JPH01121756A - Method for measuring antigen-antibody reaction - Google Patents

Abstract

PURPOSE:To rapidly and easily measure many specimens by shielding a part of the base of a vessel with a freely attachable and detachable shielding material and executing antigen-antibody reaction, precipitation of agglutinated matter and optical reading within the same optical cell. CONSTITUTION:A part of the base in the bottom of the vessel forming a microplate 3 is shielded with the shielding material 2 of a freely attachable and detachable cap 2 and the antigens or antibodies deposited on insoluble carrier particles 3 are brought into reaction with the antibodies or antigens under heating in a liquid solvent which serves as a buffer soln. The agglutinated matter of the positive specimen precipitates on the non-shielded part at the base of the plate 3 and the insoluble carriers of the negative specimen are suspended in an unreacted state. The positive specimen is, therefore, discriminated by a decrease in optical density if the cap 1 is removed and optical reading is executed. As a result, the reaction of the antigens and antibodies, the precipitation of the agglutinated matter and the optical reading are executed within the same optical cell. The many specimens are thus rapidly and easily measured.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the treatment of AIDS (AIDS: Acquired Immune Deficiency Syndrome).
The present invention relates to a method for rapidly measuring antigen-antibody reactions that can measure the presence of antibodies, herpesvirus antibodies, ATL (adult T-cell leukemia) antibodies, etc. in a short time and can be widely used in AIDS diagnosis and the like.

[Conventional technology]

Various methods for detecting antibodies against various viruses have been proposed. For example, enzyme immunoassay, gelatin particle agglutination method, and Western blot method are methods for determining whether a person is infected with the AIDS virus by measuring antibodies produced in the body of a person infected with the AIDS virus. The method requires 30 minutes to 3 hours for determination, regardless of the number of specimens, that is, even if only one specimen is used, and in the case of a long test, it takes more than one day. However, when urgently checking blood for transfusion, etc., these methods have the problem of being too time consuming.

Therefore, in order to solve the above problem, one of the inventors of the present invention made synthetic rubber latex carry an AIDS antigen and reacted it with the serum of a subject. We developed a latex agglutination method that allows rapid AIDS testing by agglutinating the latex, and presented it at the 35th Japan Society of Blood Transfusion (March 26, 1988).

On the other hand, complement fixation reaction is commonly used to measure anti-herpes simplex virus antibodies, but this method has problems in terms of specificity. Therefore, virus neutralization reaction or fluorescent antibody staining are excellent ways to increase specificity, but both require tissue culture techniques and equipment, and have the problem of being difficult to implement in a membrane testing laboratory.

Furthermore, a rapid measurement method for ATL is also desired.

[Problem that the invention seeks to solve]

One of the inventors of the present invention previously developed a method for rapidly measuring antigen-antibody reactions that can be applied to various antigen-antibody reactions and can measure a large amount of analytes in a short time. We have proposed a method for measuring antigen-antibody reactions, which is characterized by reacting antibodies in a liquid medium at room temperature, precipitating a reaction product, and then measuring the optical change in the reaction solution.

According to this method, for example, reactions in a 96-well microplate and precipitation of aggregates can be easily carried out. However, when optically reading the supernatant, it is necessary to transfer the supernatant to another container, and further improvements have been desired as a method for processing multiple samples.

[Means for solving problems]

The present invention aims to solve this problem and provide a measurement method that allows reaction, sedimentation of aggregates and optical reading to all be carried out in the same porous optical cell. be.

According to the studies conducted by the present inventors, the aggregates produced by the reaction of antigens or antibodies supported on insoluble carriers with the corresponding antibodies or antigens have the property of being easily sedimented, and can be obtained by centrifugation or natural When sedimented by a sedimentation method or the like, the sediment tends to stick to the bottom of the container and does not easily diffuse once it has settled.

On the other hand, unreacted insoluble carrier particles, on the other hand, are difficult to settle, and even if they do settle, they do not exhibit the above-mentioned fixing properties.

Therefore, if sedimentation is performed using a container equipped with a removable shield that shields a portion of the bottom of the container, in the case of a sample that has formed aggregates, the sedimentation will remove the aggregates from the bottom of the container. The sample settles in the shielded area and does not settle in the shielded area, creating an optical difference between them, but in the case of a sample that does not form aggregates, there is no optical difference between them. , and learned that it is possible to easily determine whether an antigen-antibody reaction has occurred.

The present invention has now been completed.

