JPS63221245A - Method for measuring antigen-antibody reaction - Google Patents

Abstract

PURPOSE:To prevent an increase in the amt. of agglutination with time and to improve the accuracy of measurement by adding a reaction stopping agent to a reactive liquid mixture of the antigen or antibody corresponding to a latex deposited with an antigen or antibody and measuring the quantity of the agglutination reaction of this time. CONSTITUTION:A surfactant as the reaction stopping agent is added to the reactive liquid mixture prepd. by bonding a spacer group to latex particles and mixing the antigen or antibody with the latex having such spacer group. After the agglutination reaction is stopped by the addition of such agent, the quantity of the agglutination reaction is measured. Polystyrene, polymethyl methacrylate, etc., are used as the latex to be deposited with the antigen or antibody. The latex agglutination reaction generated by immune reaction is stopped upon lapse of arbitrary time in the measurement method and, therefore, the change of the amt. of the agglutination with time is prevented and the measurement with high accuracy is permitted.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to a method for measuring an antigen-antibody reaction, and more particularly, to a method for measuring an antigen-antibody reaction using a latex agglutination method with good measurement accuracy and reproducibility. It is something.

<Prior Art> In recent years, so-called immunoassay methods have generally been adopted in the field of clinical testing as a method for detecting antigens or antibodies present in blood, urine, and other body fluids.

Among such immunological measurement methods, test reagents have been developed in which antigens or antibodies are supported on latex made of styrene particles or the like for ease of operation. When these reagents are mixed with the test solution, they cause a binding reaction with the antibodies or antigens present in the test solution, so the amount of reaction can be measured using the latex aggregation phenomenon that occurs in this reaction, and the Measurement involves qualitative or quantitative analysis of the presence or absence of aggregation or the amount of aggregation after a certain period of time using the naked eye or an optical measuring device (Japanese Patent Laid-Open No. 56-255
Publication No. 2, JP-A-58-102156, JP-A-5
9187264, Japanese Unexamined Patent Publication No. 61-65160, etc.).

<Problems to be solved by the invention> However, with the method of qualitatively observing the agglutination reaction with the naked eye using the latex reagent described above, the determination may take too long when a large number of samples are processed simultaneously. , a difference in determination time occurs between each sample, and the amount of agglutination gradually increases. Therefore, there are problems with measurement accuracy and reproducibility. In addition, for some reagents, attempts are being considered to provide quantitative properties by comparing, for example, the agglutination image after 2 minutes and the agglutination image after 4 minutes. When processing a sample, it takes time to make a determination, and it has the disadvantage that it is difficult to ensure measurement accuracy and reproducibility.

On the other hand, measurement methods using optical measurement equipment improve measurement accuracy and reproducibility to some extent, but not only do they require expensive measurement equipment dedicated to agglutination reactions, but they also require simultaneous reaction and measurement within the cell. Therefore, it takes a lot of time to process a large number of samples. Another method is to conduct the reaction in a separate container and only measure the amount of aggregation using an optical device. There is a difference in the determination time between each sample, and there is a risk that the reaction time cannot be set constant, so this method cannot be said to have high measurement accuracy.

Means for Solving the Problems The present inventors have made extensive studies to solve the above problems, and have found that by adding a surfactant as a reaction terminator during or after the aggregation reaction. We have completed the present invention by discovering that it is possible to completely stop the agglutination reaction and prevent the subsequent increase in the amount of agglutination. It is characterized by being able to process quickly and to obtain results with good measurement accuracy and reproducibility.

That is, the present invention involves adding a surfactant as a reaction terminator to a reaction mixture prepared by mixing latex carrying an antigen or antibody and the corresponding antibody or antigen, thereby stopping the agglutination reaction. , relates to a method for measuring an antigen-antibody reaction characterized by measuring the amount of agglutination reaction.

