JPH02195258A - Immunological measurement method - Google Patents

Abstract

PURPOSE:To determine very small amount of components contained in living body fluid or the like with high accuracy by using a solid phase coupled with an antibody via 'factor to be coupled with solid phase.' CONSTITUTION:As a method to stabilize a 'factor to be coupled with solid phase' to a solid phase, a method for coupling sufficient amount of said factors with the solid phase will do wherein physical adsorption and chemical coupling such as covalent binding is normally utilized. In physical adsorption method for example, polystyrene beads or the like are put into solution of said factors having excessive concentration and if the solution is left for certain amount of time, sufficient amount of said factors can be physically fixed on the polystyrene beads or the like. By constantly moving the solution of said factors at the time of stabilizing, said factors can be uniformly fixed on the surface of the solid phase.

Description

[Detailed Description of the Invention] [Field of Application] The present invention relates to an immunoassay method, and more particularly to a method for quantifying an antigen using a labeling substance for an antigen and a solid phase on which an antibody corresponding to the antigen is immobilized. , concerning an immunoassay method using a so-called competitive method. According to the present invention, hormones and other trace components contained in biological body fluids can be quantified with high sensitivity, and can be widely applied to basic medicine and clinical medicine.

[Prior art]

In recent years, various immunoassay methods have been developed and widely applied as methods for quantifying biological components and other trace components. In particular, the development of immunoassay methods using insoluble carriers, ie, solid phases, is remarkable. However, in immunoassays using antibodies immobilized on a solid phase, the so-called sandwich method is mainstream, and immunoassays using competitive methods are relatively rarely applied. The reason for this is that in immunoassays using the Sand-Deutsche method, it is sufficient to insolubilize a sufficient amount of antibody on the solid phase, whereas in immunoassays using competitive methods, the antibody-immobilized solid is required to carry out the measurement in an appropriate measurement area. This is thought to be because there is a technical difficulty in preparing the phase in that a specific amount of antibody must be insolubilized in the solid phase, and the sensitivity is also not fully satisfactory.

[Problem that the invention seeks to solve]

The present inventors have completed the present invention after intensive studies to solve the problems in manufacturing the antibody-immobilized solid phase and the measurement sensitivity in the competitive immunoassay as described above. That is, the present invention relates to an immunoassay method that solves the above-mentioned problems in competitive immunoassay methods. More specifically, the present invention relates to an immunoassay method using a competitive method, which is characterized by using an antibody-immobilized solid phase in which an antibody is bound to the solid phase via a "factor that binds to the solid phase."

[Means for solving the problems and their effects] The immunoassay method of the present invention competitively binds an antigen to be measured and a labeling substance for the antigen to an antibody immobilized on a solid phase, and finally This is a method of measuring a labeled substance bound to a solid phase, a so-called competitive immunoassay method in the solid phase method.

Labeling substances include radioactive isotopes, enzymes, fluorescent substances 1
There are luminescent substances, metals, etc. More specifically, IJ, 1
11, peroxidase, β-D-galactosidase, alkaline phosphatase, 4-methylumbelliferone, and fluorescein. A known method is used for binding the antigen and the labeling substance. For example, when using a radioactive isotope, reagents such as chloramine T and iodogen (manufactured by Pierce, Inc., registered trademark) are used, and when using an enzyme, a coupling method using a bifunctional reagent is used.

Periodic acid oxidation method etc. can be applied.

Antigens to be measured include various hormones, proteins, vitamins, and drugs, such as thyroid hormone, insulin, steroid hormone L, various carcinoembryonic antigens, vitamin D, and various antibiotics 1 and antiepileptic drugs.

As the "factor that binds to the solid phase," an antibody against the antibody, that is, a second antibody, is often used.1. Other examples include protein A, protein G, and various lectins derived from living organisms. Considering the purpose of the present invention, it is preferable that the factor bound to the solid phase be highly purified.

The solid phase used to immobilize this ``factor that binds to the solid phase'' includes various polysaccharide gels, particles made of synthetic resins such as polystyrene, balls, and test tubes.

In addition to other small containers, similar forms of glass, metal, etc.
Synthetic thin films such as nitrocellulose and nylon are suitable.

A method for immobilizing a "factor that binds to a solid phase" may be any method that binds a sufficient amount of the factor to the solid phase, but usually chemical adsorption such as physical adsorption or covalent bonding is used. Joins are available. For example, in the physical adsorption method, a sufficient amount of the factor can be physically immobilized on the polystyrene beads by placing polystyrene beads etc. in a solution of the factor at an excessive concentration and leaving it for a certain period of time. The reaction temperature is preferably between 2°C and 40°C, and the reaction time is sufficient between 10 minutes and 24 hours. The reaction p)l is preferably from 4 to 8. At this time, the factor can be uniformly immobilized on the solid phase surface by constantly moving the solution of the factor during immobilization. In the chemical bonding method, various coupling reagents such as cyanogen bromide, glutaraldehyde, sodium periodate, carbodiimide derivatives, silane agents, maleimide derivatives, and succinimide esters are used. Oxirane compounds, tosyl chloride, carbonylimidazole, etc. are used.