That is, 1. The gist of the present invention is to use a container that can be provided with a removable shield that covers a part of the bottom surface of the container, and to transfer antigens or antibodies supported on insoluble carrier particles to the antibodies or antibodies in a liquid medium. The present invention is a method for measuring an antigen-antibody reaction, which comprises reacting with an antigen under heating and precipitating the generated aggregate, then removing the shield and measuring an optical change.

The present invention will be explained below.

The present invention uses antigens or antibodies supported on insoluble carrier particles. Here, the insoluble carrier particles include, for example, a particulate organic substance 1 that does not substantially dissolve in the liquid medium in which the antigen-antibody reaction occurs.

Polymer latex obtained by emulsion polymerization such as polystyrene and styrene-butadiene copolymer, various inorganic compounds widely used as carriers, such as silica,
Examples include metal powders such as alumina, aluminosilicate, and kaolin, as well as bacteria such as staphylococcus and cell membrane fragments. Among these, polymer latex is preferred.

The carrier particles may have any shape, but the average particle size is 0.0! -/, 4μ, preferably Q, /~o, trμ.

Examples of the antigen supported on the carrier include AIDS antigen, herpesvirus antigen, ATL antigen, hepatitis virus, cytomegalovirus-EB virus, varicella-zoster virus, influenza virus, rubella virus, rotavirus, and other viruses. , cell-parasitic microorganisms such as chlamydia and chlamydia, and various bacteria. Antibodies are composed of proteins, whereas antigens are, for example, proteins, polypeptides, steroids, polysaccharides,
It consists of various things such as lipids and pollen. These antigens or antibodies are physically and/or chemically adsorbed onto the carrier by a known method. In the present invention, the above-mentioned antigen or antibody can be directly supported on the carrier, but 1. For example, the HIV protein constituting the AIDS antigen is reduced with a surfactant and/or a chelating agent and a mercaptan, so that the antigen or antibody has a molecular weight of 20,000 or less.
Preferably, the carrier supports a protein from which about 1,000 to 1,000 host cell-derived components have been removed.

When low molecular weight proteins are removed in this way, a non-specific agglutination reaction occurs.<<1 When the antigen-antibody reaction of the present invention is used for AIDS testing, etc., highly accurate test results can be obtained.

Examples of the surfactant used here include nonionic surfactants (for example, Triton and Tween (both registered trademarks) and anionic surfactants (for example, sodium dodecyl sulfate CSDS)), and chelating agents include Aminopolycarboxylic acid salts and phosphonates such as EDTA, and mercaptans include β-mercaptoethanol, dithiothreitol, and the like.

The above-mentioned reduction treatment is preferably carried out in a buffer solution, and as the buffer solution, it is preferable to use a Tris-HCl buffer, a glycine buffer, a phosphate buffer, or an acetate buffer. Furthermore, the HIV protein concentration was 0. θ/m9/ml ~/Om9/me,
The concentrations were adjusted so that the surfactant concentration was 0.07 to 10% by weight (hereinafter referred to as 1%), the chelating agent concentration was 0.01 to 10%, and the mercaptan concentration was O,OS to 30%. to remove low molecular weight proteins.

The antigen or antibody used in the present invention may be supported on an insoluble carrier in an arbitrary amount, but the antigen or antibody used in the present invention may be supported on an insoluble carrier in an amount of 5 to 50μ per gram of the insoluble carrier. , preferably g~30μ? It is better to carry it. In addition, when latex is used as a carrier, it is preferable to use water or a Tris-HCl buffer solution with a concentration of /rriVI or less as a liquid medium for storing the latex.

In the present invention, the above antigen or antibody is reacted with the corresponding antibody or antigen in a liquid medium under heating.

Here, examples of the liquid medium include water and various buffer solutions such as Tris-HCl buffer. Furthermore, add gelatin, albumin, skim milk, etc. to water at 0.000/~O,S%
, preferably in an amount of about 0.00/-0.05%, since the reaction sensitivity is improved. Although the antigen-antibody reaction can be carried out at any ratio, the concentration of the inert carrier particles carrying the antigen or antibody in the liquid medium is from 0.07 to 0.07, preferably from 0.07 to 0.07. /~O,O
Dispersed and suspended at a relatively dilute concentration of about S%,
It is preferable to add a sample containing about 5 to 2000 .mu.2/- of the corresponding antibody or antigen for reaction. The heating conditions are 30°C or higher, preferably 35 to 70°C, particularly preferably 35 to 60°C. What is the reaction time between antigen and antibody?