The latex supporting the antigen or antibody used in the present invention is made of synthetic polymers, such as polystyrene, polymethyl methacrylate, styrene-butadiene copolymer, acrylonitrile-methyl methacrylate copolymer, and carboxyl polymers of these polymers. Chemically modified products, aminated modified products, epoxidized modified products, etc. can be used. There is no particular restriction on the particle size of the particles forming the latex, but from the viewpoint of ease of adjusting the amount of antigen or antibody supported and stability of the latex particles, the particle size should be appropriately reduced to allow the antigen or antibody to be prepared. It is preferable to increase the allowable amount that can be supported, and it is desirable to prepare the particles to have a particle size in the range of 0.05 to 2.0 μm.

Conventional methods can be used to support the antigen or antibody on the latex. That is, it can be supported by a physical adsorption method or a chemical bonding method such as a covalent bond or an ionic bond using functional groups present on the surface of latex particles.

When supporting the antigen or antibody using an adsorption method, the antigen or antibody and latex are simply mixed, stirred, and incubated for a desired period of time, and then the antigen or antibody that is not adsorbed is removed by means such as centrifugation. It is possible to obtain latex carrying .

In addition, when supporting using a covalent bond, it is preferable to use a condensing agent such as water-soluble carbodiimide or a crosslinking agent such as geltal V-rudehyde as necessary, and support via a spacer such as polyethyleneimine. This method is a more reliable method since it is possible to prevent the supported antigen or antibody from being detached from the latex particles during the measurement of the antigen-antibody reaction and impairing the reproducibility of the measurement results.

In the conventional method for measuring an antigen-antibody reaction using a latex agglutination reaction, when the latex carrying the antigen or antibody obtained as described above is mixed with a test liquid, the corresponding antibody or antibody is mixed in the test liquid. When an antigen is present, an immune reaction occurs and an aggregation phenomenon between latex particles occurs. Therefore, the antigen-antibody reaction can be measured qualitatively or quantitatively by measuring the amount of aggregates produced in the reaction solution after a certain period of time has elapsed.

However, in the measurement method of the present invention, it is important to effectively stop the latex agglutination reaction caused by the immune reaction after an arbitrary period of time and to prevent changes in the amount of agglutination over time. This method is characterized in that a surfactant is added as a reaction terminator to the reaction mixture solution.

That is, when a surfactant as a reaction terminator is not added, the aggregation reaction proceeds gradually over time, so after reacting for an arbitrary period of time, immediately measure the amount of aggregation visually or using an optical device. However, it has been virtually impossible to process a large number of test liquids at the same time. However, in the present invention, by adding a surfactant, it is possible to effectively stop the latex aggregation reaction, so even if a large number of test liquids are reacted at the same time, the amount of aggregation does not increase over time. , it becomes extremely easy to measure the amount of aggregation without impairing measurement accuracy or reproducibility.

The reason for this is not clear, but in the latex agglutination reaction, the antigen-antibody reaction causes the latex particles to bind to the antibody or antigen in the test solution by sandwiching them, which deteriorates the stability of the latex particles and causes the bonds between the particles to form. It is thought that fusion occurs through small aggregation and large aggregation. but,
In the method of the present invention, by adding a surfactant,
It is presumed that the destabilized latex particles are stabilized and the progress of the antigen-antibody reaction and agglutination reaction after the addition of the active agent is prevented. In addition, by adding a surfactant, it is possible to prevent the so-called non-specific aggregation phenomenon, which is the aggregation of latex particles that occurs regardless of strong aggregation such as antigen-antibody reactions, and prevent specific aggregation caused only by the immune reaction. Ensure the phenomenon.

The type of surfactant mentioned above is not particularly limited, but anionic surfactants, which are said to have a high surfactant ability, are preferably used as they have an excellent reaction-terminating effect. Such anionic surfactants include:
Examples include alkylsulfonic acids, alkylbenzenesulfonic acids, alcoholsulfonic acids, and alkylsulfosuccinic acids. Among these, sodium lauryl sulfate is particularly preferably used as the alkylsulfone M'M. Moreover, nonionic-anionic surfactants can also be suitably used.

