JPH0943239A - Method for measuring immunity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the wide utilization wherein general usage of the method for measuring immunity is enhanced and nonspecific reaction is lessened by making agglutination accelerator and serum or its modified substance of a normal animal present in a measuring system of antigen-antibody reaction. SOLUTION: An agglutination accelerator is, for instant, preferably polyethylene glycol of mean molecular weight of 1000-50000, and concentration is preferably 0.1-5.0% in a measuring system. Serum of a normal animal does not immunologically have antigen and antibody activity for measuring material, and the serum of the animal, human, a rabbit or the like having received no medicine dosage, and healthy is preferable. It is sufficient only if they exist in the measuring system at suitable concentration of 0.1-10.0%, for instance, the agglutination accelerator and animal serum are previously added to a latex reagent supporting syphilis treponema antigen or the like. Antigen-antibody reaction is performed in a normal condition, the pH of the measuring system is preferably 4.5-10.0, temperature is 0-50 deg.C, for instance, latex agglutination reaction is utilized, and the produced agglutination is measured optically or by visual observation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay method for measuring an antigen or an antibody by an antigen-antibody reaction, and more specifically to an immunoassay method for measuring an anti-syphilis treponemal antibody by an agglutination reaction by the antigen-antibody reaction.

[0002]

2. Description of the Related Art As one of the methods for detecting anti- Treponema pallidum antibody, the antigen of Treponema pallidum is carried on an insoluble carrier, and the degree of aggregation of the insoluble carrier generated by the antigen-antibody reaction with the anti- Treponema pallidum antibody in the sample is detected. There is a method of measuring by doing. Hemagglutination and latex agglutination methods are known as such measurement methods.

As the above-mentioned detection methods, there are a method of visually judging the presence or absence of aggregation, and a method of irradiating the reaction solution with light to measure scattered light or transmitted light. The naked eye is used as a qualitative method or a semi-quantitative method. Has been.

In the measurement of these agglutination reactions, polyethylene glycol (JP-A-58-47256) is used for the purpose of improving the measurement sensitivity or promoting the antigen-antibody reaction.
Polyglycosyl ethyl methacrylate (Japanese Patent Laid-Open No. 4-12
No. 2858), a method of adding a water-soluble polymer to the reaction system has been proposed. However, with water-soluble polymers such as polyethylene glycol and polyglycosyl ethyl methacrylate, there is a problem that aggregation due to reaction of coexisting substances other than the target substance, so-called non-specific aggregation, gives an erroneous measurement result.

In order to prevent the above problems, therefore, a method has been proposed in which immunologically inactive albumins such as bovine serum albumin and equine serum albumin are added to the reaction system (JP-A-58-144748). Gazette). However, the addition of albumin as described above is not sufficient to suppress nonspecific aggregation, and an immunoassay method in which nonspecific aggregation does not occur has been desired.

Further, a method of adding γ-globulin of normal animal or a modified product thereof to the reaction system has been proposed (Japanese Patent Publication No. 61-942). This method is effective in suppressing non-specific aggregation, but in order to purify γ-globulin from serum, column chromatography operation and concentration operation are required, which complicates the work and increases the cost. It had the drawback of not being highly versatile.

[0007]

SUMMARY OF THE INVENTION The present invention is intended to solve the above problems, and an object of the present invention is to provide an immunoassay method with high versatility and low nonspecific reaction.

[0008]

The immunoassay method of the present invention comprises:
An agglutination promoter and normal animal serum or its modified product are present in the measurement system.

The immunoassay method of the present invention 2 is an immunoassay method in which the syphilis treponemal antigen is carried on an insoluble carrier and the degree of aggregation produced by the antigen-antibody reaction with the anti-treponema syphilis antibody is detected to measure the antibody. At
An aggregation promoter and the serum of a normal animal or a modified product thereof are present in the assay system for the antigen-antibody reaction.

The above-mentioned aggregating promoter is added for the purpose of improving the measurement sensitivity and promoting the antigen-antibody reaction in the measurement of agglutination reaction.

The type of the aggregation accelerator is not particularly limited, but for example, polyethylene glycol and polyglycosyl ethyl methacrylate are preferable. The polyethylene glycol has an average molecular weight of 1,000 to 5,000.
The thing of 0 is preferable and the thing of 6000-20000 is more preferable. The polyglycosylethyl methacrylate preferably has an average molecular weight of 100,000 to 1,500,000, more preferably 500,000 to 1,200,000.

If the concentration of the agglutination promoter in the assay system is lowered, the effect of promoting the antigen-antibody reaction becomes insufficient, and if it is increased, the non-specific reaction with a substance other than the target component increases. 0.0% (weight to volume percentage: w / v) is preferable, and more preferably 0.2 to 3.0% (w / v).
It is.

