JPH0617915B2 - Method for measuring antigen-antibody reaction - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring an antigen-antibody reaction.

Immunoassay method using antigen-antibody reaction (ImmunoAsei)
Have recently made rapid progress. This immunoassay is
It is roughly classified into a labeling method and a non-labeling method.

Labels in the labeling method include radioactive isotopes, enzymes, fluorescent substances, electron spin resonance substances, luminescent substances,
There are metals, bacteriophages, electrochemically active substances, etc., and for the measurement of labeled antigens or antibodies, radioactivity, optical analysis of absorption / fluorescence / luminescence, electron spin resonance, polarography, etc. are used.

On the other hand, in the non-labeling method, for example, an insoluble particle preliminarily bound with an antigen (such as latex) is used to optically detect a change in turbidity associated with an agglutination reaction, an immunoturbidimetric method using no insoluble particle, The laser nephelometry method and the like are known.

All of these immunoassays are consistent in that they directly or indirectly measure the amount of a binding substance formed by a reaction between an antigen and an antibody.

However, in such an immunoassay, there is a problem in the measurement in the so-called "antigen excess region" in which the antigen is present at a certain concentration or more.

That is, a case of an example of the non-labeling method will be described.

For example, an antibody or an antigen supported on insoluble carrier particles is reacted with an antigen or an antibody in a liquid medium, and the decrease in the transmittance of the reaction mixture (that is, the increase in the absorbance) accompanying the progress of the reaction causes the antigen-antibody reaction. Is known, and the concentration of the antigen or antibody in the sample is measured from the measured speed. Then, according to this method, the concentration of the antigen or the antibody can be quantified rapidly with high accuracy.

However, for example, in the case of an antibody-sensitized insoluble carrier, in the region where the number of antigen molecules is excessive compared to the number of antibody molecules, the excess antigen blocks antibodies that would otherwise contribute to particle aggregation. Apparently, there is a phenomenon known as so-called antigen excess region in which the progress of antigen-antibody reaction is inhibited, and in such a case, there is a concentration of the mandrel corresponding to one reaction rate. become.

In clinical tests, not only the above-mentioned non-labeling method, but the frequency of appearance of antibodies that go through the above-mentioned antigen excess region is small, and in such cases, precautionary statements are added from other clinical findings, It was common to dilute and use for inspection.

However, due to the advent of automatic machines, when a large number of specimens are processed in a short time, although the appearance frequency is extremely low, a technique for discovering this kind of antigen-excess specimen that is clinically important is required.

For example, in order to perform accurate measurement in such a case, it is necessary to always perform a double dilution method or the like in which the dilution rate is changed for the same sample twice or more.

Therefore, the present inventors have variously studied to find a more suitable measurement method for rapid processing of a large number of samples by automation,
The present invention has been reached.

That is, the gist of the present invention is to make an insoluble carrier particle support an antibody or an antigen, and to cause the supported antibody or antigen to react with the antigen or the antibody or a mixture thereof in a liquid medium, and after starting the reaction with the reaction mixture. In the method of irradiating with light at two or more time points and measuring the decrease in the transmittance of the reaction mixture within a certain period of time, the amount of the reaction conjugate produced or obtained by using a sample with a known concentration of the antigen or antibody In the corresponding curve of the measured value of the velocity and the concentration, when a plurality of concentrations correspond to one measured value, in determining the concentration of the antigen or antibody in the unknown sample from this corresponding curve, (i) the antigen or antibody Of a reaction conjugate obtained by reacting a sample of unknown concentration with the corresponding antibody or antigen to measure the maximum value of the production rate (V _max ).
The determination criterion of the concentration measurable region that can uniquely determine the concentration depending on whether or not exists, and (ii) when the maximum value (V _max ) exists, it is determined that the concentration belongs to the concentration measurable region. Determining the concentration of the antigen or the antibody in the unknown sample from the above-mentioned corresponding curve.

Hereinafter, the present invention will be described in detail.

First, examples of immunoassays to which the present invention is applied include latex agglutination reaction and immunoturbidimetric method.

In the following, as an embodiment of the present invention, an insoluble carrier particle is supported with an antibody or an antigen, the supported antibody or antigen is reacted with the antigen or the antibody in a liquid medium, and the reaction mixture is reacted with 2 The case where the method is applied to the method (including the latex agglutination reaction) of irradiating light at the above-mentioned time point and measuring the decrease in the transmittance of the reaction mixture within a certain time will be described.

First, in this method, insoluble carrier particles having an average particle size of about 1.6 μ or less, preferably 0.1 to 1.0 μ are used,
An antibody or an antigen is carried (sensitized) on this, and the antigen or the antibody in the subject is reacted therewith, and the transmittance of the reaction mixture is usually 0.3 to 2.4 μ, preferably 0.6 to The concentration of the antigen or antibody in the test sample is measured by measuring with a light beam having a wavelength in the range of 1.4μ and determining the reaction rate.

As the insoluble carrier particles, an organic polymer substantially insoluble in the liquid medium used when performing the measurement and having the average particle size, for example, polystyrene, a latex obtained by emulsion polymerization such as styrene-butadiene copolymer,
Alternatively, an inorganic oxide such as alumina is used.

Such insoluble carrier particles (preferably latex particles) are loaded (sensitized) with an antibody or an antigen capable of reacting with the antigen or the antibody in the analyte to be measured by a conventional method.

The concentration of insoluble carrier particles sensitized with antibody or antigen is usually 0.
It is used as a suspension of 01 wt% or more, preferably about 0.1-1 wt%.

This sensitized carrier is allowed to react with an antigen or an antibody in a liquid medium under a certain condition, and the amount of decrease in the permeation rate of the reaction mixture after a lapse of a certain time after the initiation of the reaction is measured to measure the antigen-antibody reaction It can be measured quantitatively. It is desirable to measure this reduction rate after the start of the reaction between the sensitized carrier, which is a constituent of the reaction mixture, and the antigen or antibody in the test solution, at least after the progress of the antigen-antibody reaction is stabilized.

For this purpose, it is preferable to mix the sensitized carrier and the test liquid, preferably with stirring, and to measure the transmittance thereof, preferably at a time after, for example, 2 to 3 seconds after mixing.

Such an antigen-antibody reaction is measured, for example, as follows.

First, a sensitized carrier is prepared by sensitizing a certain antibody or antigen to insoluble carrier particles having a certain average particle diameter.
On the other hand, using the same antigen or antibody as the antigen or antibody contained in the test liquid (sample) to be actually measured, it is used at substantially the same concentration as the medium of the test liquid at various known concentrations. Standard test solutions with various concentrations contained in the liquid medium are prepared.

Next, the sensitized carrier and the standard test solution are mixed together, and the transmittance of the reaction mixture is measured over time when the progress of the antigen-antibody reaction is stable. For example, at the stage where the transmittance is constantly decreased, the decrease rate of the transmittance of each reaction mixture per unit time is measured for each of the test solutions having various concentrations.

Next, this reduction rate is plotted, for example, in a graph with the concentration of the antigen or antibody in the standard test solution as the abscissa and the reduction rate as the ordinate, and is plotted as the antigen or antibody concentration in the standard test solution. A corresponding curve with the rate of decrease of the transmittance of the reaction mixture per unit time (reaction rate) is obtained.

Therefore, for a specific antigen or antibody, a corresponding curve as described above is prepared in advance, and for a test liquid of unknown concentration containing the same antigen or antibody, a reaction rate similar to the above is measured, and By comparing with the corresponding curve, the concentration of the antigen or antibody contained in the test solution can be quantitatively measured.

The present invention, in the above-mentioned measuring method, in the reaction rate-concentration correspondence curve obtained using a sample of known concentration, when a plurality of concentrations correspond to one reaction rate, the concentration of the unknown sample from this correspondence curve Is useful for measuring.

That is, for example, in order to determine the concentration by measuring the reaction rate of a sample in which the concentration of the antigen is unknown, (1) the corresponding curve of the antigen concentration and the reaction rate is obtained in advance by measuring the sample in which the concentration is known. It is necessary that (2) the concentration be uniquely determined from the reaction rate of the unknown sample and this correspondence curve, but (2) does not generally hold. That is, as the antigen concentration increases, the reaction rate also increases at first, but a region where the degree of increase in the reaction rate decreases appears in the middle, and when the antigen concentration further increases, a region where the reaction rate decreases rather appears. , Also after the decrease
It may increase or decrease again.

In such a case, the concentration of the unknown sample cannot be uniquely determined because a plurality of concentrations correspond to one reaction rate. In the antigen excess region where such a phenomenon occurs, it cannot be expected that an agglutination reaction and a change in transmittance commensurate with the amount of existing antigen can be expected, and thus it can be said that the region is unsuitable for measuring the concentration of an unknown sample. Therefore, when measuring the concentration of an unknown sample, it is first necessary to measure whether or not the antigen concentration is in the concentration measurable region.

As a result of the determination, if it is known that the concentration is within that region, the concentration can be uniquely determined from the reaction rate and the corresponding curve.
If it is known that the concentration cannot be measured, it is possible to dilute the sample so that the sample becomes a measurable region for measurement.

Therefore, the measuring method of the present invention in the above latex agglutination reaction will be further described.

First, the time change of the production rate of a reaction conjugate obtained by reacting a sample of unknown antigen or antibody concentration with a latex reagent sensitized with the corresponding antibody or antigen is measured. For example, the transmittance from the initial point of time of the reaction (for example, about 30 seconds after the start of the reaction) to a certain time is 1 to
Measured at intervals selected from about 40 seconds, the average reaction rate obtained by dividing the magnitude of the slope by the first-order approximation by the initial transmittance is preferably measured, or the average reaction rate in the vicinity of a certain time point,
Etc. are measured.

The present invention responds well to the presence or absence of the maximum value (V _max ) of the production rate and whether or not it belongs to the concentration measurable region capable of uniquely determining the concentration of the antigen or antibody of the unknown sample. And has arrived at the present invention.

The concentration range in which this maximum value (V _max ) exists varies depending on the concentration particle size of latex, temperature, measurement wavelength, capture interval of velocity, etc., but in order to obtain the clinically necessary concentration range, these conditions must be met. It can be appropriately selected.

When V _max is present and it can be determined that it belongs to the concentration measurable range, the concentration of the antigen or antibody in the unknown sample can be determined from the corresponding curve. In this case, as the corresponding curve,
The concentration can be determined in a higher concentration range by using the corresponding curve of the maximum value V _max and the concentration prepared for the sample in which the concentration of the antigen or the antibody is known.

On the other hand, when the maximum value V _max does not exist, it is determined that the sample does not belong to the concentration measurable range, and the sample can be diluted for further concentration measurement.

The method for measuring an antigen-antibody reaction according to the present invention is particularly useful when a large amount of sample is treated in a short time as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 [Measurement of AFP (α-fetoprotein)] Measurement conditions (latex agglutination reaction) (a) Latex (LTX): Particle size 0.24 μm Concentration 1% AFP sensitization (b) Measurement system standard substance (Std.) 20μ diluent stabilizing solution and select 9 seconds after the start of the reaction as a measurement initial point of the buffer 500μ sensitized latex 20μ wavelength 0.94 .mu.m (c) speed, and measuring the transmittance at 8-second intervals, than this maximum value V _max I checked the existence.

The results are shown in FIGS. V _max was obtained up to a concentration of about 4000 ng / m (Fig. 6).

That is, up to a concentration of 4,000 ng / m, it is determined that the concentration can be measured.

Example 2 CRP (C-reactive protein) was prepared in the same manner as in Example 1 except that the latex particle size was 0.24 μm and the concentration was 0.73%.
About the maximum value V _max of 5,000 ng /
V _max was obtained up to m.

[Brief description of drawings]

1 to 7 are diagrams showing the relationship between the transmittance (T), the production rate (V) of the reaction conjugate and the time for AFP.

Front Page Continuation (56) References Japanese Patent Laid-Open No. 53-13492 (JP, A) Extraordinary Special Issue on Clinical Pathology No. 53 Immunoassay for Clinical Testing-Technology and Applications- (February 28, 1983) Japanese Society for Clinical Pathology P. 71-81 CLINICAL CHEMISTRY, Vol. 23, No. 8 (1977) P. 1456-1464

Claims (1)

[Claims] 1. An insoluble carrier particle is supported with an antibody or an antigen, and the supported antibody or antigen is allowed to react with the antigen or the antibody or a mixture thereof in a liquid medium, and the reaction mixture is reacted with two or more after initiation of the reaction. A method for measuring the decrease in the transmittance of the reaction mixture within a certain period of time by irradiating with light, and measuring the production amount or rate of the reaction conjugate obtained by using a sample with a known concentration of the antigen or antibody. When a plurality of concentrations correspond to one measured value in the curve of correspondence between value and concentration, in determining the concentration of the antigen or antibody in the unknown sample from this correspondence curve, (i) the concentration of the antigen or antibody is An unknown sample is reacted with the corresponding antibody or antigen to measure the time change of the production rate of a reaction conjugate, and the concentration is determined depending on whether or not the maximum value (V _max ) of the production rate exists. And (ii) when the maximum value (V _max ) is present, it is determined to belong to the concentration measurable region, and the antigen in the unknown sample or A method for measuring an antigen-antibody reaction, which comprises determining an antibody concentration.