JPS61175549A - Immunological reaction measurement by fluctuations in intensity of light - Google Patents

Abstract

PURPOSE:To elevate the analyzing accuracy and processing efficiency, by determining white levels of the power-spectrum density separately before and after the antigen-antibody reaction. CONSTITUTION:A radiant ray is projected to a reaction liquid containing antigens and antibodies to detect scattered light due to fine particles 9 therein. Then, white levels in the power spectrum density are determined separately before and after the antigen-antibody reaction. Then, the frequencies giving a value of the power spectrum density lowered by about 3dB from the white levels are obtained as relaxation frequency respectively. Moreover, the radio of the relaxation frequencies is determined before and after the antigen-antibody reaction to obtain the concentration of the antigen or antibody from the resulting ratio. Here, the view of a photodetector 11 is limited using a collimator 10 having a pinhole of specified dimensions to enable the detection of fluctuations with a high sensitivity. Thus, the analyzing accuracy and the processing efficiency can be elevated.

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 (
RIA), enzyme immunoassay (E IA), fluorescence immunoassay (
FIA> etc. are well known and have high sensitivity, but there are various limitations such as handling of isotopes and waste disposal.
Further, there are disadvantages such as a long time required for measurement and high testing costs because the labeling reagent is expensive.

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. The disadvantage is that the measurement time is long. Regarding such immunoassay methods, please refer to "Clinical Test Methods Summary" (authored by Izumihara Kanai, edited by Masamitsu Kanai, Metal Publishing) and "Clinical Testing JVoJ2.22".
．． No. 5 (1978), pages 471-487.

Also, [l ma+unochemistryJ,
Vo 12, 12° No. 4 (1975), No. 34
On pages 9 to 351, 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. Disclosed is a method for quantitatively analyzing an antigen or antibody by determining 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. Also,
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. 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. Scattered light generated by the antigen-antibody reaction of microparticles with immobilized antigens is detected in a homodyne or heterogain 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, 1. The intensity of scattered light generated by microparticles as a result of an antigen-antibody reaction or the scattered light generated by an antigen-antibody reaction of microparticles on which antibodies or antigens are immobilized fluctuates due to light interference, so the power spectrum of this intensity fluctuation By focusing on the fact that density depends on the shape and size of particles, and detecting the power spectrum density of intensity fluctuations, antigen-
A lot of useful information can be obtained, such as the presence or absence of antibody reaction, the quantification of antigen or antibody, and the state of aggregation (particle size distribution) of microparticles due to antigen-antibody reaction. 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 used since the spectrum analysis of the scattered light is not performed. There's no need. 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. A method has been proposed in which the ratio of relaxation frequencies before and after the reaction is determined and the antigen-antibody reaction is measured from the value of this ratio.

In such an immune reaction measurement method using fluctuations in light intensity, the problem is how to find the relaxation frequency of the power spectral density, but a method that can be adopted is to draw a curve representing the power spectral density and read the relaxation frequency from this. It is being However, the power spectral density curve fluctuates widely, and it is very difficult to read the relaxation frequency from it, and the reading accuracy deteriorates, resulting in a reduction in analysis accuracy. Furthermore, when analyzing a large number of samples, the introduction of such manual processing or manipulation is extremely disadvantageous in terms of processing time, accuracy, cost, etc.

(Object of the Invention) The object of the present invention is to eliminate the above-mentioned drawbacks and to be able to automatically determine the relaxation frequency of the power spectral density accurately and in a short time, thus making it possible to determine the concentration of antibodies or antigens in a large number of analytes. The purpose of the present invention is to provide an immune reaction measurement method that can accurately and efficiently detect.

(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 white levels of the power spectral density before and after the antigen-antibody reaction are determined, and the frequencies that give the power spectral density values that are approximately 3 decibels lower than these white levels are determined as relaxation frequencies. The method is characterized in that the ratio of the relaxation frequencies before and after the antigen-antibody reaction is determined, and the concentration of the antigen or antibody is determined from the ratio of the relaxation frequencies.

(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, H with a wavelength of 632.8 nl is used as a light source that emits coherent light.
An e-Ne gas laser 1 is provided.