The above-mentioned normal animal serum means a serum that does not immunologically possess an antigen or antibody activity against a target substance to be measured, and is preferably a serum obtained from an animal which is healthy and has not been administered with drugs or the like. . In the present invention, warm-blooded animals,
For example, human, rabbit, goat, sheep, mouse or guinea pig serum is preferable, and rabbit, goat or sheep serum is more preferable.

In the present invention, a modified product of such animal serum can also be used. Various methods such as heat denaturation, ultrasonic denaturation, and organic solvent denaturation can be used to obtain the above-mentioned modified serum product, and the method is not particularly limited. However, heat denaturation is preferable because of its good yield and large effect. Examples of the heat denaturation method include a method of heating the serum of a normal animal without diluting it or diluting it with a buffer solution. Examples of the buffer solution include a phosphate buffer solution, a Tris buffer solution, a glycine buffer solution, an acetate buffer solution, and a citrate buffer solution. The heating temperature is preferably 40 to 80 ° C., and 50
-65 degreeC is more preferable. The heating time depends on the heating temperature. When the temperature is high, the temperature is short, and when the temperature is low, the temperature is long, but the temperature is not particularly limited and may be appropriately determined in consideration of operability.

When the concentration of the serum of the normal animal or its modified product in the assay system is lowered, the effect of suppressing the non-specific reaction is not sufficient, and when it is high, the desired antigen-antibody reaction is suppressed, so the concentration is 0.1-10. 0.0% (volume to volume percentage: v / v)
Is preferable, and 0.5 to 5.0% is more preferable.

In the immunoassay method of the present invention, the aggregation promoter and the serum of normal animal or its modified product may be present in the assay system. For example, in the case of antigen-antibody reaction in a reagent using latex as a carrier. , A latex reagent carrying Treponema pallidum antigen, etc. in advance with an aggregation promoter and
Serum of normal animal or its modified product is added.

It is also possible to use a latex reagent which does not contain these, and in this case, these may be present in the reaction system during the antigen-antibody reaction. For example,
There is a method of adding an aggregating promoter and a serum of normal animal or a modified product thereof to a sample in advance, a method of adding these to a buffer solution to be used, etc., and there is no particular limitation.

In other words, the measuring reagent of the present invention is, for example, a one-component type reagent containing an insoluble carrier carrying syphilis treponemal antigen and the like and the above substance; a first reagent containing an insoluble carrier carrying syphilis treponemal antigen and the like. And a second reagent composed of a second reagent composed of a buffer solution containing the above substance;

The above-mentioned antigen-antibody reaction is carried out under normal conditions,
Examples of the reaction medium include a phosphate buffer, a citrate buffer, a glycine buffer, a Tris buffer and the like, as well as a PIPES buffer which is a buffer using an aminoethanesulfonic acid derivative or an aminopropanesulfonic acid derivative, or HEP
ES buffer is used. The pH of the measurement system is 4.5-1
0.0 is preferable, and 5.8 to 8.0 is more preferable. The temperature of the measurement system is preferably 0 to 50 ° C, more preferably 20 to 40 ° C. The reaction time is appropriately determined.

Examples of the insoluble carrier include organic polymer powder, inorganic powder, microorganisms, blood cells and cell membrane pieces,
Examples include plastic microtiter plates. Examples of the organic polymer powder include natural polymer powders such as insoluble agarose, cellulose and insoluble dextran, polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid. Ester copolymer,
Examples thereof include synthetic polymer powders such as vinyl acetate-acrylic acid ester copolymers, and a latex obtained by uniformly suspending the synthetic polymer powder in water is preferable.

As the above-mentioned inorganic substance powder, for example, gold,
Metal pieces such as titanium, iron, nickel, silica, alumina,
Carbon powder etc. are mentioned. The average particle size of the insoluble carrier varies depending on the measuring method and measuring instrument, but is 0.05 to
Those having a thickness of 1.0 μm are usually used.

Examples of the method of supporting Treponema pallidum antigen on the insoluble carrier include a method of sensitizing by chemical or physical binding. Examples of the above-mentioned antigens include crushed bacterial cells, purified products, artificially synthesized by gene recombination, and the like, and these may be used alone or in combination.

As a measuring system for measuring an antigen-antibody reaction using the above-mentioned reagent, for example, latex agglutination reaction, hemagglutination reaction and the like are used. Examples of the method for measuring the aggregation generated by the above reaction include a method of optically observing the degree of aggregation and a method of visually observing the degree.

