becomes a problem.

On the other hand, in order to facilitate the separation of antigen-antibody reactants and non-reactants in heterogeneous immunoassays, and to substantially increase the reaction rate by having a concentration effect, a method using a porous membrane on which antibodies are immobilized has been proposed. It has been proposed (Japanese Unexamined Patent Publication No. 6O-57
257). This method requires efficient measurement of labeling substances, such as fluorescent substances, present inside and outside the membrane.

[Purpose of the invention]

An object of the present invention is to provide a method for increasing the sensitivity of fluorescence photometry in fluorescence immunoassay using a porous antibody-immobilized membrane as a site for antigen-antibody reactions.

[Summary of the invention]

In order to improve the sensitivity of fluorescent immunoassays, it is necessary to use superior labeled fluorescent substances. Characteristics required of labeled fluorescent substances include factors such as fluorescence quantum yield, fluorescence wavelength, and Stokes shift. Quantum yield is the most fundamental factor and is preferably close to 1. Generally, frequently used fluorescein isothiocyanate has a pH of 0.65 (pH 7.0) in aqueous solution.
, 0.92 (0, IN NaOH).

Furthermore, it is desirable that the fluorescence wavelength be as long as possible in order to avoid the influence of interfering fluorescence (generally mostly short-wavelength fluorescence) from coexisting substances contained in the sample. Furthermore, it is advantageous for the Stokes shift to be as large as possible in order to remove stray light caused by excitation light. In general, those with fluorescence on the long wavelength side and those with a large Stokes shift have relatively low quantum yields. Particularly when water is used as a medium, small size is a problem.

The present invention provides a method that enables highly efficient fluorescence photometry even when labeled with a fluorescent substance whose quantum yield decreases in water.

When performing an antigen-antibody reaction in a porous membrane and measuring the amount of labeled fluorescent substance captured in the membrane by fluorescence photometry, the refractive index is It is necessary to fill the pores of the membrane with a large liquid. Since fluorescent immunoassay is carried out using water as a medium, water is suitable as the liquid. However, as mentioned above, many fluorescent materials have lower quantum yields in polar media such as water than in less polar media. Table 1 shows the fluorescence properties of fluorescent substances often used in the biological science field. In the table, λ68 is the excitation wavelength, λ1. indicates the fluorescence wavelength. The table shows a comparison of fluorescence wavelength and quantum yield when using water and ethanol as the medium. With some exceptions, replacing water with ethanol significantly improves quantum yield.

Table 1 Characteristics of fluorescent substance
-(2-benzimidazolyl)phenyl)maleimide,
PNM: N-(3-phenanthryl)maleimide, PR
, M: N-(pyrene)maleimide, BTPM: N-(p
-(2-benzothiazolyl)phenyl)maleimide, C
8M: N-(6-crisyl)maleimide, ACM: N-
(3-acridyl)maleimide, PNA: N-phenyl-1-naphthylamine, FAM: N-(3-fluorancyl)maleimide, DACM: N-(7-dimethylamino-4-methylcoumarinyl)maleimide, TNS: 2
-p-toluisonylnaphthalene-6-sulfonic acid, AN
S: 1-anilinonaphthalene-8-sulfonic acid, IAE
ANS: 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, ANM: N-(1-anilinonaphthyl-4)maleimide.

That is, in a fluorescence immunoassay using a porous membrane, very good results can be obtained if the water in the membrane is replaced with a medium less polar than water after the antigen-antibody reaction is completed and the fluorescence is measured. Furthermore, it is desirable that this medium has low polarity and high refractive index.

The refractive index of water at 20°C for the D(Na) line is 1.33:
3, the dipole efficiency is 1.94. Water is easily replaced with highly compatible acetone, alcohol, etc.

Ethanol has been mentioned as a substance that easily replaces water and has physical properties suitable for the present invention, but of course, substances having such properties are not limited to ethanol. Other alcohols and other organic solvents having the above-mentioned properties can be similarly used.

