JPH0750108B2 - Immunoreactivity assay - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunoreaction assay method with high sensitivity and less nonspecific reaction.

(Prior Art) An antigen-antibody reaction is used to detect or quantify a specific substance to be measured from various biological components. For example, the substance to be measured is measured by measuring the agglutination reaction using an antibody against the substance to be measured, which is an antigen or an antibody, or latex carrying an antigen as a reagent. As a method of measuring such an immune reaction, a method of visually observing the agglutination reaction visually or visually as described above,
Various methods such as enzyme immunoassay (EIA) and radioimmunoassay (RIA) are known.

In these methods for measuring an immune reaction, various additives are used for the purpose of promoting an antigen-antibody reaction or effectively measuring a trace component. For example, a method of adding polyethylene glycol or dextran to the reaction system has been adopted. These are water-soluble or hydrophilic polymers, and the addition of these accelerates the antigen-antibody reaction that proceeds by hydrophobic interaction. For example, the agglutination reaction of the latex reagent is promoted by adding these compounds. However, these compounds also promote non-specific reactions in the reaction system, thus increasing the background value. Therefore, it cannot be detected unless the measured value exceeds the background value. That is, a large amount of sample is required. Therefore, although this method can advance the reaction in a short time, it cannot be said to be a method sufficient for suppressing the nonspecific reaction. Furthermore, the commercially available polyethylene glycol used is 290
It often contains impurities with absorption around nm. Therefore, it is necessary to purify before use, and the handling becomes complicated.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and an object of the present invention is to provide an immunoreaction assay method capable of suppressing nonspecific reactions and obtaining high sensitivity. To do.

(Means for Solving the Problems) The immunoreaction measuring method of the present invention is a method for measuring a substance to be measured comprising an antigen or an antibody using an antibody against the antigen or the antibody or an antigen, wherein the reaction of the immune reaction Alkylated polysaccharide is present in the system at a rate of 0.1-0.5% (W / W),
Thereby, the above object is achieved.

The alkylated polysaccharide used in the present invention is a polysaccharide having an alkyl group having 1 to 5 carbon atoms such as a methyl group and an ethyl group, and a water-soluble compound is used. For example, there are methyl cellulose, ethyl cellulose and the like. A suitable molecular weight of the alkylated polysaccharide is 1,000 to 500,000. If the molecular weight is too high, the viscosity when dissolved in water becomes high, making handling difficult. It is also possible to use a combination of two or more alkylated polysaccharides so that the viscosity when dissolved in water is appropriate.

The substance to be measured by the present invention is not particularly limited, and generally, any physiologically active substance that can be measured by utilizing an antigen-antibody reaction can be measured. The substance to be measured includes proteins, lipids, and the like, and examples thereof include various antigens, receptors, enzymes, and the like. Specifically, HB _s antibody, HB _c antibody, syphilis antibody (antibody against treponemal antigen and lipid antigen), anti-HIV (human immunodeficiency virus) antibody, anti-ATLA (adult T cell leukemia-related antigen) antibody, etc.,
Antibodies associated with infectious diseases are included.

The measuring system for measuring the substance to be measured according to the present invention is not particularly limited, and a usual immunoturbidimetric method, EIA, RIA, hemagglutination method and the like can be used. For example, in the case of measuring a substance to be measured by an immunoreaction using a latex reagent, an alkylated polysaccharide is added in advance to a latex reagent containing an antibody against the antigen or antibody as the substance to be measured or an antigen. The substance to be measured can be measured by performing an immune agglutination reaction using such a reagent and optically or visually observing the degree of agglutination that has occurred. Alternatively, a latex containing no alkylated polysaccharide can be used, and in this case, the alkylated polysaccharide may be present in the reaction system during the immune reaction. For example,
There is a method in which the alkylated polysaccharide is added to the sample in advance; a method in which the alkylated polysaccharide is added to the buffer solution used is not particularly limited. In the immunoreaction measuring method of the present invention, a method in which an alkylated polysaccharide is added to a latex reagent in advance is used. Method, the alkylated polysaccharide is added to the reaction system at the time of immune reaction at 0.1 to 0.5% (W / W)
Exist at a rate of.