In addition to such a gas laser, a solid laser such as a semiconductor laser can also be used as a light source that emits coherent light. Furthermore, a light source that emits incoherent light can also be used in the method of the present invention. light source 1
The laser beam 2 emitted from
and a luminous flux 5. One of the light beams 4 is condensed by a collimator lens 6, projected onto a transparent self, and focused at one point inside the self. The other light beam 5 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 source 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. Therefore, when an antigen-antibody reaction occurs in the self and an interaction occurs between the particles, the particles adhere to each other, resulting in a change in the state of Brownian motion. Scattered light scattered by the fine particles 9 in the self is made to enter a photodetector 11 consisting of a photomultiplier tube through a collimator 10 having a pair of pinholes. The collimator 10 has a hollow structure, and has a dark box structure to remove the influence of external light, and its inner surface is treated with anti-reflection treatment. Pinholes are formed before and after the cavity.

The output monitor signal of the photodetector 8 is supplied to a data processing device 14 via a low noise amplifier 13. In addition, the output signal of the photodetector 11 is fed to the low-noise amplifier 15 and the low-pass filter 111F. ．． A fast Fourier transform section 18 and an arithmetic processing section 19 are provided to perform signal processing as described later and output measurement results of antigen-antibody reactions. This measurement result is supplied to the display device @20 and displayed.

The intensity of the scattered light from the self is normalized by the short-time average value output of the light source intensity monitor signal from the photodetector 8, and after removing fluctuations in the intensity of the laser light emitted from the light source, it is converted to the fast Fourier transform unit 18. The power spectrum Δ degree of the intensity fluctuation of the scattered light is determined, and the relaxation frequency is determined from this power spectrum density. Based on this, the aggregation state of the fine particles 9 in the self, and therefore the progress state of the antigen-antibody reaction, is determined. Take measurements.

In the present invention, as described above, the power spectral density of the intensity fluctuation of the scattered light is detected, but this power spectral density is composed of a term due to the fluctuation of the interference component due to the diffusion of fine particles over a distance of about the wavelength, and a term due to the fluctuation of the interference component due to the scattering It consists of terms due to fluctuations in the number of particles caused by the movement of particles into and out of the volume.

Of these, fluctuations in scattered light due to interference are observed as spatial fluctuations in the speckle pattern, but if this is directly incident on the photodetector 11, which has a wide light-receiving surface, the light will be scattered spatially over the area of the light-receiving surface. Since smoothing is performed,
The detected fluctuation becomes small. Therefore, by limiting the field of view of the photodetector 11 using a collimator 10 having a pinhole of a predetermined size, fluctuations can be detected with high sensitivity.

In the above 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 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 heterodyne method in which a portion of the light is incident on the photodetector 11. That is,
In the present invention, as shown in FIG. 2, the angle θ between the light beam 4 incident on the self and the optical axis of the collimator 10 can be set arbitrarily. When detecting scattered light in a homodyne manner, the output signal of the photodetector 11 consisting of a photomultiplier tube is proportional to the average value ES2 of the square of the electric field strength of the scattered light. In the case of heterodyne detection in which scattered light and incident light are detected together, if the electric field strength of the directly incident light is Ee, the output signal of the photodetector 11 is as follows. Here, Ee has no fluctuation (even if there is, it is made looser than the fluctuation of the scattered light), so the fluctuation component of the output of the photodetector 11 is almost entirely the second term 2E8.
Equal to ES. In other words, an output signal approximately proportional to the electric field strength E8 of the scattered light is obtained.

Further, the collimator 10 is not limited to the above-mentioned configuration, but may have any configuration as long as it can limit the field of view of the photodetector 11 to one speckle pattern or less.

Using the above-mentioned device, the output signal of the photodetector 11 is supplied to the data processing device 14 through the low-pass filter 16, and processed together with the monitor signal from the photodetector 8 to obtain the power spectrum of the intensity fluctuation of the scattered light. The results of determining the density will be explained below. Here, the power spectral density 5(f) of the steady state establishment process X(t) can be expressed as follows.

Based on this equation, the power spectral density is calculated using fast Fourier transform. That is, the output signal from the photodetector 11 is amplified by the low-noise amplifier 15 so that the digitization level of the A/D conversion in the data processing device 14 is covered as wide as possible, and this quantized data is The power spectral density was calculated using a microprocessor. The relaxation frequency is determined from the power spectral density determined in this way, the ratio of the relaxation frequencies before and after the immune reaction is determined, and the antibody or antigen concentration is determined from this ratio, which is displayed numerically on the display device 20.

