JPS61272658A - Immunoassay method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to an immunoassay method, and more particularly to a method for rapidly and accurately quantifying trace biological components using antigen-antibody reactions.

[Background of the invention]

Radioimmunoassay is used to quantify various biological substances and drugs, but because it uses radioactive isotopes,
There is a problem in that it requires special care in handling. Therefore, various immunoassay methods using non-radioactive labeling substances such as enzymes, fluorescent substances, and chemiluminescent substances have been investigated, and among these methods, methods using enzymes or fluorescent substances as labels have reached the stage of practical use. There is. However, the problem with both these methods and radioimmunoassay is that the entire process requires time and effort.

An example of a method to simplify the entire process is U.S. Patent No. 3,
852,157. Although this method simplifies the process by not requiring an operation to separate antigen-antibody reactants and non-reactants, the analytes to be measured are limited to low molecular weight haptens.

Similarly, methods that do not require separation of antigen-antibody reactants and non-reactants include nephelometric immunoassay, which utilizes the formation of three-dimensional aggregates of antigen-antibody reactants, and latex, which uses microparticles such as polystyrene. Immunoassays have been put into practical use. However, these methods, contrary to the above-mentioned methods, target polymeric substances such as proteins. Methods that do not require an operation to separate antigen-antibody reactants and non-reactants have a simple process, but
Very suitable for measuring extremely low concentration components. On the other hand, efforts have also been made to simplify the above separation operation.

That is, a method called a solid-phase method (by washing the carrier with water after the completion of a non-aqueous reaction) allows easy separation of reactants and non-reactants.

Although the solid-phase method simplifies the immunoassay method, there are still many deficiencies in terms of automating the entire process. In order to automate immunoassays, it is essential to shorten the time required for the entire process.

The antigen-antibody reaction process takes the most time in the immunoassay process, and apart from the aforementioned methods that do not require separation operations that measure relatively high-concentration components, this process usually takes several hours to a day. It takes. This time is typically longer when measuring lower concentrations of substances.

In order to shorten this reaction time, a method has been proposed in which a column is filled with microcrystals or microparticles to which antibodies (or antigens) are bound, and a test sample, etc. is forcibly inserted into the column (Japanese Patent Publication No. 1973- 127823). This example is a method in which a liquid such as a test sample is forcibly delivered to a portion or layer of a carrier on which an antibody or the like is immobilized.

As a method of sending only a specific component (for example, an object to be detected) contained in the liquid rather than the liquid itself, there is a method of sending the specific component by electrophoresis. One example is to replace a part of the gel layer placed on a support plate with a gel layer or a layer consisting of a porous structure on which an antibody or antigen is immobilized, and to deliver the antigen or antibody, which is the substance to be detected, to this part. (U.S. Pat. No. 3,966,897).

Another example using electrophoresis is Japanese Patent Publication No. 55-132946.
is shown. That is, a gel tube filled with polyacrylamide gel is prepared, an antibody solution or an antigen solution is injected into one end of the tube, and electrophoresis is performed to form a concentrated layer of antibodies or antigens. While a solution is injected and electrophoresis is performed, an antigen-antibody reaction is performed in the concentrated layer. Protein-impermeable membranes, such as dialysis membranes, have also been inserted into portions of the gel to facilitate the formation of a concentrated layer. Here, the antibody or antigen is not immobilized on a carrier for electrophoresis and exists in a free state in the reaction layer.

The antigen-antibody reaction method using these electrophoretic methods requires that all components to be moved by electrophoresis necessarily be electrically charged in an aqueous solution. Therefore, when it is necessary to electrophores an uncharged nonionic substance, such as a nonionic hapten, the electrophoresis method cannot be used as is.

[Purpose of the invention]

The purpose of the present invention is to shorten the time required for antigen-antibody reactions in immunoassays, thereby providing an easy-to-implement method.
The objective is to provide a highly sensitive immunoassay method with a wide range of applications.

[Summary of the invention]

The method of delivering antigens or antibodies by electrophoresis is extremely effective in achieving the above objectives.

Protein antigens, antibodies made of γ-globulin, and the like are ionized under appropriate hydrogen ion concentrations to form positive or negative ions, so they can be easily moved by electrophoresis. However, among the components currently measured by immunoassay methods, there are many □ substances that do not carry an electric charge no matter how the conditions are selected. Examples of these include various steroid hormones, drugs, and the like.

The first reason for applying electrophoresis to immunoassay methods is to transfer dilute antigen in solution to an electrophoresis carrier on which antibodies are immobilized, and substantially concentrate it, thereby increasing the antigen-antibody reaction rate. It is about increasing. Another reason is that by forcibly removing unreacted labeled antigens, etc. from the membrane,
The objective is to effectively separate antigen-antibody reactants and non-reactants.

In the present invention, an antigen-antibody reaction is performed by forcibly transferring a substance to be detected to a carrier such as a porous membrane on which antibodies are immobilized using a press-in cap, and then electrophoresis is performed to remove unreacted labeled antibodies,
This method removes labeled antigens or labeled haptens by moving them out of the carrier, and can be applied to the measurement of almost all biological substances, drugs, etc.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

[Embodiments of the invention]

Example 1 Cellulose acetate membrane for electrophoresis (approximately 120 μm thick)
) was immersed in anti-human (rabbit) IgG antibody solution for about 1 hour, and then soaked in 2.5% glutaraldehyde solution (1/15M
0.1 M phosphate buffer containing NaCtf, pH 7.4
(diluted with PBS) for 30 minutes,
After further soaking in anti-Human IgG antibody solution for about 1 hour,
By thoroughly washing with BS, a cellulose acetate membrane immobilized with anti- and IgG antibodies was prepared.

