JPS61173136A - Method for measuring immune reaction by intensity fluctuation of light - Google Patents

Abstract

PURPOSE:To improve measurement accuracy by removing the frequency component higher than the relaxation frequency of powder spectral density in the stage of measuring immune reaction by making use of the intensity fluctuation of scattered light by fine particles. CONSTITUTION:The immune reaction based on an antigen-antibody reaction is measured by making use of the intensity fluctuation of the scattered light by fine particles. A laser beam 2 from a light source 1 is made into a beam 4 by a semi-transparent mirror 3 in this stage. Said beam is condensed by condenser lens 6 and is projected to a cell 7 in which the antigen-antibody reactive liquid contg. the fine particles 9 is contained. The scattered light thereof is made incident through a collimator 10 on a photodetector 11 and the output thereof is supplied via a low-pass filter 16 which removes the frequency component substantially higher than the relaxation frequency of the detected power spectral density to a data processing unit 14. The measurement with high accuracy is thus made possible without being affected by noise, etc. even if the particle concn. and the quantity of the measuring light are low.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a method for measuring an immune reaction based on an antigen-antibody reaction using intensity fluctuations of light scattered by fine particles.

(Prior art) There are immunoassay methods that utilize specific selective reactions of immune reactions as a method for measuring trace components in living bodies such as immune substances, hormones, pharmaceuticals, and immunomodulators.They can be roughly divided into methods that use enzymes or radioactive isotopes as labeling substances. Two methods are well known: labeled immunoassay and unlabeled immunoassay, which directly measures antigen-antibody complexes.

The former labeled immunoassay is radioimmunoassay (
RIm), Engineering, Enzyme, Immunoassay (EIm).

Fluoroimmunoassay (FIM) and the like are well known. However, although these analytical methods are highly sensitive, they require a long time for measurement and the labeling reagents are expensive, so they have drawbacks such as high testing costs. There are various restrictions such as processing.

In addition, the latter non-labeled immunoassay methods include immunoelectrophoresis, immunodiffusion, and precipitation, and although they are simple analytical methods, they are insufficient for precise measurements in terms of sensitivity, quantitative performance, and reproducibility. However, there is a drawback that the measurement time becomes longer. Regarding this type of immunoassay method, please refer to the "Clinical Test Methods Recommendation" (written by Izumihara Kanai,
Edited by Masamitsu Kanai, Metal M Edition>'p, "Clinical Examination J Vol.
, z 2 t'5 (1978), pages 471-487.

Furthermore, “Immunochemistry J.
Vol, 12, &4(19)5), No. 349-8
On page 51, particles carrying antibodies or antigens on their surfaces are reacted with antigens or antibodies in a liquid to be measured, and the average diffusion constant, which is an index of Brownian motion, decreases in proportion to the size of aggregated particles. A method for quantitatively analyzing antigens or antibodies by determining from changes in the spectral width of scattered laser light is disclosed. This analysis method has the advantage of not using labeled reagents, but it uses a spectrometer to detect the broadening of the spectrum of incident light due to the Doppler effect caused by the Brownian motion of particles, so the disadvantage is that the equipment is large and expensive. In addition to this, errors occur when mechanically driving the spectrometer,
It has the disadvantage of poor accuracy and reproducibility. Furthermore, this method only calculates the average diffusion constant from the spectral width of light, and has the disadvantage that the amount of information is small.

As mentioned above, conventional immunoassay methods use expensive labeling reagents, resulting in high analysis running costs, troublesome liquid handling and processing, long processing times, and high costs. If a large spectrometer is required, the accuracy and reproducibility are poor, and the amount of information obtained is small.

In order to overcome these drawbacks, the antigen-antibody reaction can be measured by taking advantage of the fact that the intensity fluctuation of light scattered by fine particles is closely related to the antigen-antibody reaction. Moreover, it is possible to perform measurements with high precision and reproducibility without using a large spectrometer, and it is also possible to shorten the measurement time and automate antigen-antibody reaction measurements, as well as to improve the accuracy of antigen-antibody reactions. A patent application was filed in 1983 for an immune reaction measurement method that can obtain a lot of useful information.
No. 148878.