Even more organic solvents are suitable for use in the present invention if the water is not replaced directly with the target medium, but via a water-compatible liquid. For example, decalin, chloroform, cyclohexane, cyclohexene, and many other organic solvents are available.

In order for the organic solvent that replaces water to be used as one suitable for the present invention, it must have the above-mentioned properties and must not cause damage such as dissolving or deforming the porous membrane that is the object to be measured. Of course, this should not happen.

[Embodiments of the invention]

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

Cellulose acetate membrane for electrophoresis (thickness approx. 120 μm)
) in an anti-human IgG antibody (rabbit) solution for about 1 hour, and then soaked in a 2.5% glutaraldehyde solution (P B
S, i.e. 0.1 containing 1/15 M NaCQ
30% in M phosphate buffer p, diluted with H7,4)
After repeating the immersion operation for 3 minutes, the membrane was further immersed in an anti-human IgG antibody solution for about 1 hour, and then thoroughly washed with PBS to prepare an anti-human IgG antibody-immobilized cellulose acetate membrane. A circular membrane with a diameter of 8III11 was cut out from this membrane and used as a reaction membrane, and an immunoassay was performed using the Sand-Deutsch method using human IgG as a measurement target.

The immunoassay method is a known method (Japanese Patent Application Laid-Open No. 60-5725
7) was followed. That is, the membrane was sandwiched between two glass ball joints with an inner diameter of 4 ml, and Tris was placed on top and bottom of the membrane.
Filled with glycine buffer (pH 8,6).

Next, 10 μQ of a human IgG standard sample diluted 2 times with a 40% sucrose solution was gently injected onto the top of the reaction membrane using a microsyringe. Next, electrophoresis was performed for 15 minutes at an applied voltage of 50V. Next, an anti-human IgG antibody (goat) solution (Miles) labeled with ANS (Table 1) was added to the
% sucrose solution and injected onto the top of the reaction membrane, electrophoresis was performed for 15 minutes at an applied voltage of 50 V. After this, take out the reaction membrane and PB-3
After rinsing briefly with water, the fluorescence of ANS present in the reaction membrane was measured at λ, 350nrn and λ, -51-5nm. Next, this membrane was sequentially immersed in 50% ethanol, 75% ethanol, and pure ethanol to replace water with ethanol, and then λ. , 370 nm, λ, 468 nm. The fluorescence intensity ratio in area measurement was about 1:8. For comparison, anti-human IgG was used instead of ANS-labeled antibody.
A sample piece was prepared in the same manner as described above except that the antibody was injected, and fluorescence photometry was performed in the same manner as above. The fluorescence intensity of both was measured using a side-mounted W (Hitachi spectrofluorometer MPF-
4), and no significant difference was observed.

〔Effect of the invention〕

As described above, according to the present invention, a fluorescent substance whose quantum yield significantly decreases in water can be used as a labeling substance for fluorescence immunoassay, and the search range for labeled fluorescent substances can be greatly expanded. .

Claims (1)

[Scope of Claims] 1. The antigen in the sample is immobilized by an antigen-antibody reaction with an antibody immobilized on a porous membrane, and the immobilized antigen is reacted with an antibody labeled with a fluorescent substance, or In the fluorescent immunoassay method, which measures the concentration of the antigen by reacting the unreacted portion of the immobilized antibody with the antigen labeled with a fluorescent substance, after all the antigen-antibody reactions are completed. A fluorescence immunoassay method, which comprises replacing water in the porous membrane with an organic solvent having a dipole efficiency lower than that of water, and then measuring the fluorescence intensity of a labeled fluorescent substance in the porous membrane. 2. The immunoassay method according to claim 1, wherein the organic solvent that replaces water has a higher optical refractive index than water. 3. The immunoassay method according to claim 1 or 2, wherein the organic solvent to be replaced with water is compatible with water. 4. The immunoassay method according to claim 1, wherein the organic solvent to be replaced with water is ethanol.