JPS61173137A - Method for measuring immune reaction by intensity fluctuation of light - Google Patents

Abstract

PURPOSE:To increase the difference in the relaxation frequency before and after reaction by adding a reaction accelerator having high viscosity into a reactive liquid 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 a condenser lens 6 and is projected to a cell 7 in which the antigen-antibody reactive liquid contg. the fine particles 9 is contained after the reaction accelerator such as polyethylene glycol having the viscosity higher than the viscosity of said liquid is added to the liquid. 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 to a data processing unit 14. The measurement with both low and high concns. is thus made possible by increasing the difference in the relaxation frequency of the power spectral density before and after the reaction.

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 is an immunoassay method that utilizes a specific selective reaction of the immune reaction as a method for measuring trace components in the body such as immune substances, hormones, medicines, and immunomodulators.It 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 (
RIA), enzyme im/assay (EIA)
, fluoroimmunoassay CFI&), etc. 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.

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 Publishing), “Clinical Examination J vol.
22.45 (1978) I, pages 471-48.

Sarani, “Immunochemistry J vOX
, 12, 164 (1975) *pp. 849-851 describes that particles carrying antibodies or antigens on their surfaces are reacted with antigens or antibodies in a liquid to be measured, and the aggregated particles are reduced in proportion to the size of the particles. A method is disclosed for quantitatively analyzing an antigen or antibody by determining the average diffusion constant, which is an index of Brownian motion, from changes in the spectral width of scattered laser light. 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, errors occur when the spectrometer is mechanically driven, resulting in 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. The disadvantages are that it requires a large spectrometer, has poor accuracy and reproducibility, and provides only a small amount of information.

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. Measurements can be performed with high precision and reproducibility without using a large spectrometer, and antigen-antibody reaction measurements can be automated, and a lot of useful information about antigen-antibody reactions can be obtained. A method for measuring an immune reaction that can obtain the following is proposed in Japanese Patent Application No. 148878/1983.

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. Scattered light generated by the antigen-antibody reaction of microparticles with immobilized antigens is detected in a homodyne or heterodyne manner, and the antigen-antibody reaction is measured based on the power spectrum density of the intensity fluctuation of this detection output.

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 aggregation type III (particle size distribution) of microparticles based on 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 there is no need to use a spectrometer as there is no 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 one antigen-antibody concentration. A method of determining the ratio of relaxation frequencies before and after the reaction and measuring the antigen-antibody reaction from this ratio value, the ratio of the change in the integrated value of the low frequency band of the power spectral density before and after the antigen-antibody reaction, and the method of measuring the low frequency band A method has been proposed for measuring an antigen-antibody reaction from the value of a relative fluctuation ratio expressed as a ratio of power spectral density levels at a specific frequency in a band.

However, in this immune reaction measurement method, as in the other immune reaction measurement methods mentioned above, in order to obtain highly reliable measurement results, it is necessary to ensure a sufficient antigen-antibody reaction. Therefore, there is a problem that the reaction time takes a long time and the processing speed becomes slow.

(Objective of the Invention) An object of the present invention is to provide an immune reaction measurement method using source intensity fluctuation, which solves the above-mentioned problems and provides highly reliable measurement results in a short reaction time.

(Summary of the Invention) The present invention projects radiation of 1 m 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 this detection output. In measuring the antibody reaction, a reaction accelerator having a higher viscosity than the reaction solution is added to the reaction solution.

(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 is an H
An e-Ne gas laser is used. As the light source 1 that emits coherent light, a solid laser such as a semiconductor laser can also be used in addition to such a gas laser. Furthermore, a light source that emits incoherent light can also be used in the method of the present invention. A laser beam 2 emitted from a light source 1 is separated into a beam 4 and a beam 5 by a semi-transparent mirror 8, one beam 4 is focused by a condensing lens 6 and projected onto a transparent self, and the other beam 5 is is made incident on a photodetector 8 made of a silicon photodiode and converted into a monitor signal representing fluctuations in the output light intensity of the light i1!1.

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. A reaction accelerator having a higher viscosity than that of PEG, such as polyethylene glycol (PEG), is injected at a concentration of about 5%. Therefore, the antigen-antibody reaction is promoted in the self, interaction occurs between the particles, particles adhere to each other, and the state of Brownian motion changes. Scattered light scattered by the fine particles 9 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 1114 via the low noise amplifier 13 and
The output signal of 1 is supplied to a data processing device 14 via a low noise amplifier 15 and a low pass filter 16. The data processing device 14 is provided with an A/D conversion section 17, a high-speed 7-channel conversion section 18, and an arithmetic processing section 19. After the intensity is normalized by removing fluctuations in the intensity of the laser light emitted from the light source 1 using the short-time average value output of the light source intensity monitor signal from the photodetector 8, the power spectrum of the intensity fluctuation of the scattered light is calculated. The relaxation frequency of the density is determined, and based on this, the aggregation state of the fine particles 9 in the self, and therefore the antigen-antibody reaction, is measured, and the measurement results are supplied to the display device 20 for display.