This immune reaction measurement method involves projecting coherent or incoherent radiation onto an antigen-antibody reaction solution containing at least an antigen and an antibody, and in addition to light scattered by particles generated by the antigen-antibody reaction or the reaction solution. This method detects the scattered light generated by the antigen-antibody reaction of fine particles immobilized with antibodies or antigens in a homodyne or heterodyne manner, and measures the antigen-antibody reaction based on the power spectrum density of the intensity fluctuation of this detection output. be.

In such an immune reaction measurement method, the intensity of scattered light generated by microparticles as a result of an antigen-antibody reaction, or the intensity of scattered light generated by an antigen-antibody reaction of microparticles on which antibodies or antigens are immobilized, is determined by light interference. We focused on the fact that the power spectral density of this intensity fluctuation depends on the shape and size of the particle, and by detecting the power spectral density of the intensity fluctuation, we can determine whether there is an antigen-antibody reaction or not. It is possible to obtain a lot of useful information such as quantitative determination and the aggregation state (particle size distribution) of microparticles due to antigen-antibody reactions. In addition, since the scattered light is received by a photodetector and fluctuations in the output signal intensity are detected, there is no need to use a labeling reagent, and a spectrometer is not required since the method does not perform spectrum analysis of the scattered light. Nor. Specifically, a method for detecting antibody or antigen concentration is to detect scattered light in a homodyne manner and utilize the fact that the relaxation frequency of the power spectral density of the intensity fluctuation depends on the particle size to detect the antigen-antibody concentration. The ratio of the relaxation frequencies before and after the reaction is determined, and from this ratio value, the antigen-
Methods have been proposed to measure antibody responses.

Here, the relaxation frequency of the power spectral density is inversely proportional to the particle size, and is approximately 400 Hz for a particle size of 0.0915 μm.
, at 0.188 μm, it is approximately 200 Hz, and at 0.805 μm, it is approximately 100 H2. Therefore, when determining the relaxation frequency, it is not necessary to use a frequency much higher than the relaxation frequency, for example, if the relaxation frequency is expected to be around 200 Hz, a high frequency above about 1 o xaz is not necessary; The high frequency component contains a lot of noise from the photodetector that receives the scattered light, which causes measurement errors.

Furthermore, if the particle concentration or measurement light amount is too low, the level of the signal component may become comparable to the noise level on the high frequency side, making measurement difficult.

(Objective of the Invention) An object of the present invention is to solve the above-mentioned problems and to provide a method that can always measure an immune reaction due to light intensity fluctuation with high accuracy without being affected by noise.

(Summary of the Invention) The present invention projects radiation onto a reaction solution containing an antigen and an antibody, detects scattered light by fine particles in the reaction solution, and detects the antigen and antibody based on the power spectrum density of the intensity fluctuation of the detection output. In measuring the antibody reaction, the power spectral density is determined after removing high frequency components higher than the relaxation frequency of the power spectral density of the detection output using a low-pass filter.

(Example) FIG. 1 is a diagram showing the configuration of an example of an apparatus for implementing the immune reaction measuring method of the present invention. In this example, the light source 1 emits coherent light with a wavelength of 682.8 nH [8
- Using N8 gas laser. As the light [1 that emits coherent light], in addition to such a gas laser, a solid laser such as a semiconductor laser can also be used. Furthermore, the method of the present invention can also use a light source that emits incoherent light. A laser beam 2 emitted from a light source 1 is separated into a beam 4 and a beam 6 by a semi-transparent mirror 8,
One light beam 4 is focused by a condensing lens 6 and projected onto a transparent self, and the other light beam 6 is made incident on a photodetector 8 consisting of a silicon 7 photodiode to vary the output light intensity of the light source 1. Convert to a monitor signal representing