If necessary, after binding a sufficient amount of the above-mentioned "factor that binds to the solid phase" to the solid phase, the unreacted reactive groups and surface of the solid phase are treated with various proteins, amino acids, amines, etc. to initiate an immune reaction. It is also possible to suppress nonspecific reactions during .

Next, the antibody is immobilized on the solid phase on which the thus obtained "factor that binds to the solid phase" has been immobilized via the factor. It goes without saying that the antibodies used here must specifically bind to the antigen to be measured, but usually various animals, such as rabbits, goats, guinea pigs, pigs, and chickens, are immunized with the antigen. A purified version of the obtained antiserum is used. Furthermore, monoclonal antibodies produced by fusion cells of B cells and myeloma cells, ie, hybridomas, may also be used; monoclonal antibodies are preferable to the polyclonal antibodies mentioned above in order to obtain antibodies of a constant quality. These antibodies are purified as immunoglobulin fractions by methods such as salting out, gel filtration, ion exchange chromatography, and affinity chromatography.

The antibody used can be the immunoglobulin fraction itself prepared as described above, but it can also be used as a fraction such as F(ab')g, Fab', or Fab in which only the antigen-binding site has been isolated. can.

The amount of antibody immobilized on the solid phase bound to the "factor that binds to the solid phase" is varied in order to arbitrarily set the measurement range depending on the concentration of the antigen in the sample to be measured. The above-mentioned “
The amount of antibody to be bound to the solid phase bound to the solid phase binding factor is usually from lng to 3 ng in the reaction solution used for one reaction.
Adjust so that it becomes 00ng.

In order to bind a specific amount of the above antibody to the "factor that binds to the solid phase" immobilized on the solid phase, a specific amount of the antibody dissolved in an appropriate buffer solution is combined with the "factor that binds to the solid phase". The bound solid phase is allowed to react for a certain period of time.

The reaction temperature is preferably between 2°C and 40°C, and the reaction time is sufficient between 10 minutes and 24 hours. reaction p
H is preferably 4 to 8.

The immunoassay method of the present invention, which uses the thus produced solid phase in which a specific amount of antibody is immobilized on the solid phase via a "factor that binds to the solid phase," has the following effects. .

(1) The concentration range in which the antigen is measured can be freely adjusted.

(2) The antigen and antibody have good reactivity and can be measured in a short time.

(3) Highly sensitive measurements are possible compared to when antibodies are directly immobilized on a solid phase.

(4) Measurement accuracy is improved because a certain amount of antibody can be uniformly immobilized on the solid phase.

This will be explained in more detail in Examples below.

Method using antibodies as "factors that bind to solid phase" (a) Production of second antibody-immobilized polystyrene beads An immunoglobulin fraction was obtained from anti-mouse IgG serum by ammonium sulfate salting out and DEAE-cellulose chromatography. . This was applied to a column of mouse IgG insolubilized Sepharose, and after washing the column, 0.1M glycine-HCl buffer (p
Specific antibodies against mouse IgG were eluted using H2,5).

The obtained specific antibody was dissolved in 0.1M phosphate buffer (pl+7) so that the absorbance at 280 nm was 0.2,
Add 200 polystyrene beads (174 inches in diameter) to this
The mixture was placed at 30°C for 1 hour with occasional stirring.

The polystyrene beads were washed with the above phosphate buffer containing 0.1% bovine serum albumin and stored in the same buffer.

(b) Production of triodothyronine (hereinafter referred to as T3) antibody-immobilized solid phase (2 types) IgG fraction of monoclonal antibody against T was added to 0.1 M phosphate buffer (pH 7) at 50 ng/ml and 200
(a
) Second antibody-immobilized polystyrene beads 100
They were placed one at a time and left at 30°C for 1 hour. The polystyrene beads were then washed to obtain two solid phases for identifying T and I antibodies.

