JPS63177061A - Control of immunoreaction - Google Patents

Abstract

PURPOSE:To accurately analyze and measure an analyte without diluting a specimen, in the reaction of the analyte with a labelled ligand, by allowing a factor inhibiting said reaction to act. CONSTITUTION:In the reaction of an analyte or a modified substance thereof with the corresponding labelled ligand, a factor inhibiting said reaction is allowed to coexist in a reaction liquid or the factor inhibiting said reaction is allowed to preliminarily act on the analyte or the modified substance thereof. Thereafter, the labelled ligand is allowed to act to control the measurable concn. region of the analyte or the modified substance thereof. By this method, the analyte can be accurately analyzed and measured without diluting a specimen to a large extent.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for adjusting measurement sensitivity when quantifying a substance using an immunochemical reaction. A method for adjusting the measurable concentration range of an analyte by using a factor that inhibits the reaction with a labeled ligand. This makes it possible to measure

The present invention is effective for analysis and measurement of various substances, especially trace analysis of biological components, so it can be used for medical treatment, diagnosis, clinical test 1 analysis,
It is heavily used in various technical fields such as quantitative determination.

(Prior Art) In recent years, with the development of medicine, the physiological significance of many trace components contained in biological body fluids has become clear. Along with this, accurate methods for quantifying trace components in these biological body fluids are becoming increasingly important in the field of clinical testing. A labeled antibody method using a labeled antibody and an insoluble carrier in which the antibody is insolubilized is known as one of the methods for quantifying these trace components.

In this labeled antibody method, an antigen is bound to an antibody-insolubilized carrier, a labeled antibody is bound to the antigen bound to the carrier, and the amount of labeled antibody bound to the carrier is quantified. This method determines the amount of antigen. In addition, a method of measuring the amount of antibody using an antigen-insolubilized carrier and a labeled secondary antibody (7) is already known, in which the antigen is labeled, the antigen is made to compete with the antigen, and the antigen amount is measured by binding with the labeled antibody. It is being

On the other hand, biological body fluids contain a wide variety of components at various concentrations, and even the same component has individual differences, so clinically necessary measurements of each objective measurement component in the field of clinical testing are required. The concentration range that must be possible (hereinafter referred to as the "required range") differs for each component. However, even in the labeled antibody method, the performance of the antibody used, the performance of the labels such as enzyme radioactive substances, fluorescent substances, etc., the reaction conditions, etc., and the measurement system assembled using these (hereinafter referred to as "kit") There is a concentration range (hereinafter referred to as "measurement range") of the sample that can be measured accurately.

Therefore, the most important thing for each kit is that the required range is included in the measurement range.

To solve this problem, in the labeled antibody method, there are currently only a few methods in which the sample is diluted very thinly in advance, especially for the following components to be measured: β2-microglobulin (hereinafter referred to as “β-anal”), α1-microglobulin, C-reactive protein, immunoglobulin (IgG, IgA, IgE, etc.), complement components (C3,04, etc.), transferrin, protein C ( protein S), various hormones or hormone-like substances (triiodothyronine, thyroxine-binding globulin, cortisol-binding protein, sex hormone-binding globulin, hPL (hu an Placental Lac)
togen) etc.).

As an example, BMG is a measurement component that is currently frequently measured in the field of clinical testing as an index for determining the efficacy of diagnosis and treatment of renal dysfunction and various malignant tumors. Enzyme immunoassay (hereinafter referred to as "enzyme immunoassay") is a type of labeled antibody method that is currently widely used.

When measuring BMG by RFIAJ (sometimes referred to as rFIAJ), use a sample diluted 100 to 1000 times with a buffer solution at a constant concentration of 10 to 100 μQ'/l to match the measurement range characteristics of each kit. There is.

This extensive dilution operation is not only very complicated, but also
Even in the development of a fully automatic measuring machine that aims to quickly process a large number of samples, this increases the complexity of the machine and leads to higher costs. In addition, it is theoretically possible to measure the sample without diluting it to a large extent, which may adversely affect measurement accuracy. The amount of sample to be used is 10-1000nQ.
However, since the amount of sample liquid is too small, it is not possible to dispense the sample accurately.
Therefore, it is inevitable that the measurement accuracy will be greatly reduced, and the current situation is that it is not put into practical use.

Therefore, at present, although complicated, this method which requires a large amount of dilution operation is unavoidably put into practical use.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned complicated and time-consuming measurement method that utilizes the reaction between an analyte and a corresponding labeled ligand, such as the labeled antibody method. This was done with the aim of developing a new analytical method that does not require extensive dilution operations.