Specifically, in the optical observation method, the measurement is carried out by mixing a sample, a sample diluent and an insoluble carrier carrying an anti-syphilis treponemal antibody, and then measuring the scattered light intensity and the absorbance of the mixture. Alternatively, the transmitted light intensity is detected. The measurement wavelength may be 300 to 2400 nm, and the measurement method is according to a known method, such as the particle size and concentration of the insoluble carrier to be used,
The reaction time measures the increase or decrease in scattered light intensity, absorbance, or transmitted light intensity. Further, these methods may be used alone or in combination.

In the visual observation method, usually, a sample and a solution containing an insoluble carrier carrying an antigen are mixed on a determination plate and shaken, and then the presence or absence of aggregation is determined. The determination can be made not only by the naked eye but also by photographing with a video camera and performing image processing.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. (Example 1) [Preparation of syphilis treponemal antigen-bearing latex solution] 400 μm of 100 mM phosphate buffer (pH 7.4) containing syphilis treponemal antigen at a protein concentration of 15 μg / ml
l is 100 μl of polystyrene latex having an average particle diameter of 400 μm (solid content 10% (w / v), manufactured by Sekisui Chemical Co., Ltd.)
And stirred for 1 hour. Next, containing 1% (w / v) of bovine serum albumin (hereinafter referred to as "BSA") 1
2 ml of 00 mM phosphate buffer (pH 7.4) was added,
Stir for 1.5 hours. This solution is heated at 10 ° C for 30 minutes, 18
Centrifuged at 000 rpm. BSA was added to the obtained precipitate.
Was added to 5 ml of a 100 mM phosphate buffer (pH 7.4) containing 0.25% (w / v), and the latex was suspended to prepare a syphilis treponemal antigen-bearing latex solution (first reagent).

[Preparation of Sample Dilution Solution] 90% of 100 mM phosphate buffer (pH 7.4) contains 2.5% (w / v) of polyethylene glycol 6000 (PEG6000, average molecular weight 7500, manufactured by Nacalai Tesque). , And then add and mix 5 ml of normal rabbit serum,
A sample diluent was prepared (second reagent). [Anti-syphilis treponemal antibody measuring reagent] The anti-syphilis treponemal antibody measuring reagent of the present example is a two-part liquid consisting of a first reagent consisting of the Treponema pallidum antigen-bearing latex liquid and a second reagent consisting of the sample diluent. It is a system reagent.

[Measurement method of anti- Treponema pallidum antibody] Biochemical automatic analyzer (Hitachi 7050 type, manufactured by Hitachi, Ltd.)
The amount of anti- Treponema pallidum antibody in human serum was measured by the following method. Measurement conditions are temperature 37 ℃, wavelength 570
nm. For the measurement, 20 μl of the standard solution or the sample (human serum: the amount of anti-syphilis treponemal antibody is unknown) was dispensed, immediately after the addition and mixing of 350 μl of the sample diluent (second reagent), 5 minutes later, the latex solution (first Reagent) 50 μl was added and mixed. 80 seconds to 320 after adding latex liquid
The amount of anti- Treponema pallidum antibody in the sample was determined from the amount of change in absorbance during the second period and the calibration curve of the amount of change in absorbance and the amount of antibody in a standard solution with a known antibody amount (standard anti- Treponema pallidum antibody solution).

Example 2 Instead of dissolving polyethylene glycol 6000 at 2.5% (w / v) in the preparation of the sample diluent in Example 1, polyglycosyl ethyl methacrylate (pGEM) was used.
A, average molecular weight 1.1 million, manufactured by Nippon Seika) 1.0%
Example 1 except that it was dissolved to give (w / v)
The measurement was performed in the same manner as described above.

(Example 3) Measurement was carried out in the same manner as in Example 1 except that normal goat serum was used instead of normal rabbit serum as the sample diluent.

Example 4 Instead of dissolving polyethylene glycol 6000 to 2.5% (w / v) in the preparation of the sample diluent in Example 1, polyglycosyl ethyl methacrylate (pGEM) was used.
A, average molecular weight 1.1 million, manufactured by Nippon Seika) 1.0%
The measurement was carried out in the same manner as in Example 1 except that the solution was dissolved so that it had a (w / v) ratio, and that normal goat serum was used instead of normal rabbit serum.

(Example 5) Measurement was carried out in the same manner as in Example 1 except that normal rabbit serum denatured by heating at 60 ° C for 30 minutes was used instead of the normal rabbit serum of the sample diluent. .

(Example 6) Measurement was carried out in the same manner as in Example 1 except that normal goat serum which had been denatured by heating at 60 ° C for 30 minutes was used instead of normal rabbit serum as a sample diluent. .

Example 7 Instead of dissolving 2.5% (w / v) of polyethylene glycol 6000 in the preparation of the sample diluent in Example 1, polyglycosyl ethyl methacrylate (pGEM) was used.
A, average molecular weight 1.1 million, manufactured by Nippon Seika) 1.0%
(W / v) was dissolved, and instead of normal rabbit serum, normal rabbit serum denatured by heating at 60 ° C. for 30 minutes was used. went.

