JPH01123150A - Reaction container and immunoassay - Google Patents

Abstract

PURPOSE:To simultaneously measure a plurality of items, by providing regions, to which a plurality of different specific bondable substances are fixed, to the bottom surface of a container receiving a specimen containing a substance to be measured. CONSTITUTION:When a blood type is judged, the interior of the well 2 provided to the spherical bottom surface arranged to a microplate 1 is degreased with acetone and a partitioning member is set while 25mg/ml of anti-serum A is added to a region 3 in an amount of 10mul and 25mg/ml of anti-serum B is added to a region 4 in an amount of 10mul and both of them are allowed to stand. Next, the microplate 1 is washed and 3% gelatin is injected in the well 2 to allow to stand in a wet chamber and, after the removal of gelatin, the regions 3, 4 are washed and dried by air to obtain a reaction container for ABO examination. A serum turbid liquid diluted to 5% is added to the well 2 in an amount of 25mul as a sample and reaction is performed for 30min at room temp. and a blood type is judged from the antigen-antibody reactions generated in the regions 3, 4. Therefore, a plurality of items can be simultaneously analyzed in the reaction container 1.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention belongs to the field of measurements using cell biology and immunology, and further relates to reaction vessels and The present invention relates to an immunological analysis method using this reaction vessel.

[Conventional technology]

As a conventional technique, there is Japanese Patent Application Laid-Open No. 62-8056.

This involves using a microplate with anti-AFP antibodies bound to each person by physical adsorption, adding a sample to each person, allowing the reaction to proceed for 1 hour, washing each person, adding rabbit anti-AFP-conjugated sheep red blood cells to each person for 3 hours. After reacting for a period of time, the agglomerated image formed on the bottom of each person was determined.

[Problem that the invention seeks to solve]

In the conventional technology, one reaction vessel is used to measure one item, so in order to measure a plurality of items, the same number of reaction vessels as the number of items are required.

Another drawback is that the amount of samples required is equal to the number of items.

The present invention has been made in view of these drawbacks, and its purpose is to measure a plurality of items using one reaction vessel.

[Means and actions for solving problems]

The first invention is the invention of a reaction container, which is composed of a reaction container and a plurality of zones each consisting of a different specific binding substance immobilized on the bottom of the reaction container. When a sample containing a substance is added to a reaction vessel, the sample comes into contact with an area formed on the bottom of the vessel and consisting of a plurality of different specific binding substances.

The second invention is an invention of an analysis method for measuring an antibody using the reaction container of the first invention, and a step of adding a sample containing the antibody to be measured into the reaction container of the first invention and reacting for a certain period of time. , a step of separating a solid phase and a liquid phase, a step of adding carrier particles on the surface of which antibodies against the antibodies on the solid phase are immobilized into a reaction vessel and reacting for a certain period of time, and a step of separating each area provided on the bottom of the reaction vessel. The method consists of a step of detecting an aggregated image consisting of a specific binding substance-antibody-antibody/carrier particle complex.

The third invention is an invention of an analysis method for measuring cells using the reaction container of the first invention, and a step of adding a sample containing cells to be measured into the reaction container of the first invention and reacting for a certain period of time. and a step of detecting an aggregated image consisting of a specific binding substance-cell complex in each area provided on the bottom of the reaction vessel.

Specific binding substances used in the present invention include substances with antigenicity, typified by ligands and receptors, antibodies that bind to antigens, and immunoglobulins.

Substances that are involved in specific binding reactions such as immune reactions include antibodies against immunoglobulins, cell membrane proteins, and antibodies against cell surface antigens.

Carrier particles used in the present invention include human or animal blood cells, cells, latex particles, gelatin particles, ribosomes, and the like.

The reaction vessels used in the present invention are preferably test tubes or microplates, and are roughly divided into those containing glass as a main component and those containing plastic as a main component. The former is silicate glass, the latter is polystyrene, polyvinyl chloride,
Examples include polyacrylate.

〔Example〕

The present invention will be explained based on examples.

11 Insert a partition member having a tip shape that matches the shape of the bottom of the reaction container into the reaction container, and bring the tip into close contact with the bottom of the container.0 Add antibody solution or antigen solution to the area partitioned by the partition member. Add and leave at room temperature for 1 hour. Next, after removing the partition member, it is washed with phosphate buffer (PBs).

After washing, add protein or gelatin solution such as BSA to the reaction container and incubate for 1 hour at room temperature to perform profking. Wash with PBS to remove residual gelatin solution. Reaction container that can be used for analysis by performing 0 or more operations. is obtained.

When increasing the number of measurement items, the shape of the partition member may be changed to increase the partitioned area.

Although the specific binding substance is immobilized on the bottom of the reaction vessel by physical adsorption as described above, it is of course possible to immobilize the specific binding substance by covalent bonding. In the case of reaction vessels made of silicate glass, epoxysilane ((CHsO
) After zsi, the specific binding substance can be immobilized on the bottom of the container by adding it to the area partitioned by the above-mentioned partition member. In the case of a microplate made of acrylic, add 5N hydrochloric acid to the wells, incubate at 60°C for 4 hours to perform hydrolysis, and after washing with PBS,
5% polyethyleneimine (CH*CHtNH)-, MW
・Add an acetone solution containing 60,000 to 80,000 and incubate at 40°C for 2 hours. Pus
After washing with water, a 5% glutaraldehyde solution is added into the well to aldehyde the well surface. After introducing the aldehyde group to the surface, a specific binding substance can be added to the area partitioned by the above-mentioned partition member to immobilize it on the bottom of the container.