(Means for solving the problem) As a result of conducting research from various angles to achieve the above purpose, it was completely unexpected that the analyte, which is the component to be measured, and its corresponding By applying a factor that inhibits this reaction during the reaction with a labeled ligand, it is possible to move the sample to a higher concentration range, in other words, without significantly diluting the sample.
We discovered that analytes can be measured accurately.

As a result of further investigation, the reaction inhibitory factor is the analyte and ml! It was also confirmed that the same excellent effect can be obtained even if the analyte is reacted with the analyte before reacting with the 'l ligand.

The present invention was completed as a result of further study and research based on these new findings.

In the present invention, an analyte is an object to be measured, and a ligand is a substance that has a biological correlation with the analyte. This serves as a ligand. These analytes (ligands) include a wide variety of substances, including the following biological components: antigens (antibodies), antibodies (secondary antibodies), antibodies (anti-idiotypes). antibodies), hormones (receptors), DNA
(sRNA), yeast l! 4 (substrate 1 reaction product, coenzyme, competitive inhibitor).

The analyte may be modified to further increase its reactivity with the ligand by binding it with avidin, biotin, protein A, etc., or conversely, it may be modified with avidin, biotin, protein A, etc. to further increase its reactivity with the ligand. It is also possible to carry out modifications by removing parts that do not exist or by forming subunits.

The ligand is labeled to facilitate measurement after the reaction, and this labeled ligand (hereinafter also referred to as "labeled ligand") is used in the present invention. The labeling treatment may be carried out according to a conventional method, and enzymes, fluorescent substances, radioactive substances, ferritin, etc. are appropriately used as labels according to a conventional method.

In the present invention, a factor that inhibits the reaction between the analyte (and/or a modified substance thereof) and a labeled ligand acts on the reaction, thereby allowing the analyte to be accurately reacted without diluting the sample to a large extent. (modified substance) analysis, 311
This makes it possible to determine the Therefore, the factor can be any substance as long as it inhibits the above reaction, for example, it reacts with the analyte (specifically, the anti-JM antibody to be measured) or its modified substance. In addition to antibodies, antigens, or modified fragments thereof, substances containing these (blood, serum, body fluids, etc.) can also be used in some cases. In addition, it is also possible to use a substance that weakens the immune response, such as an eluent such as ethylene glycol, and buffers, lysis solutions, substrates, and reaction inhibitors can also be used in some cases. . It should be noted that these substances are merely mentioned for illustrative purposes, and it goes without saying that any substance that inhibits the above reaction can be used. The factor is adjusted and added to an appropriate concentration, taking into consideration the amount of analyte, the binding property with the labeled ligand, the type of specimen, and other conditions.

This reaction inhibitory factor only needs to act on the above reaction, and the timing of its addition does not matter. Therefore, the factor may be present in the reaction solution during the reaction between the analyte and the labeled ligand, or it may be added in advance. After the analyte and the factor are allowed to act, a labeled ligand may be made to act.

Next, embodiments of the present invention will be described.

Example 1 An antibody against BMG obtained from a rabbit was immobilized on the inner surface of a polystyrene test tube (hereinafter referred to as a "immobilized" tube). A glucose oxidase-labeled antibody was obtained from a rabbit. "Enzyme immunoassay" 2nd edition (Igaku Shoin) P, 105-P. using anti-BMG antibody and glucose oxidase.

It was produced according to the method described in 108.

The same antibody used for immobilization was used as the inhibiting factor, and the antibody concentration was 10 μg/IQ containing 0.1% albumin and 100 mM sodium chloride.
It was prepared using ffiM phosphate buffer (hereinafter referred to as "dissolution solution").

On the other hand, 300 ma.
= 1-base of 50 N phosphate buffer (pH 7.5) containing 10 g of peroxidase and 50% glycerol;
Then, add 10 m of 15 N of 4-aminoantivirine solution.
fl were sequentially added to prepare a substrate solution.

First, add 0 solution to each of the 9 in-phase tubes.
．． Add 5IQ and 0.1+l of inhibitory factor solution
0th order with 1fl added, BNG in advance by 0.01°0.03.0.1.0.3.1.0.3.0.10.3
Each solution prepared at a concentration of 0.1100 u/atfi was dispensed in an amount of 10 μΩ and stirred. 2 at 37℃
After reacting for an hour, the reaction solution was removed, and after washing with phosphate buffer, the glucose oxidase-labeled antibody was added at 0.1 u/m
0.6 Q of the diluent dissolved in the diluent was added and stirred.

After standing at 37° C. for 2 hours, 1 mff1 of the substrate solution was added and stirred. After being left at 37° C. for 90 minutes, the absorption of the reaction solution at 505 nm was measured using a spectrophotometer. The relationship between the BNG concentration and the absorbance of each standard solution obtained was plotted on a logarithmic scale graph, and the results shown in FIG. 1 were obtained.

