JPH01288767A - Enzyme immunity sensor - Google Patents

Abstract

PURPOSE:To dispense with removal and fitting operations of a film and reproduction thereof and to enable the precise and simple analysis of immunity, by using room temp. superconducting particulates prepared by the covalent linkage of antibodies for an immune substance to be detected. CONSTITUTION:Antibodies 4 for an immune substance to be detected are covalent- bonded to a room temp. superconducting particulate 5 by using a chemical modification method, and material thus prepared is suspended in a buffering liquid. Next, a solution containing an immune substance being labeled with catalase and the same with the immune substance to be detected is mixed in a prescribed quantity in a sample liquid containing said immune substance to be detected. The suspension of the antibody- covalent-bond superconducting particulates is mixed in said mixed solution and an immune reaction is made to proceed for a prescribed time. After this immune reaction, the solution is introduced into a flow cell 6 and the superconducting particulates are collected on the surface of a selective permeation film on an oxygen electrode 1 by the Meissner effect. Subsequently, components other than ones bonded to the superconducting particulates are removed by washing, a prescribed quantity of buffering liquid containing a hydrogen peroxide is introduced into the flow cell 6 and oxygen produced by decomposition of the hydrogen peroxide is detected by the oxygen electrode 1.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an enzyme immunosensor used for quantifying immune substances.

[Prior Art] Measurement of immune substances such as antigens and antibodies is very important for medical diagnosis, such as confirming the presence or absence of infection. Conventionally, radioimmunoassays, enzyme immunoassays, and the like have been used, but in recent years, biosensor-type immunoassays have been developed from the standpoint of ease of operation. FIG. 3 is a schematic cross-sectional view of a conventional enzyme immunobiosensor as shown in, for example, "Ion Electrodes and Enzyme Electrodes J (published by Kodansha, 1981), 176-182, edited by Tsutomu Suzuki. In the figure, (1) is the oxygen electrode that serves as the base electrode,
(2) is an oxygen permeable membrane attached to the oxygen sensitive part, (3) is an organic polymer membrane attached to the oxygen permeable membrane (2), and (4) is an oxygen permeable membrane attached to the oxygen sensitive part. The antibody is covalently immobilized on the surface of the organic polymer membrane (3) by chemical modification.

Next, its operation will be explained. A solution containing a predetermined amount of a catalase-labeled antigen is mixed at a predetermined ratio with a sample solution containing the antigen to be detected. The enzyme immunosensor shown in FIG. 3 is immersed in the mixed solution and left for a certain period of time to allow the antigen to be detected and the catalase-labeled antigen to bind to the antibody (4). This sensor is then washed to remove antigen molecules that are not bound to the antibody (4). The sensor thus treated is immersed in a solution containing a certain amount of hydrogen peroxide, and the response of the oxygen electrode (1) is measured. Hydrogen peroxide is decomposed by antigen-labeled catalase to generate oxygen, so the response at the elementary electrode increases in proportion to the amount of catalase bound to the antibody (4) through the antigen.

Since the catalase-labeled antigen and the antigen in the sample solution competitively bind to the antibody, the response of the oxygen electrode decreases in inverse proportion to the amount of antigen in the sample solution.

In this way, the amount of antigen in the sample solution can be quantified using this sensor.

[Problem to be solved by the invention] Since the conventional enzyme immunosensor is configured as described above, there is a limit to the amount of antibody or antigen that can bind to the organic polymer membrane (3), resulting in low sensitivity. There is also a problem in that, when used, it is necessary to remove the membrane and regenerate the bound antibody.

This invention was made to solve the above-mentioned problems, and aims to provide an enzyme immunosensor that can improve detection sensitivity and does not require membrane desorption or regeneration.

[Means for Solving the Problems] The enzyme immunosensor of the present invention is configured to quantify the immune substance to be detected using room-temperature superconducting particles to which antibodies against the immune substance are covalently bonded.

[Operation] In this invention, an antibody molecule for the antigen or antibody to be detected is covalently bonded to room-temperature superconducting particles, and after mixing with a test sample solution, reaction, and washing, the particles are attached to a substrate using the Meissner effect. After collecting on the surface of the sensitive part of the electrode, the labeled enzyme is reacted. Therefore, unlike organic polymer membranes, there is no limit to the amount of antigen or antibody that can be bound, improving sensitivity, and there is no need for membrane desorption or regeneration of bound antibodies when reusing the membrane.