The self contains an antigen-antibody reaction solution, which is a mixture of a buffer solution in which fine particles 9 having antibodies or antigens bound to their surfaces are dispersed, and a test solution containing the antigen or antibody. Therefore, an antigen-antibody reaction occurs in the self, interactions occur between fine particles, and fine particles adhere to each other, resulting in a change in the state of Brownian motion. The scattered light scattered by the microparticles 〇 in the self passes through a collimator 10 having a pair of pinholes and is made incident on a photodetector 11 consisting of a photomultiplier tube. The collimator 10 has a dark box structure to eliminate the influence of external light, and its inner surface is subjected to antireflection treatment, and pinholes are formed at the front and rear of the dark box.

The output monitor signal of the photodetector 8 is supplied to the data processing device 14 via the low-noise amplifier 13, and the output signal of the photodetector 11 is supplied to the low-noise amplifier 15 and the low-pass filter 1.
6 and then supplied to the data processing device 14. The data processing device 14 includes an A/D conversion section 17. High-speed 7-layer converter 1
8 and an arithmetic processing section 19, this data processing device 1
4, the output signal of the photodetector 11, that is, the intensity of the scattered light from the self, is used to remove fluctuations in the intensity of the laser light emitted from the light source 1 by outputting the short-term average value of the light source intensity monitor signal from the photodetector 8. After normalization, the relaxation frequency of the power spectrum density of the intensity fluctuation of the scattered light is determined, and based on this, the aggregation state of the fine particles 9 in the self,
Therefore, the antigen-antibody reaction is measured, and the measurement results are supplied to the display device 20 for display.

In this embodiment, the output signal of the photodetector 11 is supplied to the data processing device 14 through a low-pass filter 16, but this low-pass filter 16 removes frequency components sufficiently higher than the relaxation frequency of the detected power spectral density. The cutoff frequency is approximately 10 K11z when latex particles having a particle size of about 0.1 to 0.8 μm are used as the fine particles 9.

In this way, if the frequency component sufficiently higher than the relaxation frequency of the output signal of the photodetector 11 is removed by the low-pass filter 16, the noise of the photodetector 11 can be suppressed even when the particle concentration or the amount of measured light is low. The relaxation frequency can be detected with high accuracy without being affected by

Figures 2 and 3 show that in the measuring apparatus shown in Figure 1, a buffer solution adjusted to pH 7 with Tris-110t was used.
Immunoglobulin G antibody was immobilized on the surface of latex particles with a diameter of 0.3 μm, and l O''' as an antigen.
Power spectrum before the start of the antigen-antibody reaction and after the start of the R (15 minutes later) when the antigen-antibody reaction solution containing immunoglobulin G at f/ml and 109/ml (D concentration) is housed in Cerua. 2 and 8, the cutoff frequency of the power spectral density halo bass filter 16 is 1.
It drops rapidly from 0KHz. Therefore, it is possible to detect each relaxation frequency with high accuracy without being affected by high frequency components, not only in the case of high concentration as shown in Fig. 2, but also in the case of low concentration as shown in Fig. 8. In the case of an antigen concentration of 10-'9/ml shown in FIG. 2, the relaxation frequency before the reaction is approximately 50 Hz, whereas the relaxation frequency 15 minutes after the reaction has changed to 10 Hz. On the other hand, the antigen concentration IQ-9 shown in FIG.
In the case of 9/mj, the relaxation frequency before the reaction starts is about 95 Hz, and the relaxation frequency after the reaction is about 40 Hz. Therefore, the ratio F of the relaxation frequencies before and after the antigen-antibody reaction is defined as the relaxation frequency after the antigen-antibody reaction, and this value is determined for several known antigen concentrations to calculate the ratio F of the antigen concentration and relaxation frequency. If a calibration curve representing the relationship is determined in advance, the antigen concentration can be determined from the calibration curve by detecting the ratio F of relaxation frequencies at an unknown antigen concentration.