Example 2 Solid-phase tubes and enzyme-labeled antibodies prepared in the same manner as in Example 1 were used. The inhibiting factor is different from the rabbit from which the antibody used for immobilization was obtained. An antibody against BMG obtained from another rabbit was used, and its concentration was 0.
10.100.500ug/+12 was prepared.

First, 0.4raQ of diluted solution was added to each of the 9 solid phase tubes.
First, 0.1 ml of the inhibitory factor at a concentration of Oug/mR was added.

Thereafter, in the same manner as in Example 1, the same operation was repeated for nine types of BMG standard solutions, changing the concentration of the zero-order inhibiting factor solution whose absorbance was determined to 100.500 ug/+aR. The relationship between the concentration of the standard solution and the absorbance with respect to the concentration of each inhibiting factor obtained was plotted on a graph on a logarithmic scale, and the results shown in FIG. 2 were obtained.

Example 3: Immobilized tubes and enzyme-labeled antibodies prepared in the same manner as in Example 1 were used. For the inhibitory factor, an antibody against BMG obtained from goats was used, and the concentration was adjusted to 50%.
It was adjusted to ug/mfl. Thereafter, in the same manner as in Example 1, the absorbance of 505rv+ for each standard solution was determined. The obtained results are plotted on a logarithmic scale graph and shown in FIG.

Example 4 An antibody against BMG obtained from a goat was immobilized on the inner surface of a polystyrene test tube by a conventional method.

As the inhibitory factor, an antibody against BMG obtained from a goat was used, and the concentration was adjusted to 0.1 mg/an.

First, add 0.5 mQ of the solution to the 9 in-phase tubes mentioned above.
Further, the same standard solution as in Example 1 was dispensed in 10 μΩ portions each, and 100 μΩ portions of the 6th order inhibiting factor with a concentration of Omg 7 mfi were added and stirred. The following operations were performed in the same manner as in Example 1.

Next, the same operation as above was performed for the inhibitory factor having a concentration of 1 ragl-0. The results obtained ・
The results are plotted on a logarithmic scale graph and shown in FIG.

(Effects of the Invention) As shown in these examples, if the method of the present invention using this inhibiting factor is used, measurement can be performed without the large amount of dilution of the sample, which has been complicated up until now, and measurement accuracy can also be maintained. This makes it ideal for rapid processing of large quantities of specimens. And also in pushing forward with fully automated measurement using machines.

There is no need for pre-treatment operations such as extensive dilution of the specimen.
This is advantageous because it can be designed more simply.

Furthermore, in the method of the present invention, the measurement range can be finely adjusted by changing the amount of the inhibiting factor. In other words, as the amount of the inhibiting factor to be acted on increases, the measurement range moves to a high concentration range, which is convenient because it can optimally accommodate the slightly different required ranges for each biological body fluid.

[Brief explanation of the drawing]

Figure 1 shows the B of the standard solution obtained in Example 1.
The relationship between MG concentration and absorbance is plotted on a logarithmic scale graph, and each point is connected by a curved line (hereinafter referred to as a "calibration curve"). FIG. 2 is based on the results obtained in Example 2. A calibration curve is drawn for each concentration of the added inhibitory factor.
100u/lIQ is 10-, 500ug/mQ
Those shown are -Δ-, respectively. ) FIG. 3 depicts the results obtained in Example 3 as a calibration curve. (In order to make it easier to judge the effect of the inhibiting factor, the calibration curve obtained when the inhibiting factor is not added (see Example 2) is also appended with...)

Claims (6)

[Claims] (1) In the reaction between the analyte or its modified product and the corresponding labeled ligand, a factor that inhibits this reaction is allowed to coexist in the reaction solution; or a factor that inhibits this reaction and the analyte or its modification 1. A method for adjusting the measurable concentration range of the analyte or its modified substance, the method comprising: reacting the analyte or its modified substance with a labeled ligand in advance; (2) The method according to claim 1, wherein the factor that inhibits the reaction is an antibody corresponding to the analyte. (3) Claim 1, characterized in that the analyte is an antigen and the ligand is an antibody corresponding to the antigen.
The method described in section. (4) The method according to claim 1, wherein the analyte is an antibody and the ligand is a second antibody against the antibody. (5) The method according to claim 1, wherein the analyte is an antibody and the ligand is an anti-idiotypic antibody against the antibody. (6) Claim 1, characterized in that the analyte is an antibody and the ligand is an antigen that reacts with the antibody.
The method described in section.