The surfactant as a reaction terminator is added in an amount of 0.01 to 10% by weight, preferably 0.05 to 5% by weight, in the reaction mixture of latex and test liquid. However, it is usually added to the test liquid in the form of an aqueous solution with a concentration of 0.1 to 10% by weight. The amount of surfactant added is 0.01
If the amount is less than 10% by weight, depending on the type of surfactant, the effect of stopping the reaction may be weak and the agglutination reaction may not be stopped sufficiently, and if it exceeds 10% by weight, reactants ( Aggregates) may begin to dissociate, creating turbidity and making it difficult to accurately measure the amount of aggregation, or the aggregates may become so crumbled that measurements using optical measuring instruments may no longer be possible. .

As described above, the measurement method of the present invention involves adding a surfactant as a reaction terminator to a reaction mixture prepared by mixing latex carrying an antigen or antibody with the corresponding antibody or antigen. The amount of agglutination reaction is measured by effectively stopping the reaction, and the method for measuring the amount of agglutination may be either a measuring method using the naked eye or a measuring method using an optical measuring device. However, as an accurate quantitative analysis method, it is preferable to use a spectrophotometer or turbidity meter, and in particular, a microplate molded from a resin such as polystyrene and having a plurality of wells (for example, 96 wells) is used. A microplate spectrophotometer that measures absorbance using wells as cells can be suitably used.

<Effects of the present invention> As described above, the method for measuring an antigen-antibody reaction of the present invention involves adding a reaction terminator to a reaction mixture obtained by mixing latex carrying an antigen or antibody and the corresponding antibody or antigen. By adding a surfactant as a surfactant, the agglutination reaction of latex particles can be effectively stopped, and the antigen-
Since the method measures the amount of antibody reaction, a large number of samples can be processed quickly without an increase in the amount of aggregation over time, and the measurement accuracy and reproducibility are good.

<Example> The present invention will be described in more detail with reference to Examples below.

Example 1 (al latex Δ Gosai Methyl methacrylate 8.0 g, isobutyl methacrylate 5.0 g, acrylic acid 1.0 g, methacrylonitrile 3
．． 0 g and triethylene glycol dimethacrylate 0.7 g, and 370 g of distilled water were added, and an aqueous solution of 0.1 g of potassium persulfate dissolved in distilled water Log was added, and the temperature was maintained at 75 ° C. under a nitrogen stream. Polymerization was carried out for about 8 hours, with a polymerization rate of about 99% and an average particle size of 0.30 μm.
of latex was obtained.

This latex was washed with distilled water by centrifugation four times, and then 0.01111 ot/l borate buffer (p
After washing twice with H7.5), the obtained latex particles were redispersed in 0.01 mol/l borate buffer (pH 7°5) so that the solid content concentration was 5% by weight. Ta.

(b) Binding of spacer group to latex grains 100 ml of the latex obtained above was mixed with 100 ml of ε-aminocaproic acid aqueous solution (0.02 mol/I),
The pH was adjusted to 7.5 with IN aqueous sodium hydroxide solution. Next, add 0.01 mo to 200 ml of this aqueous solution.
Add 20 ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (25 μ/ml) dissolved in l/I borate buffer (pH 7,5),
The reaction was allowed to proceed at room temperature for 3 hours with stirring. Thereafter, after being left at 10°C day and night, the particles were washed by centrifugation three times with 0.01 mol/l boric acid buffer (pH 7.5) to obtain latex particles bound with spacer groups. It was redispersed in a buffer solution so that the solid content concentration was 5% by weight.

(C) Preparation of anti-human albumin Human albumin was dissolved in distilled water and mixed with an equal amount of complete Freund's adjuvant to form an emulsion, which was used to immunize rabbits. The mice were immunized three times every two weeks at a dose of 0.1 μ/dose, and then blood was collected from a vein. After separating the serum from the collected saliva, the serum was salted out with 35 deuterated ammonium sulfate aqueous solution, and the precipitate was dialyzed against O, OI mol/I phosphate buffer (pH 7.5).

After dialysis, 0.01 mof/I phosphate buffer (pH 7
The mixture was developed on a DEAE (diethylaminoethyl)-cellulose column equilibrated with 5), and the eluted fraction was designated as an anti-human albumin rabbit IgG fraction.

