JPS61271457A - Immunological analysis - Google Patents

Abstract

PURPOSE:To enable analysis of a specified object to be inspected with a simple and small apparatus without any B.F separation (separation of a labeled antigen or antibody involved in antigen-antibody reaction from those not involved therein), by sucking a free labeled substance after an emulating reaction into a flowcell to make an enzyme reaction. CONSTITUTION:A specific amount of an antibody 2 which is subjected to an antigen-antibody reaction specifically with alpha-fetoprotein (AFP) is arranged on the inner wall of a reactor 1 as converted to a solid phase, a serum sample is injected into the container 1 and the AFP3 done into a specified amount of a labeled reagent with luciferine 4 and the AFP5 in the sample and the AFP3 labeled with luciferine 4 in the labeled reagent are subjected to a emulating reaction with the antibody 2. The, the reaction liquid in the container 1 is sucked into a flowcell 7 having luciferase 6 converted to a solid phase. Thus, the AFP5 and the AFP3 not involved in the antigen-antibody reaction are sucked into the flowcell 7, where the luciferine 4 bonded to the AFP3 is oxidized with the luciferase 6 as catalyst to emit light. The amount of the emission is received with a photodetector 8 to identify and assay the AFP5.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] This invention relates to an immunological analysis method that utilizes competitive reactions.

[Conventional technology]

Conventionally, immunoassay methods have been proposed that take advantage of the high specificity and detection sensitivity of antigen-antibody reactions and use antigens or antibodies labeled with a predetermined substance to analyze a predetermined antigen or antibody in a sample. PT using isotopes
A (radioimmunoassay), EIA (enzyme immunoassay) using enzymes, FIA using fluorescent substances (
Fluoroimmunoassay) etc. are known.

[Problem that the invention seeks to solve]

However, conventionally known immunoassay methods usually require so-called B/F separation, which separates labeled antigens (antibodies) that participated in the antigen-antibody reaction from those that did not, by washing. There is a problem in that the analysis operation is relatively troublesome, and the apparatus is therefore large and complicated.

This invention was made by focusing on these conventional problems, and allows a given test substance to be analyzed with simple operations without performing 8F separation, and therefore can be carried out using a simple and compact device. The purpose of this study is to provide a method for immunological analysis that can be performed.

[Means and effects for solving the problem] In order to achieve the above object, the present invention uses a sample and a sample labeled with a predetermined substance capable of enzymatically reacting the same substance as the analyte to be analyzed in the sample. A fixed amount of the labeled reagent is competitively reacted with the immobilized antigen or antibody in the reaction container to cause an antigen-antibody reaction, and the liquid containing the labeled reagent that did not participate in the antigen-antibody reaction is removed from the reaction container. The sample is sucked into a flow cell containing a predetermined enzyme and subjected to an enzyme reaction, and the test substance in the sample is analyzed based on the enzyme reaction.

〔Example〕

FIG. 1 is a schematic reaction diagram showing a first embodiment of the present invention. In this example, AF
P is identified and quantified using a labeling reagent labeled with luciferin. As is known, luciferin is excited by free energy when oxidized by oxygen molecules in the presence of luciferase, emits visible light, and returns to the ground state, as shown in the reaction formula below.

Therefore, in this example, a predetermined amount of antibody 2 that specifically reacts with antigen and antibody with AFP was immobilized on the inner wall of reaction vessel 1,
Serum as a sample and a predetermined amount of a labeling reagent in which AFP3 is labeled with luciferin 4 are injected into this reaction container 1, and AFP5 in the sample and AFP3 labeled with luciferin 4 in the labeling reagent are converted into antibody 2. make a competitive reaction. As a result, amounts of AFP5 and AFP3 corresponding to the amount of AFP5 in the sample bind to antibody 2 through antigen-antibody reactions, and the remaining AFP5 and AFP3 float in the reaction solution.

Next, the reaction solution in the reaction container 1 is sucked into the flow cell in which the luciferase 6 is solidified using a suction pump or the like.

As a result, AFP5 and AFP3 that did not participate in the antigen-antibody reaction are sucked into the flow self, where AFP
Luciferin 4 bound to 3 is oxidized by oxygen molecules in the liquid using luciferase 6 as a catalyst, resulting in luminescence.

Since this amount of light emission corresponds to the amount of AFP5 in the sample, it is received by the photodetector 8, and based on the output, AFP5 in the sample is identified and quantified.

As described above, according to this example, free luciferin 4 after the competitive reaction is sucked into the flow cell without performing B-F separation by washing, and the enzyme reaction is caused to occur, and the luminescence is measured. Since AFP5 is identified and quantified, the analytical operation becomes extremely simple. therefore,
If you are configuring a device that automatically performs this, B-
Since the washing device for F separation becomes unstable, the entire device can be made simple and compact.