(c) Measurement of T. Mix 50 μl of a standard solution of T (0,37,5,75,150,300°600 ng/a) and 400 μl of a solution of T labeled with peroxidase, and (b)
The two kinds of T3 antibody-immobilized solid phases obtained in step 1 were added, and an immunoreaction was performed at 37°C for 30 minutes. After washing the solid phase, the peroxidase activity bound to the solid phase was measured using orthophenylenediamine and H,0□ as enzyme substrates (A4qzn
(Measure the absorbance of m). The resulting calibration curve is shown in FIG. This shows that the measurement range can be adjusted by changing the amount of T and antibody immobilized on the solid phase. That is, when using T prepared using a solution dissolved at a concentration of 50 ng/-, and using an antibody-immobilized surface phase, the measurement range was O to 300 ng/d1 (
(marked 0 in Figure 1), and the measurement range was 37.5 to 600 ng/mff1 when using T prepared using a solution dissolved at a concentration of 200 ng/mff1 and an antibody-immobilized solid phase.
di (marked with * in Figure 1). In other words, the measurement range can be selected by using a T prepared by specifying the amount of antibody to be bound and an antibody-immobilized solid phase.

! Measurement Accuracy in LifLL T3 Measurement The Ts monoclonal antibody was directly immobilized on polystyrene beads (hereinafter referred to as direct immobilization solid phase) using the T3 monoclonal antibody dissolution Kl (30 μs/1ffi). ). U
T was measured by the ±1 method. T s Ong/ml
! When the standard solution was measured 10 times using each solid phase, the results shown in Table 1 were obtained.

(Left below) As shown in Table 1, the average absorbance of the directly immobilized solid phase is 0.4.
10. The absorbance variation (CV) was 9.92%,
In the case of the indirect immobilization solid phase, the average absorbance was 0.880°, and the absorbance variation (CV) was 2.04%. In other words, the method using an indirect immobilized solid phase has smaller measurement variations than the measurement using a directly immobilized solid phase, and it is also possible to manufacture the T and antibody immobilized solid phase using a low concentration T and antibody solution. Despite this, higher absorbance can be obtained than measurements using directly immobilized solid phases.

This means that the reactivity of the T3 antibody on the solid phase toward the antigen or antigen labeling substance in the measurement using the indirectly immobilized solid phase according to the method of the present invention is better than that in the method using the T3 antibody on the directly immobilized solid phase. I understand that.

Step 1 - Measurement of thyroxine T4 Add 100 anti-heron IgG antibody-immobilized polystyrene beads prepared according to (a) of W soil to a solution (500 ng/d) of rabbit-derived I4 antibody purified by affinity chromatography. An I4 antibody-immobilized solid phase was prepared according to (b) of Example 1.

Add I4 antibody-immobilized solid phase labeled with β-D-galactosidase to 50 μl of I4 standard solution (0, 1, 25, 2, 5, 5, 0, 10° 20 μg/d1), and add m soil (C).
The reaction was carried out in the same manner. After washing the solid phase, the enzyme activity bound to the solid phase was measured using orthonitrophenyl-β-D-galactozide as an enzyme substrate, and the calibration curve shown in FIG. 2 was obtained. Note that the measurement was repeated twice.

Measurement of I3 using M''''I Using I3 labeled with I25!, T was measured according to the method used in Family 1 history (however, T, antibody 200ng/d
(using a solid phase prepared in ). When radioactivity bound to the solid phase was measured using a gamma counter, the minimum measurement sensitivity was 2.
It was possible to measure up to a maximum concentration of 300 ng/4e at 0 ng/d1.

m↓ Method using protein A as a "factor that binds to the solid phase" Protein A-immobilized polystyrene beads were prepared using a solution of protein A (0,1/d) according to the method of (a) in the previous study. Manufactured. Using this, I4 according to the leader of the group
An antibody-immobilized solid phase was prepared, and I4 was measured in the same manner as in Sample I. As a result, a calibration curve almost the same as that shown in FIG. 2 was obtained.

〔Effect of the invention〕

According to the present invention, an immunoassay method using a competitive method can be set according to the required measurement range of trace components contained in biological body fluids and other fluids, and has the effect of enabling more accurate measurement and greatly increasing the reactivity of antibodies to antigens. be.

[Brief explanation of the drawing]

Figure 1 shows the calibration curve of triodothyronine in IL Example 1, and in the figure, 10- means 50n.
The calibration curve is shown when using a T3 antibody-immobilized solid phase prepared using a solution dissolved at a concentration of g/m, and -・- is 2
A calibration curve is shown when using T and an antibody-immobilized solid phase prepared using a solution dissolved at a concentration of 00 ng/Id. FIG. 2 shows the calibration curve for thyroxine in chiasmitis 1.

Claims (1)

[Claims] 1) In an antigen quantitative method using an antigen labeling substance and a solid phase on which an antibody corresponding to the antigen is immobilized, an arbitrary amount of antibody is immobilized on a solid phase on which a sufficient amount of "factor that binds to the solid phase" is immobilized. An immunoassay method characterized by using a solid phase immobilized via the factor.