(d) Preparation of antibody-bound latex 5m Latex with spacer group obtained in (b) above
0.01 mol/I borate buffer (pH 7,
5 > 2 ml and 11 ml of distilled water were mixed, and further 2 nil of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (2 nv/m+) was added, and after 10 minutes, the mixture obtained in (C) above was added. Antibody solution (5■/ml
) 5 ml was added and reacted at 101°C for 2 hours.

Then, 10% by weight of arginine water-soluble tffl (pII
7.5) After adding 5 ml and incubating for 1 hour to consume excess water-soluble carbodiimide in the reaction mixture, add 0.01 mol/1 borate buffer (pH 8,2
) was washed three times by centrifugation, and then redispersed in borate buffer (pH 8, 2) to a solid concentration of 5% by weight to obtain an antibody-bound latex.

(e) Measurement of human albumin Human albumin solution (lμg/ml and 0.1p
g/ml) to 0.01 mol/1 borate buffer (
Dilute 100 times with pH 8°2) and add 100μ of the diluted sample.
0.1 was dispensed into the wells of the microplate, and then 0.1. O
Add 50 μl of the carrier-bound latex of (d) above diluted with 1 mol/I borate buffer (pH 8,2) to a fixed concentration of 0.05%, perform a shaking reaction for 100 seconds, and then % sodium lauryl sulfate solution (pH 8.2
, borate buffer) was added.

On the other hand, instead of sodium lauryl sulfate, the same buffer was used for 7 nights at 50
A control was prepared by adding μl.

Thereafter, changes in absorbance at 630 nm were measured at regular intervals until 1 hour later using a microplate spectrophotometer, and the results are shown in Table 1.

Table 1 As is clear from the results in Table 1 above, the antigen of the present invention
It has been found that the surfactant added in the method for measuring antibody reactions can completely stop the progress of the reaction. Therefore, it is possible to prevent the antigen-antibody reaction from progressing over time, so there is no increase in the amount of aggregation (it is clear that the measurement accuracy is good).

Example 2 Human albumin solution 1 μg/ml was added to O, Ol mol
Diluted 100 times with /l borate buffer (pH 8,2), dispensed 100 μl of the diluted sample into the wells of a microplate, and then diluted with 0.01 mol/l borate buffer (pH 8,2).
50 μm of the (d+ carrier-bound latex of Example 1 diluted to a fixed concentration of 0.05% with H8,2) was added.

After performing a shaking reaction for 100 seconds, 50 μl of each of the various surfactants listed in Table 2 (pH 8.2, borate buffer) was added, and the absorbance at 630 nm was measured for one hour using microplate spectrometry. Measured using a photometer,
The results are shown in Table 1. In addition, the values in Table 2 are obtained by subtracting the values measured by performing the same experiment by adding borate buffer 100111 instead of the 100-fold diluted human albumin solution above from the measured height measurements. It is a value.

Table 2 *1: Neogen R is an anionic interfacial agent that is sodium alkylbenzene sulfonate.

*2: Plysurf used in the examples is Blysurf^212E, which is a nonionic-anionic surfactant that is polyoxyethylene alkylphenyl ether phosphate.

From Table 2 above, it is clear that the antigen-antibody reaction is effectively stopped by the addition of a surfactant.
It is clear that when measuring the amount of aggregation, addition of a surfactant can prevent changes over time and provide good measurement accuracy.

In addition, the amount of surfactant added is o, oot% and 15%.
In this case, the change in absorbance is slightly larger than in other cases, suggesting that the amount added should preferably be within a suitable range.

Claims (2)

[Claims] (1) After stopping the agglutination reaction by adding a surfactant as a reaction terminator to a reaction mixture prepared by mixing latex carrying an antigen or antibody and the corresponding antibody or antigen, the agglutination reaction is stopped. A method for measuring an antigen-antibody reaction, which comprises measuring the amount of reaction. (2) The method for measuring an antigen-antibody reaction according to claim 1, wherein the surfactant is an anionic surfactant.