It may be carried out for about 7 to 1S minutes, depending on the particle size of the insoluble carrier, the concentration of the antigen or antibody, the temperature, etc.

In the present invention, the reaction product generated by the above-mentioned antigen-antibody reaction aggregates the latex and precipitates in the liquid medium, and it is particularly preferable to force the precipitate. Examples of the method include a method using a magnetic field and a centrifugation method. Among these, centrifugation is preferred. The centrifugation conditions vary depending on the particle size of the insoluble carrier and the concentration of antigen and antibody, but the range is -1000 to 5.
o o o r, p, m, preferably 2000-S-
It is preferable to carry out the treatment at 1700 Or, p, m for 3 to 20 minutes.

In the present invention, the above-mentioned reaction and precipitation of the reactants are preferably carried out using a microplate, for example, a 96-sized one, and according to this method, a large number of specimens can be processed in a short time.

A removable lid 1 equipped with a plate 2 as shown in FIG. 1A is prepared so as to fit into each small hole of the microplate 3 used at this time. Immediately after the start of the reaction, or after the completion of the reaction in the case of a short reaction, the lid is attached and the flocculate sedimentation operation is performed. The conditions for this sedimentation operation vary depending on the size of the insoluble particles to be supported, but for example, in the case of latex particles of 0.2 μm.

If using a centrifuge, 200 Or, p, m, /
A time of about S minutes is sufficient, and if sedimentation under gravity is desired, a time of about 12 to 1 g/g hours is sufficient. in any case,
Sedimentation conditions can be determined by simple preliminary tests.

After the sedimentation is complete, in the case of a positive sample, aggregates settle on the unshielded part of the bottom of the container as shown in Figure H, whereas in the case of a negative sample, the insoluble carrier floats in an unreacted state as shown in Figure IC. ing.

When you remove the cover with the shield, each small hole is
When viewed from above, it shows images like Figure/[) and Figure/E,
This can be optically read using a normal microplate reader or the like. .

At this time, the optical density of the positive sample decreases, but the negative sample shows the same optical density as when a blank sample containing no reactant is added, so it can be easily distinguished from the printed data.

In addition, the above-mentioned positive specimens can be accurately determined by visual inspection (1), and can be very easily determined using, for example, Immunopure (2), which shines light from below the container.

The shape of the optical cell used in the present invention is preferably a cylindrical cell with a flat bottom from the viewpoint of optical reading, but the cell is not limited thereto and may be in the shape of a box. The structure of the shield does not have to be cylindrical as shown in FIG. 7; for example, if the cross section is made into a ten shape, a positive reaction can be displayed as a ten.

In addition, when using a normal microplate reader, it is generally preferable to place a shield in the center, but if you make it semicircular and shield half of it as shown in Figure 2, you can avoid turbidity on the left and right sides after sedimentation. Judgment becomes easier by comparing the degrees.

If the area to be shielded is too large, it will reduce the volume of the cell, so it should be less than half, preferably half the diameter of the bottom of the cell (
The area should be about y4). Further, the shield need not be integrated with the lid as shown in FIG. 1, but may be a simple one as shown in FIG. 3.

As is obvious from its effect, the height at which the shield is provided should be closer to the bottom of each cell, and it is most convenient if it is in close contact. However, as long as the distance from the bottom is negligible compared to the height of the liquid level, there is no problem even if they are far apart.

〔Effect of the invention〕

According to the method of the present invention, antigen-antibody reaction, precipitation of aggregates, and optical reading can all be performed within the same porous optical cell.

A large number of specimens can be measured quickly and easily.

EXAMPLES The present invention will be further explained in detail by giving examples below.

〔Example〕

Reference example 1 Ric Mori of the Pasteur Institute as an antigen = -f
- (Luc Montagnier) Using the AIDS virus LAV-t given to me by the professor, I made it into a CD.
! After infecting I cells (OEM/C1/3) and culturing for 2 days, the supernatant was removed and subjected to ultracentrifugation using a silica density gradient to obtain a virus fraction.

This fraction sooμg-7iooμt'? The cells were suspended in 00 μt of Tris-HCl buffer (pHg, 3), and 100 μt of a solution containing 5 DSJ% and Cormel Kabutoethanol S% was added thereto.

Heat treatment was performed at lθθ°C for 7 minutes. At this time.