FIG. 2 is a schematic reaction diagram showing a second embodiment of the present invention. In this example, whether or not AFP is produced in serum is identified and quantified using a labeling reagent that labels AFP with glucose (hereinafter abbreviated as GLLI). For this reason, in this example, a predetermined amount of antibody 12 that specifically reacts with antigen and antibody with AFP is immobilized on the inner wall of reaction container 11,
This reaction vessel 11 contains 4-aminoantipyrine (hereinafter abbreviated as 4.^A) and phenol as a coloring reagent. Next, the sample serum and AFP13 were
A predetermined amount of a labeling reagent labeled with Lυ14 is injected to cause a competitive reaction between AFP15 in the sample and AFP13 labeled with GLU14 in the labeling reagent against the immobilized antibody 12. As a result, antibody 12 has 8 F in the sample.
AFP15 and AFP13 in amounts corresponding to the amount of P15 are combined by antigen-antibody reaction, and the remaining AFP15 and AFP13 float in the coloring reagent.

Next, the reaction solution in the reaction container 11 is sucked into the flow cell 17 having the immobilized enzyme 16 using a suction pump or the like to cause an enzyme reaction. Note that the immobilized enzyme 16 uses glucose oxidase (COD) and peroxidase (POD). Here, AFP1, which was not involved in the antigen-antibody reaction,
GLU14 bound to 3 is an oxidoreductase C
Using OD as a catalyst, it is decomposed into gluconic acid and hydrogen peroxide as shown below, and the hydrogen peroxide is injected as a coloring reagent using POD, an oxidoreductase, as a catalyst.
It reacts with A and phenol to form its oxidized condensate.

4・AA+phenol+HzOz ---1) 1 (20+
4.AA, phenol (oxidized condensate) In the above enzymatic reaction, the oxidized condensate of 4.AA and phenol exhibits a color with a wavelength of 500 nn+, and the amount and therefore the concentration of the color corresponds to the amount of AFP15 in the sample. , this is photometered by a light projector 18, a filter 19 that transmits light with a wavelength of 500 nm, and a photodetector 20, and the AFP 15 in the sample is identified based on the output of the photodetector 20.
Quantify.

According to this embodiment, since color development by an enzyme reaction is finally utilized, there is an advantage that by immobilizing a predetermined enzyme on the flow cell 17, analysis can be performed using a normal colorimeter.

Note that the present invention is not limited to the above-described embodiments, and can be modified in many ways. For example, the test substance is not limited to AFP, but other antigens or antibodies can also be analyzed, and the labeling substance is not limited to luciferin or GLU, but other enzyme-reactive substances can be used.
Corresponding enzymes may be immobilized in the flow cell accordingly.

Furthermore, in the second embodiment, the coloring reagent was injected before the sample and the labeling reagent, but it can be injected at any timing before the sample and the labeling reagent are injected into the flow cell 17, at the same time as the sample and the labeling reagent, or after the sample and the labeling reagent. You can. Furthermore, in the second embodiment, a coloring reagent was used and the immobilized enzyme 16 was provided in the flow cell 17 for photometry. GL, which is a labeling substance that did not participate in the antigen-antibody reaction,
AFP15 in a sample can also be identified and quantified by measuring the concentration of Ul4.

〔Effect of the invention〕

As described above, according to the present invention, a free labeled substance after a competitive reaction is sucked into a flow cell and subjected to an enzymatic reaction, and a predetermined analyte in a sample is identified and quantified based on the enzymatic reaction. This eliminates the need for B-F separation by washing, which simplifies analytical operations.
This has the advantage that the device that implements this can be configured easily and compactly.

[Brief explanation of drawings]

FIG. 1 is a reaction schematic diagram showing a first embodiment of the present invention. FIG. 2 is a reaction schematic diagram showing a second embodiment.

Claims (1)

[Claims] 1. A sample and a predetermined amount of labeling with a predetermined substance capable of enzymatically reacting the same substance as the analyte to be analyzed in the sample are placed in a reaction container containing a predetermined amount of a predetermined immobilized antigen or antibody. Reagents are injected to cause these samples and labeled reagents to compete with the immobilized antigen or antibody to cause an antigen-antibody reaction, and then the immobilized antigen or antibody is removed from the reaction vessel, and the antigen-antibody reaction is performed on it. The liquid containing the unreacted labeling reagent is sucked into a flow cell having a predetermined solid-phase enzyme and subjected to an enzymatic reaction, and the test substance in the sample is analyzed based on the enzymatic reaction. immunological analysis method.