JPH0529065B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immunoassay device, and particularly to an immunoassay device that prevents sample diffusion during sample introduction and improves accuracy.

[Conventional technology]

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 the immobilized antibody and antigen are moved in this direction by electrophoresis. An antibody reaction is caused and immobilized, and then the antibody labeled with the immobilized antigen in the above process is moved by electrophoresis and reacted, or the unimmobilized antibody immobilized on a membrane-like carrier is The labeled antigen in the sample is immobilized on a membrane-like carrier by either electrophoretic movement of the labeled antigen and reaction, and the concentration of the labeled substance is measured. A method for measuring the concentration of antigen has been proposed (Japanese Patent Application Laid-open No. 57257-1983).

[Problem that the invention seeks to solve]

Although this innomassay method has many effects such as shortening the measurement time and facilitating device integration, it is desirable to give further consideration to the reproducibility and accuracy of the measurement results.

An object of the present invention is to provide an improved immunoassay device that uses an electrophoresis method that applies a potential gradient perpendicular to the surface of a membrane and can provide highly accurate results.

[Means for solving problems]

The above object is achieved by an apparatus that minimizes mixing of the sample and electrolyte during or immediately after injection of the sample. Specifically, the sample is not poured directly onto the top of the carrier to be electrophoresed, but rather a thin tube is placed at the top close to the electrophoresis carrier, the sample liquid is poured into this thin tube, and then an electrode is placed inside the tube. A DC voltage is applied between the sample and the electrode provided at the bottom of the electrophoresis carrier to guide the sample to the top of the electrophoresis carrier.

[Effect]

The above-mentioned thin tube suppresses the reaction between the sample and the electrolyte as much as possible, and good accuracy can be obtained.

〔Example〕

Embodiments of the present invention will be described below with reference to FIG.

Example 1 A tris-glycine buffer solution is poured into an upper electrolytic cell 3 and a lower electrolytic cell 4 through an upper electrolyte injection nozzle 1 and a lower electrolyte injection port 2 to form an upper electrolyte 5 and a lower electrolyte 6. . Inhale 10μ of the sample whose pH has been adjusted by adding a high concentration Tris-glycine buffer into the sample introduction tube 7, move it into the upper electrolytic tank 3, and transfer it to the polyacrylamide gel immobilized with antibodies against the component to be measured. It is stopped near a certain antibody-immobilized porous membrane 8. Using a power source 11, a DC voltage is applied between the sample introduction electrode 9 placed inside the sample introduction capillary 7 and the lower electrode 10 in the lower electrolytic tank 4 so that the electrode 10 becomes the anode and the electrode 9 becomes the cathode. do. At this time, each component in the sample is introduced into the antibody-immobilized porous membrane 8. Turn off the power supply 11 and remove the upper electrode 12 and electrode 10 provided inside the upper electrode tank 3.
A DC voltage is applied between them using a power supply 13 so that the electrode 10 becomes an anode and the electrode 12 becomes a cathode. Next, 10μ of a luminol-labeled antibody solution (containing 20% sucrose) was gently injected onto the antibody-immobilized porous membrane 8 using the nozzle 14, and the electrode 1
A DC voltage is applied between the electrode 2 and the electrode 10 so that the electrode 10 becomes the anode and the electrode 12 becomes the cathode. At this time, an amount of luminol-labeled antibody corresponding to the amount of the measurement component previously trapped in the membrane 8 remains on the membrane 8, and excess luminol-labeled antibody moves to the lower electrolyte 6.

Next, the upper electrolytic solution 5 is removed from the upper electrolytic cell 3 using the suction nozzle 15, and the lower electrolytic solution 5 is also removed from the lower electrolytic cell 4 using the lower electrolytic solution outlet 16. Next, using the luminescent reagent injection nozzle 17, 0.1M
of H ₂ O ₂ aqueous solution and 0.1N NaOH aqueous solution.
Solution containing sodium hypochlorite at a concentration of 10mM
200μ is injected onto the film 8, and the amount of light emitted at that time is measured through the optical glass window 18, and the light receiving section 19 is placed below it.
Measurement is performed using a photo counter 20 equipped with a.

As an example of the measurement results, human immunoglobulin G was selected as the measurement target, and the applied DC voltage was 250 V for sample introduction, sample reaction, and labeled antibody reaction, and the application times were 5 minutes, 25 minutes, and 30 minutes, respectively. It was measured. As a result of measuring the same sample 25 times using this device, the average concentration in the sample was 3.3 x 10 ^-3 g/, and the labeling deviation was 0.23 x 10 ^-3 g/. Sucrose was added to the same sample as in this measurement at a ratio of 20%, and the sample was introduced by injecting it onto the membrane 8 in the same manner as the luminol-labeled antibody, using the same equipment as described in the example. It was measured. Results are the average of 25 measurements.
It was 3.0×10 ⁻³ g/, standard deviation 0.85×10 ⁻³ g/. By providing the sample introduction capillary 7 in this way, it was possible to obtain measurement results with significantly improved accuracy.