Note that the present invention is not limited to the above-mentioned example, and can be modified or changed in many ways.

For example, in the above-mentioned embodiment, the direction of the light beam 4 incident on the self and the optical axis direction of the collimator 10 are set at 90 degrees, and a homodyne method is adopted in which the incident light beam does not directly enter the photodetector 11, but the incident light beam It is also possible to adopt a heterodyne method in which a part of the light is incident on the photodetector 11. Furthermore, the display on the display device 20 is not limited to the relaxation frequency, but may also display power spectral density data.
The antigen concentration may be displayed by further performing necessary calculations based on the relaxation frequency obtained by calculation. Furthermore, although the above explanation has been given as an example of immunoglobulin G (I9G), immunoglobulin A (IgA) is used.

XyM, IgD, XriE, Australian antigen, syphilis antigen 1. It can be applied to the measurement of all substances that cause agglutination due to antigen-antibody reactions, such as insulin. Furthermore, in the above-mentioned embodiments, antibodies were immobilized on the surface of microparticles to detect antigens in the specimen, but it is also possible to immobilize antigens on the surface of microparticles and detect antibodies in the specimen. can. Furthermore, although polystyrene latex particles were used as the fine particles in the above embodiments, other organic particles or inorganic particles such as glass may also be used.

Furthermore, in the above-mentioned example, fine particles were present in the antigen-antibody reaction solution from the beginning, but instead of using such fine particles, the scattered light by the fine particulate products generated as a result of the antigen-antibody reaction could be absorbed. You can also use it. An example of such an antigen-antibody reaction is a reaction using human chorionic gonadotropin (HOG) as an antigen and anti-human chorionic gonadotropin (anti-HOG) as an antibody, and the antigen-antibody complex generated by this reaction is The body can be treated as a particle. Furthermore, the antigen itself can also be used as particles. In such an antigen-antibody reaction, Candida albicans (yeast) is used as the antigen.
Examples of using anti-Candida albicans as an antibody,
In addition, blood cells, cells, microorganisms, etc. can also be used as particles. Furthermore, in the embodiment shown in FIG. 1, a patch method was adopted in which the antigen-antibody reaction solution was contained in a cell and the measurement was carried out.
Measurement is performed while continuously flowing the antigen-antibody reaction solution 7
Of course, it is also possible to use one type.

(Effects of the Invention) As described above, in the present invention, the power spectral density 1 degree is obtained after removing the high frequency component of the detection output higher than the relaxation frequency of the power spectral density using a low-pass filter. Even when the concentration and light intensity are low, immune reactions due to intensity fluctuations can always be measured with high precision.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the configuration of an example of an apparatus for implementing the immune reaction measuring method of the present invention, and FIGS. 2 and 3 are diagrams showing an example of measurement using the apparatus shown in FIG. 1. 1... Laser light source 2, 4, 5... Luminous flux 8.
... Semi-transparent mirror 6 ... Condensing lens 7 ... Cell 8 ... Photodetector 9 ... Fine particles
10... Collimator 11.1. photodetector
18.15...Low noise amplifier 14...Data processing device 16...Low pass filter 17.-A
/D conversion unit 18...Fast Fourier transform unit 19...Arithmetic processing unit 20...Display device 2nd
Figure 3 Figure 1: Original wave number (Hz)

Claims (1)

[Claims] 1. In projecting radiation onto a reaction solution containing an antigen and an antibody, detecting light scattered by fine particles in the reaction solution, and measuring the antigen-antibody reaction based on the power spectrum density of the intensity fluctuation of this detection output, A method for measuring an immune reaction using light intensity fluctuation, characterized in that the power spectral density is determined after removing high frequency components higher than the relaxation frequency of the power spectral density of the detection output using a low-pass filter.