As schematically shown in FIG. Various concentrations of human IgG solutions (prepared in PBS) were placed on this membrane.
After injecting 500μ of 5, it was gently suctioned in the direction of arrow 6 to send HiI-IgG into the membrane along with the liquid. Then commercially available fluorescein isoteocyanate (FIT
C) Labeled anti-human) IgG antibody (rabbit) solution (diluted 50 times with PBS) 500 μtf was injected in the same manner and passed through the membrane. Next, the glass tubes 3 and 3' were filled with Tris-glycine buffer (pH 8, 6) 7, and electrophoresis was performed at an applied voltage of 50 V for 10 minutes as shown in FIG. After the electrophoresis was completed, the membrane was removed, briefly rinsed with PBS, and then the total fluorescence intensity at 52Q nm was measured using excitation light of 485 nm. The measurement results are shown in Figure 3.

In the above experiment, the IgG solution was immobilized with the antibody (passed through the human membrane), and then electrophoresed according to the method described above.
The other operations were the same as above, and the effect of electrophoresis during this intermediate process was investigated. No significant difference was observed in the results.

Example 2 Anti-thyroxine antibody (rabbit) was immobilized on a cellulose acetate membrane for electrophoresis in the same manner as in Example 1. This membrane was sandwiched between glass tubes as shown in Figure 1, and thyroxine solutions of various concentrations (prepared in PBS) and FITC-labeled thyroxine solutions (prepared in PBS) were placed on the membrane.
(adjusted to an appropriate concentration with S) was injected in 300 μt portions, and the solution was gently sucked through the membrane in the same manner as before. Next, electrophoresis was performed at 50 V for 10 minutes in the same manner as in Example 1. The membrane was rinsed briefly with PBS, and the fluorescence intensity of the membrane at 520 nm was measured. The measurement results are shown in Figure 4.

FITC has a carboxyl group which is a dissociative group in the molecule. Therefore, under conditions of pH 8.6, it is negatively charged. Since thyroxine also has a carboxyl group, FITC-labeled thyroxine has a high electrophoretic mobility, and FITC-labeled thyroxine that is not bound to the antibody is easily removed from the membrane.

When FITC is used as a label, FITC-labeled molecules such as steroid hormones and drugs that do not have a dissociative group can be easily moved through the membrane during electrophoresis due to the action of the dissociative group of FITC.

Therefore, it goes without saying that the present invention can also be applied to these molecules. Also, as a label, FITC
In addition, many other fluorescent substances having a dissociative group, enzymes, or labeling substances in which fluorescent substances, chemiluminescent substances, etc. are bonded to molecules having a dissociative group can be used.

In the above example, the object to be detected, etc. was transferred into the antibody-immobilized membrane by suction, that is, a depressurization operation, but the solution may be transferred by applying pressure from the sample side, mechanical force such as centrifugation, or other methods. Any method is fine as long as it is suitable.

It is clear that various organic and inorganic membranes can be used as the antibody-immobilized membrane.

〔Effect of the invention〕

As explained above, according to the present invention, the reaction time required for one antigen-antibody reaction in the conventional immunoassay method can be shortened from 3 to 4 hours to 1 day to less than 1 hour.

It goes without saying that the time required for this reaction in the present invention varies depending on the type of reaction membrane, the type of component to be detected, the type of labeling reagent, the method of transferring the sample liquid, the electrophoresis conditions, etc.

Furthermore, according to the present invention, unreacted substances and excess labeled antibodies are removed by passing through the reaction membrane, thereby eliminating the need for operations such as washing with water, which are required in conventional immunoassays based on in-phase reactions.

Furthermore, since the present invention can be applied even if the object to be detected does not have a dissociative group, it can be applied to almost all objects to be measured, and has the characteristic of having an extremely wide range of applications.

[Brief explanation of the drawing]

Fig. 1 is a schematic diagram showing the method of feeding a sample solution into the antibody-immobilized membrane, Fig. 2 is a schematic diagram showing the method of performing electrophoresis, and Figs. 3 and 4 are diagrams showing the results of the present invention. It is. 1... Antibody-immobilized membrane, 2... Glass filter, 3.
3'...Glass tube, 4,4'...OIJing, 5.
...sample solution, 6...arrow indicating the direction of movement of the solution, 7
...buffer solution.

Claims (1)

[Scope of Claims] 1. In an immunoassay method in which an antigen or hapten in a sample is bound to an immobilized antibody by an antigen-antibody reaction and the concentration of the antigen or hapten is measured, (a) substantially the entire area of a porous carrier; (b) In the process of moving the antigen or hapten of the sample to be measured by mechanical force, an antigen-antibody reaction occurs with the immobilized antibody and the antibody is immobilized, or (c) any of the steps of causing an antigen-antibody reaction competitively with the immobilized antibody in the process of simultaneously moving the antigen or hapten and the labeled antigen or labeled hapten by mechanical force, and fixing the antigen or hapten; Either a labeled antibody is moved by mechanical force to the immobilized antigen in a competitive reaction and reacted, or a labeled antigen or a labeled hapten is transferred to an unreacted area of the immobilized antibody in the non-competitive reaction by mechanical force. (d) a step of moving residual unreacted substances from the above reaction to the outside from the porous carrier by electrophoresis; and (e) a step of causing the above-mentioned label to move. An immunoassay method characterized by measuring the concentration of an antigen or hapten in a sample by measuring the concentration of an antibody, a labeled antigen, or a labeled hapten. 2. The immunoassay method according to claim 1, wherein the carrier on which the antibody is immobilized is in the form of a membrane. 3. The immunoassay method according to claim 1, wherein the labeling substance has a dissociative group.