(Example 8) In the preparation of the sample diluent in Example 1, instead of dissolving polyethylene glycol 6000 to 2.5% (w / v), polyglycosyl ethyl methacrylate (pGEM) was used.
A, average molecular weight 1.1 million, manufactured by Nippon Seika) 1.0%
(W / v) was dissolved, and instead of normal rabbit serum, normal goat serum denatured by heating at 60 ° C. for 30 minutes was used. went.

Comparative Example 1 Measurement was carried out in the same manner as in Example 1 except that normal rabbit serum was not added in the preparation of the sample diluent in Example 1.

Comparative Example 2 Measurement was carried out in the same manner as in Example 1 except that polyethylene glycol 6000 was not added in the preparation of the sample diluent in Example 1.

Comparative Example 3 Example 1 was repeated except that polyethylene glycol 6000 and normal rabbit serum were not added in the preparation of the sample diluent in Example 1.
The measurement was performed in the same manner as described above.

[Measurement of Nonspecific Reaction Inhibition Rate] When human serum of unknown anti-syphilis treponemal antibody amount was measured according to Comparative Example 1, the anti-syphilis treponemal antibody amount was 10 T. U. (Titer unit) Serum showing the above value is positive for syphilis, 10
T. U. Serum showing a value of less than is judged to be negative for syphilis.
The nonspecific reaction inhibition rate was measured by the following method. First, according to Comparative Example 1, the amount of anti- Treponema pallidum antibody was determined according to Comparative Example 1 with respect to 200 normal human serum samples confirmed to contain no anti- Treponema pallidum antibody with the existing reagent for measuring Treponemal anti-syphilis antibody (Cellodia TPHA manufactured by Fujirebio Inc.). Was measured. As a result, 10 T. U.
The above values are shown, and 10 samples (Sample No. 1, Sample No. 2,
Sample No.3, Sample No.4, Sample No.5, Sample No.6, Sample No.7,
Sample No.8, Sample No.9, and Sample No.10) were selected.

The amount of anti- Treponema pallidum antibody was measured in accordance with Examples 1 to 8 for the 10 samples selected as described above. The results are shown in Table 1. The measurement results obtained according to the above Comparative Example 1 are also shown in Table 1.

For each example, 10 out of 10 specimens
T. U. The ratio of the specimens showing the measured value of less than was determined and shown in Table 1 as the nonspecific reaction inhibition rate.

[0042]

[Table 1]

From Table 1, when measured according to Comparative Example 1,
It can be seen that a sample which is apparently positive for syphilis due to the non-specific reaction is highly suppressed for the non-specific reaction when measured according to Examples 1 to 8 and is correctly determined as negative for syphilis.

Since Comparative Examples 2 and 3 did not contain an aggregation promoter, the sensitivity required for the measurement was not obtained by this measuring method, and the amount of anti- Treponema pallidum antibody could not be measured. Therefore, the measurement results for Comparative Example 2 and Comparative Example 3 are omitted.

[0045]

The immunoassay method of the present invention is as described above, and it is not necessary to purify γ-globulin from serum, and an appropriate one is selected from aggregation promoters and normal animal serum or its modified products. As a result, it can be widely used as an immunoassay method with high versatility and low nonspecific reaction. The present invention
For example, a carrier such as latex or erythrocyte is used and can be widely applied as a diagnostic agent by a latex agglutination method or a hemagglutination method utilizing an antigen-antibody reaction. The immunoassay for detecting the anti- Treponema pallidum antibody of the present invention 2 is as described above,
Widely used as a syphilis assay with high versatility and low non-specific reaction by selecting appropriate one from aggregation promoter and normal animal serum or its modified products without the need to purify γ-globulin from serum. it can. INDUSTRIAL APPLICABILITY The present invention 2 can be applied as a syphilis diagnostic agent by a latex agglutination method utilizing an antigen-antibody reaction or a hemagglutination method using a carrier such as latex or erythrocyte, and the reagent is a clinical test for the diagnosis and treatment of syphilis. Can be widely used in fields such as.

Claims (2)

[Claims] 1. An immunoassay for measuring an antigen or an antibody by an antigen-antibody reaction, wherein an aggregation-promoting agent and serum of a normal animal or a modified product thereof are present in the assay system for the antigen-antibody reaction. Immunoassay. 2. An immunoassay in which the antibody is measured by carrying the syphilis treponemal antigen on an insoluble carrier and detecting the degree of aggregation of the insoluble carrier produced by the antigen-antibody reaction with the anti-treponemal syphilis antibody. The immunoassay method according to claim 1, wherein an agglutination promoter and serum of a normal animal or a modified product thereof are present in the assay system for antibody reaction.