JPS628056A - Specific bonding analysis - Google Patents

Abstract

PURPOSE:To make it possible to satisfy various conditions such as simplicity, high sensitivity, stability and low cost etc., by contacting a liquid medium with a U-shape or V-shape microplate (MP), to which the specific bonding homologue of a ligand was bonded, for a predetermined time. CONSTITUTION:A liquid medium is contacted with a U-shape or V-shape MP, to which the specific bonding homologue of a ligand was bonded, for a predetermined time. After washing, a carrier particle, to which the ligand or the specific bonding analogue thereof was bonded, is contacted with MP for a predetermined time and the agglutination image on the bottom part of MP is subsequently observed. Because the bottom part of MP is inclined in a U-shape or V-shape, when reaction is negative, the carrier particle is fallen to the center of the bottom part according to gravity to show a button shaped agglutination image. When reaction is positive, the carrier particle is adhered to the inclined surface of MP without being fallen to the bottom part to show a diffused agglutination image. Therefore, by observing the agglutination image, it can be judged whether reaction is positive or negative and various conditions such as simplicity, stability and low cost etc. can be satisfied.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a specific binding analysis method, and more specifically, to a specific binding analysis method that can easily and sensitively detect a ligand in a liquid medium such as serum. Concerning combination analysis methods.

[Prior art and its problems] Conventionally, as a specific binding analysis method for measuring a ligand in a liquid medium, a passive hemagglutination method has been used.

Radioimmunoassay and enzyme antibody method are known.

The passive hemagglutination method is a method in which antigens or antibodies are artificially bound to a carrier such as fixed red blood cells, and the corresponding antibodies or antigens are detected by an agglutination reaction.This method is a simple and stable method; It has the drawbacks of low sensitivity and non-specific agglutination occurring up to 8-fold dilution from the test serum stock solution, requiring further dilution.
-Radioimmunoassay involves binding a radioactive isotope to an antigen or antibody to make the corresponding antibody or antigen gamma-
The zero method, which is a method of measuring with a counter or beta counter, is the most sensitive method among the current immunoassay methods, but it requires special testing facilities because it uses radioactive isotopes. It has the disadvantage of being expensive due to the short half-life of the elements.

Enzyme-antibody method is a method in which an enzyme such as peroxidase or alkaline phosphatase is bound to an antigen or antibody, and the corresponding antibody or antigen is detected by a color reaction using a chromogenic substrate.This method is almost similar to radioimmunoassay. Although it shows comparable sensitivity, it has the disadvantages that the reaction conditions are strict, it requires equipment such as a colorimeter, and the cost of reagents is expensive, although it is not as expensive as radioimmunoassay.

[Object of the Invention] It is an object of the invention to provide a specific binding analysis method that overcomes the drawbacks of conventional methods and satisfies conditions such as simplicity, high sensitivity, stability, and low cost.

[Structure of the Invention] The specific binding analysis method of the present invention is a specific binding analysis method for measuring a ligand in a liquid/medium, and is a specific binding analysis method for measuring a ligand in a liquid or medium. After contacting the liquid medium with a type or Vfi microplate for a predetermined time, the microplate is washed, and then carrier particles to which the ligand or its specific binding analog is bound are brought into contact with the microplate for a predetermined time. This method is characterized by observing the agglomerated image at the bottom of the microplate.

In the present invention, the ligand to be analyzed is α
- Antigens such as fetoprotein, HBg antigen, HBe antigen: antibodies, drugs, poisons, bacteria, viruses, cancer markers; Hormones such as erythroboetin: receptors, etc.

The microplate used in the present invention may be U-shaped or V-shaped.
Any type of material may be used, but materials whose main component is plastic, such as polystyrene, polyvinyl chloride, or polyacrylate, are usually used. Such microplates include NSC plate (manufactured by Biotic Co., Ltd.) and Ray 88U (manufactured by Terumo Corporation).
, immunoassay plates (manufactured by Dainippon Pharmaceutical Co., Ltd.), etc. are commercially available.

Bind to the microplate. The specific binding counterpart of the ligand may be anything as long as it specifically binds to the ligand; for example, if the ligand is an antigen or hapten, the corresponding antibody; if the ligand is an antibody, the corresponding antibody; refers to the corresponding antigen or idiotype antibody; if the counterpart is an antibody, a monoclonal antibody may be used.

The microplate and the counterpart can be coupled, for example, as follows. That is, when the counterpart is a protein, it is usually used in physiological saline or phosphate buffered saline (hereinafter fPB).
sJ), borate buffer, etc., preferably 0.15M
Using borate buffer (p) 18.4), the corresponding
Drop 25 ILl into each hole at a concentration of ILg/aJ,
- This can be done by leaving it at 4°C overnight. In addition, if the corresponding body is other than protein, this can be done by mixing and bonding with the corresponding body albumin, bovine albumin, etc., dropping this into each hole, and allowing it to stand at 4°C overnight. can.