The conditions of the above-mentioned immunoreaction are the same as in the usual case, and various buffers depending on the type of the substance to be measured are used as the reaction medium. The buffer solution may have any ionic strength or pH that does not inactivate the substance to be measured and does not inhibit the antigen-antibody reaction. During the reaction, choline chloride, EDTA, etc. can be added to further enhance the specificity of the measurement system.

According to the present invention, the action of the alkylated polysaccharide suppresses the nonspecific reaction in the immune reaction, and the substance to be measured can be measured with high sensitivity. Examples of the present invention will be described below, and the effects thereof will be specifically described.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 (Measurement of syphilis antibody by a manual method using an agglutinating plate) An antibody against the Treponema pallidum antigen was measured.
In this example, the following reagents and measurement samples were used. Also in Examples 2 to 6 and Comparative Examples 1 to 6 described below, the same reagents and samples having the same names were used unless otherwise specified.

PBS (Phosphate buffer): monosodium phosphate (dihydrate), disodium phosphate (dihydrate) and sodium chloride at final concentrations of 0.02M and 0.15M of phosphoric acid and sodium chloride, respectively. Preparation was performed by adding to purified water so that the pH was 7.4.

It was prepared by dissolving reagent grade BSA (bovine serum albumin) in 1% BSA / PBS: PBS at a concentration of 1% (W / W).

1% Triton X-100: Triton X-100 in phosphate buffer
Was dissolved to be 1% (W / W).

0.3% methyl cellulose: Methyl cellulose (manufactured by Hanai Chemical Co .; average viscosity 100 cps) was dissolved in 1% BSA / PBS at 0.3% (W / W).

0.3% ethyl cellulose: Ethyl cellulose (manufactured by Hanai Chemical Co., Ltd .; average viscosity 40 cps) was dissolved in 1% BSA / PBS at 0.3% (W / W).

3% polyethylene glycol: 1% BSA / PBS with polyethylene glycol (manufactured by Hanai Chemical Co .; average molecular weight 6000) 3%
It was dissolved so as to become (W / W).

3% Dextran: Dextran (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 1% BSA / PBS at 3% (W / W).

Syphilis antigen solution: Treponema pallidum (CDC (Center folate) cultured in rabbit testis for 10 to 14 days.
r Disease Control, Public Health Service, USDepart
ment of Health, Education and Welfare, Atlanta, Georg
ia) was used to inoculate the rabbit testicles and subcultured, and 1 ml of a cell suspension containing 10 ⁹ cells / ml suspended in physiological saline was taken. It was washed by centrifugation (6,000 rpm × 5 minutes, 3 times) in a phosphate buffer. Then, 1% Triton X-100 was added to the obtained precipitate.
ml was added and incubated at 37 ° C for 30 minutes. Then, this was subjected to an ultracentrifuge (50,000 rpm × 1 hour) to collect the supernatant, which was diluted 1,000 times with 1% Triton X-100 and used.

Syphilis-positive rabbit serum: Serum was collected from rabbits raised for 45 days after inoculation of Treponema paris dam into testes. This serum was diluted 100-fold, 200-fold and 400-fold with 1% BSA / PBS before use.

Normal Rabbit Serum: Treponema sera collected from rabbits not inoculated with Paridam was used. When the titer was measured using a commercially available TPHA kit (Cerodia TP; made by Fujirebio), the result was negative. This serum is 1% BS
It was diluted 100 to 400 times with A / PBS before use.

Latex: polystyrene latex (solid content 10%) having a particle size of 0.23 μm manufactured by Sekisui Chemical Co., Ltd. was used.