Figure 3 shows that 5xio-'g 7m 12 antigen (
It shows the change in power spectrum density before and after adding CRP). Both curves represent Lorentzian power spectral densities, and are due to interference effects in the power spectral densities of intensity fluctuations of scattered light. It can be seen that the power spectrum density before and after the immune reaction is inversely proportional to the relaxation frequency fr+ and the diameter of the fr2 microparticle. In other words, the intensity fluctuation of the scattered light is a combination of a component due to the interference of coherent light that exposes the movement of fine particles as described above, and a component due to fluctuations in the number of particles within the scattering volume, but in this example, the interference The components are mainly detected, and the relaxation frequency of the power spectral density is the reciprocal of the time it takes for particles to travel the distance of the wavelength of light, so if the particle size increases isometrically as aggregation due to immune reaction progresses, the travel time increases. becomes longer, and the relaxation frequency fr2 decreases. That is, the antigen-antibody reaction causes particles to aggregate with each other, the Brownian motion changes, and the relaxation frequency of the Lorentzian curve shifts to the lower side.

In the present invention, the antigen or antibody concentration is determined by utilizing this shift of the relaxation frequency to the lower side. Generally, the relaxation frequency fr of the Lorentzian spectrum is given by the following equation.

where 5(f) is the power spectral density at frequency f, and N is the number of particles in the scattering volume.

Therefore, if we calculate the ratio of the relaxation frequencies before and after the antigen-antibody reaction, this ratio R will have a certain relationship with the concentration of the antigen or antibody. Alternatively, the concentration of the antibody can be determined. However, as shown in Figure 3, the power spectral density fluctuates greatly over all frequencies, so
The problem is how to find the relaxation frequency.

As mentioned above, the power spectral density of the light intensity of the scattered light becomes a Lorentzian spectrum, and the relaxation frequency of this Lorentzian spectrum is the frequency when the white level (flat part) is lowered by 3 dB in the low frequency region. You can ask for it. This value of 3 dB is uniquely determined regardless of the shape of the Bowers vector density curve, that is, the presence or absence of an antigen-antibody reaction, the diameter of the microparticles used, etc.

FIGS. 4A to 4D schematically illustrate a method for determining relaxation frequencies using the method of the present invention. FIG. 4A shows the power spectrum density drawn from the measured data, which includes large fluctuations not only in the white level in the low frequency region but also in the falling portion in the high frequency region.

First, the measured data supplied from the fast Fourier transform section 18 is computer-processed in the arithmetic processing section 19 and smoothed to form a smooth curve as shown in FIG. 4B. Next, the average value of the values in the low frequency region, for example from 1 to 107, is determined to determine a flat white level value WL as shown in FIG. 4C.

Next, as shown in the fourth diagram, find a reference level RL that is 3 dB lower than this flat white level WL, find the frequency at the intersection of this level and the smooth falling part of the power spectrum density, and calculate this. Let the relaxation frequency be fr. This can be determined as a frequency that provides a power spectral density that is 3 dB lower than the white level WL.

Generally, when smoothing processing is performed, the power spectrum density becomes a smooth curve as shown in FIG. 4B, but if the falling portion of this curve still contains fluctuations, there is a risk of an error.

In such a case, the above-mentioned process may be performed multiple times and the average value may be taken, or the white level WL may be lowered by about 368 dB, for example, 2.9 dB.

Find multiple frequencies that give a level reduced by 3.0 dB and 3.1 dB, and calculate 3. from these frequencies. The relaxation frequency at a position lowered by O dB can be determined by processing such as an averaging method or an interpolation method.

FIG. 5 is a graph showing the relationship between the ratio R of relaxation frequencies before and after the reaction obtained as described above and the antigen concentration C. Antibody-sensitized particles made of polystyrene latex with a particle size of 0.1μ were used as microparticles, CRP was used as the antigen, and after adding the antigen, incubation was carried out for 8 times, and the upper temperature was increased for 3 minutes for 5 minutes to 1 hour.
The ratio R of the solid wave number at the end of 0 minutes is calculated. The relationship between the relaxation frequency ratio R and the antigen concentration C is approximately expressed by an exponential function R-1ae (c-'r). Here, a is a constant determined by the reaction time, and ■ is the antigen concentration at the point where the curve intersects the horizontal axis.

The present invention is not limited to the embodiments described above, but can be modified and changed in many ways. The method of the present invention includes immunoglobulin G (IaG), immunoglobulin A (1 (I)
A), I (JM, il: ID.

It can be applied to the measurement of all substances that cause agglutination due to antigen-antibody reactions, such as 1gE, Australian antigen, m-toxin antigen, and 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. You can also do it. moreover,
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, scattered light from fine particulate products generated as a result of the antigen-antibody reaction was used. You can also. An example of such an antigen-antibody reaction is human chorionic gonadotropin (HCG>
using anti-human chorionic gonadotropin (anti-HC) as the antibody.
There is a reaction using G), 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 candida is used as an antigen.
For example, