JPS58173465A - Optical measuring method of antigen and antibody reaction - Google Patents

Abstract

PURPOSE:To shorten more greatly the measuring time than usual and to make the quantitative determination of an antigen or antibody possible with high accuracy, by irradiating a laser beam to a mixed reaction solution in an early stage of the antigen and antibody reaction and detecting the intensity of scattering light in the range of a specific scattering angle. CONSTITUTION:Scattering of light due to a composite produced by the antigen and antibody reaction is selectively detected by setting an angle theta formed by a laser beam axis passing through the center of a reaction covette 2 and by a photodetector 3 to 30 deg.-60 deg. by irradiating light flux from a He-Ne laser or a visible near infrared semiconductor laser 1 to the covette 2. An ordinary photoelectric conversion element is used for the detector 3. The raising rate of intensity in a fixed time (within about 5min) is found by measuring the intensity of the light scattering from an early stage of the antigen and antibody reaction and the measurement is made possible with high accuracy and high sensitivity in a shorter time than a method measuring the sattering intensity by setting a conventional angle theta at >=150 deg. in comparision with a calibration curve found by the antigen or antibody having already known concentration.

Description

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

More specifically, the present invention relates to a nephelometric immunoassay using a laser as a light source.

Accurately quantifying antigens or antibodies is an important issue not only in the medical field but also in a wide range of life science fields.In recent years, as the demand for quantitative measurement of antigens or antibodies has increased, high-precision and rapid measurement is becoming more and more important. The realization of this is strongly desired.

The quantitative method using the plate immunodiffusion method, which has been widely used in the past, has the drawbacks that it takes one to several days for measurement, and that the interpretation of the results is complicated and requires skill, and there are large individual differences. .

In recent years, a nephelometric immunoassay has been proposed that attempts to capture the formation reaction of antigen-antibody complexes using light scattering, and attempts have been made to improve operability and quantitative accuracy. The reality is that we need time,
This measurement method is still insufficient for emergency testing or high-speed processing of multiple samples.

In view of the above drawbacks, the present inventors have discovered that in nephelometric immunoassay using a laser as a light source, light scattered by antigen-antibody complexes is detected in the early stage of the reaction in which antigen-antibody complexes are formed in the test liquid. The present invention was completed by discovering the existence of a selective detection angle that changes extremely sharply and applying this to a method for quantifying antigens or antibodies.

According to the present invention, it is possible to measure an antigen-antibody reaction with good quantitative accuracy using a simple operation, and furthermore, it is possible to perform highly accurate measurements in a significantly shorter time than conventional nephelometric immunoassays.

In the present invention, a solution of an antigen or antibody to be measured is mixed and reacted with a corresponding antibody or antigen, this reaction mixture is irradiated with a laser beam, and the intensity of scattered light of the reaction mixture is measured at a scattering angle θ=30°C to 60°C. This is an optical measurement method for antigen-antibody reactions, which is characterized by selective detection of antigen-antibody reactions.

FIG. 1 shows one embodiment of the arrangement of a laser light source, a test sample serving as a light scattering source, and a photodetector according to the present invention.

Reaction cuvette 2. Test sample within 4. is laser light source 1
．． The specimen is irradiated with a constant amount of laser beam emitted from the specimen, and the incident beam is scattered depending on the state of the sample to be tested.

A photodetector 3 that measures this light scattering intensity and converts it into an electrical signal. is placed in a specific position relative to the reaction cuvette.

Laser light source 1. For example, a helium neon laser with an oscillation wavelength of 632.8 mm or an oscillation wavelength of 700 to 800 mm.
A visible and near-infrared semiconductor laser of 1.0 mm may be used, and a reaction cuvette 2. For example, a tester cuvette with an inner diameter of 5 mm, and a photodetector 3. may be a normal photoelectric conversion element.

Reaction cuvette 2. The laser optical axis passing through the center of the photodetector 3. Assuming that the angle formed by θ is θ, the temporal change in light scattering intensity accompanying the formation of an antigen-antibody complex differs depending on the scattered light detection angle θ.

A in FIG. 2(a) is the apparatus of the above-mentioned embodiment, and θ=50
This is a record of changes over time in the light scattering intensity S as the reaction progresses to form an antigen-antibody complex in the case of .

The light scattering intensity S starts from the time when the solution containing the antigen or antibody to be measured is mixed with the reagent containing the corresponding antibody or antigen.
begins to increase and maintains a constant level after time TA has elapsed.

In the so-called end point method, the final level after TA is used to quantify the antigen or antibody.

Therefore, the shorter the time it takes to reach the final level, in other words, the faster the rise speed of the light scattering intensity S is, the faster the measurement becomes possible.

In conventional nephelometric immunoassay, θ≧90
Whereas the forward scattered light of .degree. is detected, the method according to the invention makes use of the measurement area of the backward scattered light.

B in FIG. 2(a) similarly records the temporal change in the light scattering intensity S when θ=150°.

For comparison with the case of θ = 50°, the final amount of light reached was set to 10
The vertical axis is normalized as 0.

It was found that when θ=150°, the time TB required to reach a certain level is several times longer than TA.

Specifically, while TA is several minutes, TB is approximately several tens of minutes.

This indicates that backscattering has better detection sensitivity at the initial stage of antigen-antibody complex formation, and is sensitive to differences in the spatial distribution of light scattering intensity due to the size of antigen-antibody complex particles. This is thought to be due to this.

Conventional nephelometric immunoassays focused only on light scattering intensity, and as a result, forward scattered light was generally used. However, the present inventors focused on detection sensitivity for the initial process of antigen-antibody reaction. We found that backscattered light sensitively reflects the initial state of the reaction.

However, in the region of θ>30°, the scattered light signal itself becomes weak and the influence of light reflected by the reaction vessel becomes strong, resulting in a decrease in speed accuracy.

According to the present invention, the detection angle θ=30° C. to 60° C. is the optimum detection angle that is most sensitive to the rise of the antigen-antibody reaction and can realize side lighting with high accuracy.

If detection is carried out in this region, known electrical circuits can easily obtain a signal of sufficient level for subsequent processing.

Therefore, sensitive and highly accurate measurement is possible with a much shorter measurement time TA, which is a fraction of the measurement time TB of conventional nephelometric immunoassays.

Based on the knowledge that the temporal change in light scattering intensity varies depending on the detection angle of scattered light, we discovered the existence of an optimal detection angle in a specific region of backscatter that is sensitive to the initial process of antigen-antibody reaction. Regarding the application of the method as a rapid quantitative method, there is no example mentioned before the technical disclosure related to the present invention.

The results of determining the calibration curve of human immunoglobulin G (hereinafter abbreviated as IgG) using the apparatus of the above example are the results of the third experiment.
It is a diagram.

That is, 300 μl of anti-IgG serum (sheep) 12.5-fold dilution
Add 30μ■ of a 301-fold dilution of standard serum containing human IgG antigens at various concentrations to ■, incubate for 5 minutes at room temperature, read the output of the photodetector of the above device, The relationship between the scattering intensity S) - (light scattering intensity S before the start of the reaction) and the concentration was determined.

A sufficient calibration relationship for quantitative measurement was obtained with a reaction time of 5 minutes.

Conventional endpoint methods require 30 minutes to 1 hour for similar measurements.
It is clear that the effects of the present invention are significant, also from the fact that it takes a long time.

Furthermore, a method of quantifying the concentration of antigen or antibody from the rate of change in light scattering intensity (hereinafter referred to as dynamic nephelometric immunoassay) allows for even faster measurement.

FIG. 2(b) is a recorded signal (dS/dT) obtained by performing differential processing with respect to time on A in FIG. 2(a), which is the waveform of the time-dependent change in light scattering intensity according to the present invention. be.

In dynamic nephelometric immunoassay, for example, the maximum value P of the differential signal is used for quantifying antigen or antibody.

The time TC from the reaction time to the appearance of P is the time required for measurement, and since TC<TA, the measurement time is further shortened than in the end point method.

Using the device of the above example, platelet fibrinogen (
Hereinafter, it will be abbreviated as FIB. Figure 4 shows the results of a calibration curve obtained by dynamic nephelometric immunoassay using P.

That is, anti-FIB serum (rabbit) 12.5-fold diluted solution 300
50μ■ of standard plasma diluted 21 times containing FIB antigen at each concentration was added to μ■, and the output of the photodetector of the above device was processed by a known differential electric circuit, and the maximum value P of the differential and the concentration were I wanted a relationship.

A sufficient calibration relationship for quantitative measurement was obtained within 2 minutes of measurement time.

Dynamic nephelometric immunoassay requires high detection sensitivity in the initial stage of the reaction, and it is clear that the present invention, which allows sensitive detection immediately after the start of the reaction, is significantly effective.

FIG. 5 is a block diagram of an actual antigen-antibody reaction measuring device according to the present invention.

The light scattering intensity signal converted into an electric signal by the photodetector is sent to a signal preprocessor 5. is input.

Signal preprocessor5. In dynamic nephelometric immunoassay using P, electrical amplification is performed, and differential processing is further performed, and the results are sent to an A/D converter 6. Enter.

Computer 7. calculates the concentration of the antigen or antibody by processing the information of the light scattering intensity signal converted into a digital quantity by the A/D converter.

It is well known that various procedures other than the above-mentioned examples have been proposed as a method for calculating the concentration of an antigen or antibody, and that various programs corresponding to each procedure can be created, but this does not limit the embodiments of the present invention. do not have.

Furthermore, there is a display/print section 8 for displaying data processed by the computer. Recorder to continuously observe the reaction process 10. and reagent dispensing mechanism automatic sample preparation mechanism9. The device is configured by:

As described above, it is clear that an automated and simplified antigen-antibody reaction measuring device can be easily realized by combining the present invention and existing techniques.

Although the embodiments of the present invention have been described above, the present invention is not limited to these embodiments, but can be applied to various specific examples within the scope of the present invention.

As mentioned above, the present invention can capture the initial process of antigen-antibody reactions with high sensitivity and is also excellent in quantitative relationships, so it is highly accurate and can significantly shorten measurement time compared to conventional methods. This makes it possible to realize measurements and greatly contributes to the abbreviation and automation of measurements.

Further, according to the automatic measuring device according to the method of the present invention, emergency testing or high-speed processing of a large number of samples can be performed quickly, accurately, and with high sensitivity.

[Brief explanation of drawings]

FIG. 1 is a diagram of the principle of the side light system according to the present invention, FIG.
Figure 3 is a calibration characteristic diagram for antigen concentration and light scattering intensity in IgG measurement, and Figure 4 is a calibration diagram for maximum rate of change in antigen concentration and light scattering intensity in FIB measurement. The characteristic diagram, FIG. 5, is a block diagram of an antigen-antibody reaction measuring device to which the present invention is applied. 1. Laser light source 2. Reaction cuvette 3. Photodetector6. A/D converter7. Computer patent applicant Wako Pure Chemical Industries, Ltd.

Claims (2)

[Claims] (1) Mix and react a solution of the antigen or antibody to be measured with the corresponding liquid or antigen, irradiate this reaction mixture with a laser beam, and measure the intensity of the scattered light of the reaction mixture at a scattering angle θ = 30°C to 60°C. An optical measurement method for antigen-antibody reactions characterized by selective detection. (2) The method for optically measuring an antigen-antibody reaction according to claim 1, wherein the antigen or antibody to be measured is quantified from the rate of change in intensity of scattered light of the reaction mixture.