(A) Sensitization of syphilis antigen to latex: Latex 100
μ and 400 μ of syphilis antigen solution were rapidly mixed and stirred at room temperature. 1 hour later, add 5% of 1% BSA / PBS, 15000r
It was centrifuged at pm for 1 hour. This was repeated twice to wash the latex. The washed pellet was suspended in 2 ml of 0.3% methyl cellulose or 0.3% ethyl cellulose and well dispersed to obtain a latex reagent having a solid content of 0.5%. This was stored at 4 ° C.

(B) Measurement of syphilis antibody: 50 μm each of syphilis-positive rabbit serum and the latex reagent obtained in (A) were collected on an agglutination observation plate, mixed and stirred, and then reacted for 3 minutes. As a control, the same reaction was performed with normal rabbit serum. The aggregation of the latex reagent was visually observed. Aggregated specimens were defined as positive, and specimens where no aggregation was observed were defined as negative. The results are shown in Table 1. In Table 1, + and ++ are positive (+
+ Indicates a high degree of aggregation), ± indicates a false positive, and-indicates a negative.

Comparative Example 1 Instead of 0.3% methylcellulose, 1% BSA / PBS, 3% polyethylene glycol or 3% dextran was used and a reaction was carried out in the same manner as in Example 1. The results are shown in Table 1.

It is clear from Table 1 that in the latex reagent, the use of the immunoreaction measuring method of the present invention enables accurate measurement of a specimen that produces a false positive in the conventional reagent without causing a nonspecific reaction.

Example 2 (Measurement of syphilis antibody using a fully automatic analyzer) The same samples and reagents as in Example 1 were used to perform the measurement with a Hitachi 7050 fully automatic analyzer. However, latex reagent is methyl cellulose (or ethyl cellulose)
Was added, and diluted with 1% BSA / PBS to adjust the solid content to 0.25%. 0.3% methyl cellulose or 0.3% ethyl cellulose was used as a diluting solution described later. The measurement conditions are as follows.

Sample volume 20μ Latex reagent (R2) 50μ Diluent (R1) 350μ Measurement wavelength 570nm Sample 20μ was taken, and to this was added diluent (R1) 350μ. After adding 50 μl of the latex reagent, the solution was stirred and mixed. And from the addition of the latex reagent
Difference in absorbance at wavelength 570 nm after 80 seconds and 320 seconds (ΔOD
₅₇₀ ) was read by a spectrophotometer, and the amount of change in this absorbance is shown in Table 2 (average value of n = 4). The antigen-antibody reaction was performed at 37 ° C. The relationship between the dilution ratio of syphilis-positive rabbit serum and ΔOD ₅₇₀ is shown in FIG. 1 together with the results of Comparative Example 2. In FIG. 1 and FIG. 2 described later, ● indicates the measured value when methyl cellulose was used, and ○ indicates the measured value when ethyl cellulose was used. In addition, the numerical values of the amount of change in absorbance shown in Table 2 and Table 5 described later are
This is a display of the output value from the 50-type fully automatic analyzer.

Comparative Example 2 The same as Example 2 except that 1% BSA / PBS, 3% polyethylene glycol or 3% dextran was used as the diluent. The results are shown in Table 2 and FIG.

In Fig. 1 and Fig. 2 described later, △ is 1% BSA / PBS,
▲ indicates the measured value when polyethylene glycol (PEG6000) was used, and □ indicates the measured value when 3% dextran was used.

As is clear from the results shown in Table 2 and FIG. 1, according to the present invention, even in a high dilution positive serum in which agglutination was not observed by the conventional method, high sensitivity to the extent that it can be judged as positive can be obtained. It was Furthermore, no aggregation due to non-specific reaction is observed. Thus, highly specific and highly sensitive measurements were achieved.

Example 3 (Measurement of syphilis antibody by ELRSA) In addition to the reagents and samples listed in Example 1, the following reagents and plates were used in this example.

Peroxidase labeled anti-rabbit IgG: Peroxidase labeled anti-rabbit IgG (Miles Laboratories) 1% B
It was diluted 1,000 times with SA / PBS before use.

Peroxidase substrate: o-in phosphate-citrate buffer
Phenylenediamine (dihydrochloride) was added at 2 mg / ml, and hydrogen peroxide solution was added at 0.03% (H ₂ O ₂ ). The substrate was prepared just before use.