Here, since the power spectral density of the scattered light is Lorentzian, its relaxation frequency fr is expressed by . However, 1Δ section 1 indicates the change in the wave number vector, which is expressed as , and D indicates the diffusion coefficient, which is expressed as .

In the above equations (2) and (3), n6 is the layer ratio of air, θ is the scattering angle, λ is the wavelength, k is Boltzmann's constant, T is the absolute temperature, l is the viscosity, and r is the particle size. .

In the present invention, as described above, a reaction promoter having a higher viscosity than the reaction solution is added to the reaction solution.

In this way, the reaction is promoted and at the same time the viscosity of the entire reaction liquid increases, so as is clear from equation (3) above, the diffusion coefficient becomes smaller, and therefore the relaxation frequency fr after the reaction becomes , as is clear from the above equation (1), is smaller than when no reaction accelerator is added.

That is, as shown in Figure 2, when a reaction accelerator is added, the relaxation frequency fro of the power spectrum density before the reaction is almost the same as when no reaction accelerator is added, but the relaxation frequency fr after the reaction is as shown by the broken line. The relaxation frequency fr' becomes smaller than the relaxation frequency fr' when no reaction accelerator is added, and the difference from the relaxation frequency fro before the reaction becomes large.

In this way, if the difference between the relaxation frequencies before and after the reaction becomes large, when determining the antigen concentration from the ratio of relaxation frequencies (fr/fry), the calibration curve representing the relationship between the value of the ratio and the antigen concentration becomes 10 as shown by the solid line in Figure 3.
It has a sufficient slope even in the low concentration region of ~10 (g/1Lt), and the measurement accuracy especially at low concentrations can be significantly improved compared to the case where the reaction accelerator shown by the broken line is not added.

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 embodiment described above, the direction of the light beam 4 incident on the self and the optical axis direction of the collimator 10 are set to 90', and the homodyne method is adopted in which the incident light beam does not directly enter the photodetector 11. It is also possible to adopt a heterogain method in which a part of the incident light flux is incident on the photodetector 11.Also, the display on the display device 20 is not limited to the relaxation frequency, but may also display power spectral density data. However, the antigen concentration may be displayed by further performing necessary calculations based on the relaxation frequency obtained by calculation.In addition, the antigen concentration is determined not only by the ratio of the relaxation frequencies before and after the reaction.
It can also be detected by the relative fluctuation ratio. 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, without using such fine particles, it is also possible to utilize scattered light caused by fine particulate products produced as a result of the antigen-antibody reaction. As an example of such an antigen-antibody reaction, human chorionic gonadotropin (OG
) was used as the antibody, and anti-human chorionic gonadotrivin (anti-H
OG), and the antigen-antibody complexes produced by this reaction can be treated as fine particles. Furthermore, the antigen itself can also be used as particles. In such an antigen-antibody reaction, Candida albicans (yeast) is used as the antigen and anti-Candida albicans is used as the antibody, and blood cells, cells, microorganisms, etc. can also be used as particles. Furthermore, in the embodiment shown in FIG. 1, a patch method was used in which the antigen-antibody reaction solution was contained in a cell and measurements were performed, but a flow-type method was adopted in which the measurement was performed while the antigen-antibody reaction solution was continuously flowing. Of course, it is also possible.

(Effects of the invention) As described above, in the present invention, since the reaction is carried out by adding a reaction accelerator, highly reliable measurement results can be obtained in a short reaction time, and the processing speed is improved. can be done. In addition, since the viscosity of the reaction accelerator is higher than that of the reaction solution, the difference in relaxation frequencies becomes large, especially when measuring the antigen-antibody reaction based on the ratio of the relaxation frequencies of the power spectrum densities before and after the reaction. , it is possible to obtain highly accurate measurement results even at low concentrations.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the configuration of an example of an apparatus for carrying out the immune reaction measurement method of the present invention, and FIG. 2 and FIG. 8 are diagrams for explaining the present invention in detail.

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 a reaction accelerator having a higher viscosity than the reaction solution is added to the reaction solution.