In the above-mentioned method, the areas where a plurality of specific binding substances are to be immobilized are separated by a partition member, but for example, a styrene monomer is thinly applied to all but one area at the bottom of the reaction vessel where immobilization is to be performed. Benzoyl peroxide (
BPO) solution is added to polymerize. PBS inside the container
After washing with water, a specific binding substance is added and immobilized by physical adsorption. After washing, a BSA solution is added and blocked, and the remaining residue is removed by washing with B5At-Pus.

The polystyrene film is then removed and a separate specific binding substance is added to the remaining area for immobilization, or
By repeating the process of dividing the area by applying styrene monomer, a plurality of specific binding substance immobilization areas can be formed on the bottom of the reaction vessel.

’　　　　　　　　　　　 FIG. 1 shows a reaction vessel used for ABO table testing.

After degreasing the inside of the well 2, which has a spherical bottom surface with a constant curvature and is arranged in the microplate 1, with acetone, a partition member is set to divide the inside of the well into two semicircular sections. - anti-A serum in area 3, 25 μ/
- Add 10 ttl of anti-B serum to each zone 4 and leave at room temperature for 2 hours. Next, after washing plate 1 by immersing it in PBS, 3% gelatin was added to well 2.
and leave it in a greenhouse for 1 hour at room temperature. After removing this 3% gelatin, plate 1 was
An antibody immobilization reaction container for ABO table testing is prepared by immersing it in AS, washing it, and air drying it.

Next, a method for determining blood type using this reaction container will be explained with reference to FIG.

A patient's blood cell suspension diluted to 5% is prepared as a sample, and 25 Il of each sample is added to each well 2 of the microplate 1 and allowed to react at room temperature for 30 minutes. At this time, an antigen-antibody reaction occurs between the anti-A serum and anti-B serum immobilized in zones 3 and 4, respectively, and the antigen on the red blood cells, and the ABO blood type is determined from the binding pattern of the blood cells to each zone. can. Figure 2(a) shows the A-type bonding pattern.

Figure 2(b) shows the B-type bonding pattern, and Figure 2(c) shows the A-type bonding pattern.
Figure 2(d) shows the bonding pattern of type B and the bonding pattern of type 0, respectively.

According to this embodiment, the ABO blood type can be determined from the binding pattern in one well.

' ABO engineering Prepare a microplate 1 in which type A blood cells are immobilized in area 3 and type B blood cells are immobilized in area 4 as shown in FIG. Prepare patient serum as a sample and place each sample on microplate 1.
Add 25 ttl to each well 2 and incubate for 30 minutes at room temperature.Next, wash the wells three times with PBS to remove unbound antibodies.Next, add mouse anti-human Ig-conjugated polystyrene latex particles (particle size 3-) Pour 25 ttl of suspension into each well and incubate for 30 minutes at room temperature. When the antibody in the patient's serum binds to the blood cells immobilized in each area, the mouse anti-human 1g on the latex particles binds thereon, and this can be determined by looking at the binding pattern in well 2. .

Figure 3(a) shows A-type bonding pattern, Figure 3(b) shows B-type bonding pattern.
FIG. 3(c) shows the bonding pattern of type AB, and FIG. 3(d) shows the bonding pattern of type 0.

As explained in the Examples above, the reaction vessel of the present invention can be used to determine the presence of specific antigens on cell surfaces and to determine the presence of antibodies in serum. Although the embodiments have been described using blood type determination as an example, the present invention can also be applied to the measurement of viruses or bacteria. Useful applications include, for example, isotypic, allotypic or idiotypic antibody determination and virus or cell typing.

Although antiserum was used as the antibody immobilized on the bottom of the reaction vessel in the examples, a monoclonal antibody having a single specificity or a mixture of several types of monoclonal antibodies can of course also be used.

〔Effect of the invention〕

According to the present invention, multiple items can be analyzed at once in one reaction vessel.

[Brief explanation of the drawing]

FIG. 1 is a plan view of a microplate that is an embodiment of the present invention. Figure 2 (a), (b), (c), and (d) show the A-type and B-type formed in the well in the ABO table test.
Type, AB type. FIG. 3 is a diagram showing bonding patterns of type 0. Figures 3 (a), (b), (c), and (d) show A-type and B-type formed in the well during ABO back inspection.
Type, AB type. FIG. 3 is a diagram showing bonding patterns of type 0. 1-・-・-・Microplate

Claims (1)

[Scope of Claims] 1. A reaction container for accommodating a sample containing a substance to be measured, the reaction container having a bottom surface provided with a region in which a plurality of different specific binding substances are immobilized. 2. The reaction container according to claim 1, wherein the reaction container has a conical bottom surface. 3. The reaction container according to claim 1, wherein the reaction container has a spherical bottom surface. 4. A step of adding a sample containing an antibody to be measured into a reaction vessel having a region on the bottom in which a plurality of different specific binding substances are immobilized and reacting for a certain period of time, and a step of separating a solid phase and a liquid phase. , adding carrier particles on the surface of which antibodies against antibodies on a solid phase are immobilized into a reaction vessel and reacting for a certain period of time; - An immunological analysis method characterized by comprising the step of detecting an aggregated image made of a carrier particle composite. 5. A step of adding a sample containing cells to be measured into a reaction vessel having zones on the bottom surface in which a plurality of different specific binding substances are immobilized and reacting for a certain period of time; 1. An immunological analysis method comprising the step of detecting an aggregated image consisting of a specific binding substance-cell complex. 6. The immunological analysis method according to claim 4 or 5, wherein the step of detecting the aggregated image is a visual observation. 7. The immunological analysis method according to claim 5, wherein the step of detecting the aggregated image comprises staining the cells and then measuring the stained cells visually or photoelectrically. .