(A) Immobilization of antigen: Syphilis antigen solution was dispensed into each well of a microtiter plate by 50 μm and incubated at room temperature for 1 hour. After washing the wells once with 1% BSA / PBS 200μ, add 1% BSA / PBS 200μ and incubate at room temperature for 1
Incubation was carried out for a period of time to perform blocking. Then, 1% BSA / PBS was removed by suction and immediately used for ELISA analysis.

(B) Measurement of syphilis antibody: 1% BSA / PBS was replaced with 0.3% methylcellulose or 0.3% ethylcellulose.
First, syphilis-positive rabbit serum diluted 00,200 and 400 times
An antibody solution was used. Dispense 50μ into each well,
Incubated at room temperature for 1 hour. As a control, a solution prepared by diluting normal rabbit serum in the same manner was prepared, and this was used for similar dispensing and incubation.

After 1 hour, the reaction solution was removed by suction, and 1% BSA / PBS 200μ
After washing 3 times with peroxidase-labeled anti-rabbit Ig
50 μg of G (second antibody solution) was dispensed into each well.
After incubating at room temperature for 1 hour, the reaction solution was removed by suction and washed 3 times with 1% BSA / PBS 200μ.

Immediately, 100 μl of a peroxidase substrate was dispensed into each well and incubated at room temperature for 1 minute. As a substrate blank, a well to which neither the first antibody nor the second antibody was added was prepared, and the substrate solution was added and incubated in the same manner. After the incubation, 1N sulfuric acid was dispensed in 100 μm aliquots to stop the enzymatic reaction. The operation was performed so that the enzyme reaction time in each well was constant. After stopping the reaction, the absorbance at 492 nm was measured by a microtiter plate reader (MTP-100, manufactured by Corona) using the substrate blank as a control. The results are shown in Table 3 (average value of n = 4).

Comparative Example 3 The same as Example 3 except that 1% BSA / PBS, 3% polyethylene glycol or 3% dextran was used as a diluent for syphilis-positive serum. The results are shown in Table 3.

As is clear from the results in Table 3, according to the present invention, it is possible to reduce the nonspecific reaction in normal rabbit serum, which could not be suppressed by the conventional method, between 0 and 0.005 in the absorbance. As for positive sera, as a result of specifically promoting the antigen-antibody reaction, it became possible to improve the sensitivity almost three times or more.

Example 4 In this Example (Measurement of HB _s antibody by manual methods using agglutination plate), the reagent of Example 1 and the same name Example 1
Preparation was performed in the same manner as in. The method for preparing other reagents is shown below.

HB _s antigen solution: A purified HB _s antigen purified from human plasma by affinity chromatography was used. The antigen was dissolved in phosphate buffer and the concentration was measured by the Lowry method. Immediately before use, dilute with phosphate buffer and adjust the concentration to 1
Adjusted to ~10μg / ml, which was used as a HB _s antigen solution.

HB _s- positive rabbit serum: An antiserum obtained by immunizing rabbits with purified HB _s antigen together with Freund's complete adjuvant was used.
The column was used after being absorbed by a column to which normal human serum was bound. As a column to which normal human serum was bound, CNBr-activated Sepharose CL4B (Pharmacia) was used, and the column was prepared according to the manufacturer's (Pharmacia) instructions. Absorbed anti-HB _s serum was 100 to 400 with 1% BSA / PBS.
It was used after diluting twice.

(A) Sensitization of latex with HB _s antigen: latex 100μ
And 400 μl of HB _s antigen solution were rapidly mixed and stirred at room temperature. After 1 hour, 5 ml of 1% BSA / PBS was added and the mixture was centrifuged at 15000 rpm for 1 hour. This was repeated twice to wash the latex. The washed pellet was suspended in 2 ml of 0.3% methyl cellulose or 0.3% ethyl cellulose and well dispersed to obtain a latex reagent having a solid content of 0.5%. This 4
Stored at ° C.