Example 2 Another embodiment will be explained with reference to FIG.

After injecting the upper electrolyte 5 and the lower electrolyte 6 in the same manner as in Example 1, the same sample as in the previous example was sucked into the sample introduction capillary 7 and poured into the upper electrolytic cell 3. It is moved and stopped just before the antibody-immobilized porous membrane 8. A power source 11 is used to connect the sample introduction electrode 9 and the upper electrode 21 installed near the antibody-immobilized porous membrane 8 in the upper electrolytic cell 3 so that the electrode 21 becomes an anode and the electrode 9 becomes a cathode. Apply DC voltage so that At this time, each component in the sample moves onto the antibody-immobilized porous membrane.

Note that the electrode 21 was insulated by leaving only the tip portion and applying resin coating to the other portions. After turning off the power supply 11, a DC voltage is immediately applied between the upper electrode 21 and the lower electrode 10 using the DC power supply 13 so that the electrode 10 becomes the anode and the electrode 21 becomes the cathode. Next, a luminol-labeled antibody solution was introduced onto the antibody-immobilized porous membrane using the same procedure.
Furthermore, it moves into the membrane and reacts.

Luminol luminescence detection is performed in the same manner as in Example 1. Sample introduction, sample reaction, labeled antibody introduction, labeled antibody reaction, human immunoglobulin as the measurement target.
Apply a DC voltage of 150V, respectively.
250V, 150V, 250V, application time 2, respectively
It was measured in minutes, 25 minutes, 2 minutes, and 30 minutes. As a result of measuring the same sample as in Example 25 times with this device, the average concentration in the sample was 3.3 × 10 ^-3 g/, and the standard deviation was
It was 0.24×10 ^-3 g/, and highly accurate measurement results equivalent to those of Example 1 could be obtained.

A schematic diagram of an example of a device that realizes these is shown in FIG.

A reaction section (hereinafter referred to as reaction cell 22) consisting of an upper electrolytic cell 3, a lower electrolytic cell 4, a porous membrane 8, a quartz glass window 18, an upper electrode 12, 21, and a lower electrode 10 is arranged continuously, and The reaction is carried out while proceeding in the direction of the arrow 23, and the measurement is carried out in a dark box 28.

〔Effect of the invention〕

As explained above, the present invention has the effect that highly accurate measurement results can be obtained.

[Brief explanation of the drawing]

1 and 2 are schematic diagrams showing a reaction cell portion in which the present invention is carried out, and FIG. 3 is a conceptual diagram showing the configuration of the entire mechanism portion. DESCRIPTION OF SYMBOLS 1... Upper electrolyte injection nozzle, 2... Lower electrolyte injection port, 3... Upper electrolytic tank, 4... Lower electrolytic tank, 5... Upper electrode solution, 6... Lower electrolyte, 7...
...Tube for sample introduction, 8...Antibody-immobilized porous membrane,
9... Sample introduction electrode, 10... Lower electrode, 11
...DC power supply, 12 ... Upper electrode, 13 ... DC power supply, 14 ... Nozzle, 15 ... Suction nozzle, 1
6... Lower electrolyte outlet, 17... Luminescent reagent injection nozzle, 18... Quartz glass window, 19... Light receiving section, 20... Photo counter, 21... Upper electrode, 22... Reaction cell, 23... ...Arrows indicating the moving direction of the connected reaction cells, 24, 25, 26, 2
7...Arrows indicating the movement directions of various nozzles, 28
...Dark box.

[Claims]

1. In an immunoassay in which the antigen in a sample to be measured is moved using electrophoresis, fixed by an antigen-antibody reaction with an immobilized antibody, and the concentration of the antigen is measured, (a) Different antigens to be identified are used. multiple types of antibodies,
Different membranous carriers for electrophoresis are held integrally with the solid support in the hollow part of the ring-shaped solid support, have substantially the same thickness as the solid support, and have the same composition. A step of stacking these membrane carriers for electrophoresis by fixing them substantially over the entire area; (b) combining the laminated membrane carriers with an electrolytic solution in a first electrolytic cell and an electrolytic solution in a second electrolytic bath; (c) Injecting the sample to be measured into the first electrolytic cell and bringing it into contact with the liquid.