In the present invention, specific binding analogs of ligands include:
Analogs, which refer to substances that exhibit substantially the same behavior with respect to the binding affinity of the specific binding counterpart of the ligand, can also be used in the same manner as the ligand to bind to carrier particles.

Carrier particles used in the present invention include human or animal blood cells, bacteria, latex, gelatin, ribosomes, and the like. The binding between the carrier particles and the ligand or its specific binding analog can be carried out according to a conventional method.For example, the binding between red blood cells and antibodies can be carried out using tannic acid, glutaraldehyde, chromium chloride, etc. as a coupling agent. This can be done by using Various types of carrier particles bound to ligands are commercially available, and these commercially available products may be used.

The contact time between the microplate to which the counterpart is bound and the liquid medium is usually 30 minutes to 2 hours.

After the contact, a cleaning process is performed, and the cleaning liquid used here may be tap water, deionized water, or distilled water.
Especially PBS containing Tween 20
It is preferable to use

The carrier particles to which the ligand or its specific binding analog is bound are then brought into contact, the contact time being usually 2 to 4 hours.

When observing the agglutination image at the bottom of the microplate treated as described above, the bottom of the microplate is inclined in a U-shape or V-shape, so if one reaction is negative, the carrier particles will move to the center of the bottom according to gravity. The carrier particles fall and exhibit a button-shaped aggregation image, while in the case of one positive reaction, the carrier particles do not fall to the bottom but adhere to the slope and exhibit a diffused aggregation image. Therefore, by observing the agglutination image, it is possible to determine with the naked eye whether the reaction is positive or negative.

[Effect of the Invention 1 The specific binding analysis method of the present invention satisfies the conditions of simplicity, stability, and low cost, and has a sensitivity almost comparable to that of radioimmunoassay.

[Examples of the Invention] Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example 1 Measurement of α-fetoprotein Anti-α derived from rabbits
- Dissolve the purified Fetoprotein antibody in physiological saline and use a U-type microplate (Betray 86U (manufactured by Terumo)).
Drop 25 drops at a concentration of lILg/- into each hole of
It was left standing at 4°C overnight.

After washing the plate with PBS containing 0.05% Tween 20 (hereinafter referred to as "Tween 2O-PBSJ"), 25 ml of Tween 2O-PBS was added to each well.Next, the test serum was diluted with PBS. , 25 terminals were dropped into each well and sensitized for 1 hour at room temperature.After washing the plate 5 times with Tween 2O-PBS, 0% of sheep red blood cells conjugated with anti-α-fetoprotein rabbit specific antibody (manufactured by Sankyo ■) was added. ．．
25 ILl was added to each well at a concentration of 1%, and after standing at room temperature for about 3 hours, the image of the tube bottom was determined. As a comparison, an enzyme antibody method (using an enzyme antibody method kit manufactured by Mitsui Pharmaceutical Co., Ltd.) was performed. The amount of α-fetoprotein was calculated by comparison with a standard sample. The results are shown in Table 1. In Table 1, the unit of α-fetoprotein amount is r18/-.

It can be seen from Table 1 that the method of the present invention has sensitivity and reproducibility comparable to that of the enzyme antibody method.

Example 2 Measurement of 8Bs antigen Purified anti-HBs antibody derived from guinea pig was dissolved in physiological saline and 25 ILi was dropped into each hole of a U-type microplate (Betray 9Bυ (manufactured by Terumo Corporation)) at a concentration of 1 g g/j. The mixture was left standing at 4°C overnight. Tween 2O-PB
After washing the plate with S, 25 ml of Tween 2O-PBS was added to each well.Next, the test serum was diluted with PBS, and 25 ml of Tween 2O-PBS was added dropwise to each well to sensitize for 1 hour at room temperature. did. After washing the plate 5 times with Tween 2O-PBs, add 25 ILIL of anti-HBs rabbit specific antibody-conjugated sheep red blood cells (manufactured by Yamanouchi Pharmaceutical) to each well at a concentration of 0.1%, and leave it at room temperature for about 3 hours. After that, the bottom image was determined. As a comparison, radioimmunoassay (using a radioimmunoassay kit manufactured by Guinabot) was performed. The criterion for radioimmunoassay was that a test sample was determined to be positive if it was twice or more the cutoff value (average value of 5 normal human sera x 2.1). The results are shown in Table 2.

Table 2 shows that the method of the present invention has sensitivity and reproducibility comparable to that of radioimmunoassay.

Claims (1)

[Claims] A specific binding analysis method for measuring a ligand in a liquid medium, the liquid medium being brought into contact with a U-shaped or V-shaped microplate to which a specific binding counterpart of the ligand is bound for a predetermined period of time. After washing, the microplate is contacted with carrier particles to which the ligand or its specific binding analog is bound for a predetermined period of time, and an aggregated image at the bottom of the microplate is observed. Characteristic specific binding analysis method.