[Example] Hereinafter, an example of the present invention will be described with reference to the drawings. 1st
The figure shows room-temperature superconducting fine particles (4) to which an antibody (4) against an immune substance to be detected is covalently bound, according to an embodiment of the present invention.
There is a schematic diagram showing 5). FIG. 2 is a schematic cross-sectional view showing an enzyme immunosensor according to an embodiment of the present invention. In the ward,
(1) has an oxygen electrode serving as a base electrode, (2) has an oxygen permeable membrane, (6) has a solution outlet (8) and an inlet (9),
A flow cell (7) indicates a solution for measuring the amount of labeled enzyme remaining after superconducting fine particles (5) bound with antibodies (4) are mixed with a test solution, reacted, and washed.

Next, the operation will be explained. Room-temperature superconducting fine particles (5) are prepared by covalently bonding antibodies (4) against the immune substance to be detected using an appropriate chemical modification method, and suspended in an appropriate buffer. A predetermined amount of a catalase-labeled solution containing the same immune substance as the detection target is mixed with a sample solution containing the immune substance to be detected. A suspension of antibody covalently bonded superconducting fine particles is mixed with this mixed solution, and an immune reaction is caused for a certain period of time. During this process, an alternating magnetic field can be applied to the immune reaction solution to cause the superconducting fine particles to move due to the Meissner effect for the purpose of accelerating the reaction.

After this immunoreaction, transfer this solution to the flow cell shown in Figure 2 (
6). When a constant magnetic field is applied from the bottom to the top of the flow cell (6) during the introduction process, the superconducting fine particles gather on the surface of the permselective membrane on the oxygen electrode (1) due to the Meissner effect. After all the immune reaction solution has been introduced, a washing solution is introduced to wash and remove components other than those bound to the superconducting fine particles. Next, a buffer solution containing hydrogen peroxide was added to a blow cell (6
) and then stop feeding. By detecting oxygen generated by the decomposition of hydrogen peroxide by catalase using the oxygen electrode (1), the immune substance in the sample solution can be quantified using the same principle as in the prior art. Note that after the introduction of the immunoreaction solution, the series of steps is carried out with a constant magnetic field applied.

After the immune substance has been quantified, the magnetic field is removed, the cleaning solution is discarded, and the flow cell (6) and oxygen permeable membrane (2) are cleaned.

In this invention, room-temperature superconducting fine particles are used instead of an organic polymer membrane, so like a membrane, antigens that can bind to
There is no limit to the amount of antibody, which improves sensitivity, and there is no need to remove the membrane or regenerate the bound antibody when reusing it, making the operation simple and inexpensive.

Although catalase was used as the enzyme label in the above example, any enzyme may be used as long as it causes the disappearance or production of the electrode active substance during the reaction process. Further, although an oxygen electrode was used as the base electrode, any electrode or semiconductor sensor may be used as long as it can detect the electrode active substance. Furthermore, although the blow cell method was shown in the embodiment, the same effect can be obtained even if a batch method is used. Furthermore, although the method using a competitive immune reaction has been shown, the same effect can be obtained using a non-competitive immune reaction method such as the sandwich method.

[Effects of the Invention] As described above, according to the present invention, the immune substance to be detected is quantified using room-temperature superconducting particles to which an antibody against the immune substance to be detected is covalently bound. This has the effect of providing an enzyme immunosensor that can perform analysis.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing a room-temperature superconducting particle to which an antibody against an immune substance to be detected is covalently bonded, according to an embodiment of the present invention, and FIG. 2 is a diagram showing an enzyme immunosensor according to an embodiment of the present invention. FIG. 3 is a schematic cross-sectional view showing a conventional fermentation immunosensor. In the figure, (4) is an antibody against the immune substance to be detected, and (5) is a room-temperature superconducting particle to which an antibody against the immune substance to be detected is covalently bonded. In addition, in the figures, the same reference numerals indicate the same or corresponding parts. Agent Masu Oiwa No. 1 Epidemic No. 20-6; 1t (2 to 3 antibodies Fig. 3

Claims (1)

[Claims] An enzyme immunosensor for quantifying the immune substance to be detected using room-temperature superconducting particles to which an antibody against the immune substance is covalently bonded.