JPS61272662A - Immunoassay method - Google Patents

Abstract

PURPOSE:To obtain the result of measurement with good accuracy by absorbing once a sample to be measured in a dried porous material, disposing the same near a carrier for electrophoresis and introducing a sample thereto. CONSTITUTION:The sample is dropped through a sample injecting nozzle 2 onto a sample absorber 1 consisting of dry filter paper. A trisglycine buffer soln. is then poured through an injecting nozzle 3 and an injecting port 4 into an upper electrolytic cell 5 and a lower electrolytic cell 6 to prepare an upper electrolyte 7 and a lower electrolyte 8. A DC voltage is impressed between platinum electrodes (cathode) 10 and (anode) 11 from a DC power source. The various components in the sample absorber 1 are migrated to an antibody immo bilized film 12 fixed with the antibody for the component desired to be measured and only the measuring component is trapped in the film 12. The other com ponents pass through the film 12 and migrate into the lower electrolyte 8. An antibody soln. labeling luminol is then injected from a nozzle 13 onto the film 12 and the DC voltage is impressed thereto. The electrolytes 7, 8 are discharged and a soln. of a hypochlorous acid and H2O3 is injected onto the film 12. The quantity of the light emitted from the film 12 is measured by a photocounter.

Description

[Detailed description of the invention] [Field of application of the invention] The present invention relates to immunoassay methods.

[Background of the invention]

By immobilizing an antibody on a membrane-like carrier and applying a potential gradient perpendicular to the surface of this membrane, the antigen in the sample to be measured is moved in this direction by electrophoresis, and an antigen-antibody reaction occurs with the immobilized antibody. is raised and fixed, and -F
In this process, the antibody labeled with the antigen is immobilized and transferred by electrophoresis to cause a reaction, or the labeled antigen is transferred to the unreacted portion of the antibody immobilized on a membrane-like carrier by electrophoresis. A method has been proposed for measuring the concentration of antigen in a sample by immobilizing a labeled substance on a membrane-like carrier by either reaction or reaction, and measuring the concentration of this labeled substance (Unexamined Japanese Patent Publication No. 1986-57257)
.

However, this immunoassay method has many advantages such as shortening the measurement time and making it easy to implement equipment, but when it is implemented into an equipment, the reproducibility of the measurement results is poor.

From the viewpoint of accuracy, it is desirable to give more consideration to the sample injection section.

[Purpose of the invention]

An object of the present invention is to provide an improved immunoassay method that provides highly accurate results in an immunoassay method using electrophoresis as described in JP-A-60-57257.

[Summary of the invention]

This objective is achieved by minimizing mixing of the sample and electrolyte during or immediately after sample injection. Specifically, instead of pouring the sample directly onto the top of the carrier for electrophoresis, the sample solution can be absorbed into a liquid absorber such as dry filter paper, and then this absorber can be placed on top of the carrier for electrophoresis. .

[Embodiments of the invention]

An embodiment of the present invention will be described below with reference to FIG. 10 μQ of a sample in which the amount of a specific component is to be measured is dropped onto -L of the sample absorber 1 made of dry filter paper through the sample injection nozzle 2. Next, a Tris-glycine cleaning solution is poured into the upper electrolytic cell 5 and the lower electrolytic cell 6 through the upper electrolyte injection nozzle 3 and the lower electrolyte injection port 4 to form an upper electrolyte 7 and a lower electrolyte 8. . Next, using the DC power supply 9, a DC voltage is applied between the platinum electrodes 10 and 11 so that the electrode 11 becomes the anode and the electrode 10 becomes the cathode.

At this time, various components contained in the sample absorber 1 move to the antibody-immobilized porous membrane 12, which is a polyacrylamide gel on which antibodies against the components to be measured are immobilized. When the DC voltage is further applied, only the component to be measured is trapped in the antibody-immobilized porous membrane 12, and the other components are trapped in the antibody-immobilized porous membrane 1.
2 and moves to the lower electrolyte 8. Next, an antibody solution labeled with luminol (containing 20% sugar) 10
μQ is gently injected onto the antibody-immobilized porous membrane 12 using the nozzle 13, and a DC voltage is applied again. At this time, an amount of luminol-labeled antibody corresponding to the amount of the measurement component previously trapped in the membrane 12 remains on the membrane 12, and excess luminol-labeled antibody moves to the lower electrolyte 8. Next - L part electrolyte 7
is removed from the upper electrolytic cell 5 using the suction nozzle 14, and the lower electrolytic solution 8 is also discharged from the lower electrolytic cell 6 using the lower electrolytic solution outlet 15. Next, using the luminescent reagent injection nozzle 16, 0.1M H2O2 aqueous solution 1. OOμM and 0. lNN
200 μQ of a solution containing sodium hypochlorite at a concentration of 10 mM in a0 ■■ aqueous solution was injected onto the membrane 12, and the amount of light emitted from the membrane 12 at that time was measured through the quartz glass window 17, and the light receiving unit 18 was placed below it. Measurement is performed using the first to fourth counters 19.

As an example of the measurement results, the results are shown when human immunoglobulin G was selected as the object to be measured, and the DC voltage was applied twice at an applied voltage of 250 cm and an application time of 30 minutes. When the same sample was measured 20 times with this device, the average concentration in the sample was 3.3 x 1. The standard deviation of CV”g/Q is 0.25
X 10"3 g/fl. Sucrose was added at a rate of 20% to the same sample as in this measurement, and the sample was measured using the same apparatus as described in this example, except that the sample absorber 1 was removed. The results are the average of 20 measurements: 3.OX 10
-ag/Q and standard deviation is 0.85X 10-3g/
It was Q. By providing the sample absorber 1 in this manner, it was possible to obtain measurement results with significantly improved accuracy.

〔Effect of the invention〕

As explained in Section L below, the present invention has the effect of obtaining highly accurate measurement results.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing a reaction cell portion of an apparatus for carrying out the present invention, and FIG. 2 is a conceptual diagram showing the configuration of the entire mechanical portion. ]...Sample absorber, 2...Sample injection nozzle,;3.
...-1 partial electrolyte injection nozzle, 4...lower electrolyte injection [1, 5...-upper electrolytic tank, 6...lower electrolytic tank,
7... Upper electrolyte, 8... Lower electrolyte, 9... DC power supply, 10, 1.1... Platinum electrode, 12... Antibody immobilized porous membrane, 13... Labeled antibody Injection nozzle, 14.
... Upper electrolyte suction nozzle, 15 ... ys electrolyte discharge port, 16 ... Luminescence reagent injection nozzle, 17 ... Quartz glass window, 18 ... Light receiving part, 19 ...) 1st- Counter, 20...Membrane fixer, 21...Reaction cell occupying the main part of FIG. 1, 22...Arrow indicating the direction of movement of a series of reaction cells, 23, 24° 25.26... Arrows indicating moving directions of various nozzles, 27... dark box.

Claims (1)

[Claims] An electrophoresis carrier on which antibodies are immobilized substantially over the entire area, and an antigen of a sample to be measured are caused to cause an antigen-antibody reaction with the immobilized antibodies during the electrophoretic movement process, and are immobilized. Either the labeled antibody is moved by electrophoresis and reacted with the immobilized antigen, or the labeled antigen is moved and reacted with the unreacted part of the immobilized antibody by electrophoresis. In the immunoassay method, in which the concentration of the antigen in the sample to be measured is determined by performing one of the reactions and measuring the amount of the labeled antibody or labeled antigen, the sample to be measured is dried on a porous material. An immunoassay method characterized in that a sample is introduced by absorbing the sample in the vicinity of the electrophoresis carrier.