The cloudiness caused by the virus completely disappeared. This liquid,
Ultrafiltration is performed using Millibore's 1 mol Ka) II-HK, and the fraction remaining on the filter is washed twice with the above-mentioned Tris-HCl buffer to remove the fraction with a molecular weight of 10,000 yen or less. was removed. Thereafter - the residue on the filter was taken up in 1000 μt Tris-HCl buffer and added with 5D 82% and λ-mercaptoethanol 5
Tris-HCl buffer (pHA, g) containing
After adding 0θOpL and resolubilizing at 100c for 1 minute, θ,q% NaCl-containing phosphate buffer (pH
7,2) was subjected to dialysis for 6 hours. The dialysis solution was adjusted with Tris-HCl buffer so that the protein content was 300 to 350μ54/-, and 1rr1t was mixed with an equal amount of 2
Add to Tivostyrene latex (particle size: 0.0 μm) suspension.

Stir mechanically for about 70 minutes. After completion, the latex suspension was centrifuged in the cold at 10,000 r I) m for 20 min, the supernatant was removed and then added to a 0.2% bovine albumin (BSA) solution (in Tris-HCl buffer). ) l- and stir mechanically for 2 minutes.

A latex reagent for anti-HIV antibody detection was prepared.

Example 1 Test serum Sμt diluted 70 times with physiological saline was placed in each well of a commercially available flat-bottomed 9A-well microplate, and then 0.00% diluted with physiological saline. iM) Lis-HCl buffer (pH, J
) / J sμt was added.

Next, 20μ of the anti-HIV antibody detection latex reagent prepared in Reference Example 1 (particle size 0.22μm, concentration 0.05%)
and stirred for 30 seconds using a shaker. This plate was placed in a plastic bag to prevent water evaporation and left in an air bath at 60° C. for 10 minutes to complete one reaction. Take out the plate and open the lid with a shield as shown in Figure 1/A.
The plate was covered with a lid so that the condition was as shown in Figure/A, and the plate was centrifuged for 15 minutes in an oven.

After centrifugation, remove the lid and immediately turn the
Measured using a microplate reader. The results are shown in Table 1.

For reference, the results of measuring each test serum using a Western Blot kit manufactured by Bio-Rad, which is commercially available as a reagent for detecting HIV antibodies, are also listed.

As can be seen from Table 1, the results of both measurement methods were in good agreement.

Table 7 Example 2 When measurements were carried out in the same manner as in Example 1 except that 1 μt of the original serum was used instead of 5 μt of the 10-fold diluted test serum, exactly the same Yin/Yang determination results were obtained.

Example 3 Type of herpes simplex virus (H8V) as antigen!
, Myama strain was grown using Vero cells as a host, and the virus released into the maintenance solution was purified by sucrose density gradient centrifugation, and the purified virus particles obtained were purified according to Reference Example/ A solubilized antigen from which proteins with a molecular weight of less than 1 million yen were removed was obtained.

This solubilized antigen was supported on latex in the same manner as in Reference Example 1 to prepare latex for anti-H8V antibody detection.

The anti-H8V antibody detection latex obtained above was used instead of the anti-HIV antibody detection latex used in Example 1, and purified virus particles were subcutaneously inoculated into rabbits together with Freund's complete adjuvant as a test sample. .

30 days later, the serum and intraocular fluid from the time of intraocular infection with K1ff1M virus particles alone were used.

When measurements were carried out according to Example 1, Yin and Yang could be determined satisfactorily in the same manner as in Example.

[Brief explanation of the drawing]

Figure 5 represents a schematic diagram for explaining the measurement method according to the present invention. Figure /A shows a container 3 containing a reactant DA fitted with a lid equipped with a shield 2. Figures 1B and 1C show the appearance of aggregates after they have settled, and Figures 1D and 1E show the situation after the shielding material was removed. The state observed from the top of the container is shown. In addition, Figure 1B and Figure/D show the case of a positive sample; Figure 1/C
and Figure/E show the case of a negative specimen. Figures 1 and 3 show one example of the shield of the present invention. Applicant Mitsubishi Chemical Industries Co., Ltd. Agent Patent Attorney Hase - Part 2 Procedural Master's Manual (Method) Date of February 1988

Claims (1)

[Claims] (1) Using a container that can be equipped with a removable shield that covers a part of the bottom of the container, the antigen or antibody supported on insoluble carrier particles is mixed with the antibody or antigen in a liquid medium under heating. After reacting with and settling the formed aggregate,
A method for measuring an antigen-antibody reaction, which comprises removing the shield and measuring an optical change.