(B) Measurement of HB _s antibody HB _s positive rabbit serum and the latex reagent obtained in the item (A) of this Example were placed on an agglutination observation plate by 50 μm, mixed and stirred, and reacted for 3 minutes. As a control, the same reaction was performed with normal rabbit serum. Aggregation of the latex reagent was performed as in Example 1. The results are shown in Table 4.

Comparative Example 4 A reaction was performed in the same manner as in Example 4 except that 1% BSA / PBS, 3% polyethylene glycol or 3% dextran was used instead of 0.3% methylcellulose. The results are shown in Table 4.

As is clear from Table 4, according to the present invention, it is possible to diagnose with high specificity even a specimen in which a false positive occurs in the conventional method.

Example 5 (Measurement of HB _s antibody using fully automated analyzer) HB _s antibody was measured according to the method of Example 2 using the samples and reagents of Example 4. The latex reagent was also 0.25% as in Example 2, and 0.3% methyl cellulose or 0.3% ethyl cellulose was used as the diluent. The results are shown in Table 5 and FIG.

Comparative Example 5 The same as Example 5 except that 1% BSA / PBS, 3% polyethylene glycol or 3% dextran was used as the diluent. The results are shown in Table 5 and FIG.

As is clear from the results shown in Table 5 and FIG. 2, according to the present invention, even with a high dilution positive serum in which agglutination was not observed by the conventional method, high sensitivity which can be judged as positive is obtained. It was Furthermore, no aggregation due to non-specific reaction is observed. For this reason, highly specific and highly sensitive measurements were achieved.

Example 6 (Measurement of insulin by EIA) In this example, the reagent having the same name as that of Example 1 is the same as that of Example 1.
Preparation was performed in the same manner as in. The following kits and sera were used as EIA kits and samples.

EIA kit for insulin measurement: INSULIN [MITSUI] II (manufactured by Kainos).

High-insulin serum: Serum with high insulin concentration,
Other insulin measurement kits [Insulin B-Test Wako
(Manufactured by Wako Pure Chemical Industries, Ltd.)], the serum confirmed to contain insulin.

Add methylcellulose or ethylcellulose to the enzyme-labeled antibody solution, which is a kit component, to give a final concentration of 0.3.
% (W / W). Using this enzyme-labeled antibody solution, the insulin concentration was measured for high-insulin serum in accordance with the package insert of the kit of Kainos. As a control, other insulin measurement methods (Insulin B
-Test Wako) resulted in low insulin concentration (10 μU / ml)
The low-insulin serum confirmed to be (below) was also measured at the same time. The absorbance (average value of n = 4) at 420 nm is shown in Table 6.

Comparative Example 6 In Example 6, the measurement was carried out when methylcellulose or ethylcellulose was not added to the enzyme-labeled antibody and when polyethyleneglycol or dextran was added in place of methylcellulose or ethylcellulose so that the final concentration was 3%.
The results are shown in Table 6.

As is clear from Table 6, by using the immunoreaction assay method of the present invention, it is possible to suppress the nonspecific reaction and reduce the background value.

(Effect of the Invention) According to the present invention, as described above, in the measurement of the substance to be measured using the immune reaction, the non-specific reaction can be suppressed and the substance to be measured can be measured with high sensitivity. INDUSTRIAL APPLICABILITY The present invention can be used for immunoassays using latex reagents, EIA, RIA, etc., and various physiologically active substances can be effectively measured.

[Brief description of drawings]

FIGS. 1 and 2 is, when the syphilis antibody and HB _s antibody in the serum were measured by the present invention, is a graph showing the results obtained absorbance relationship of serum dilution and measurement.

Claims (1)

[Claims] 1. A method for measuring an immune reaction in which a substance to be measured comprising an antigen or an antibody is measured using an antibody against the antigen or the antibody or the antigen, wherein an alkylated polysaccharide is added in an amount of 0.1 to 0.5 to the reaction system of the immune reaction. % (W / W), which is an immunoreaction measurement method.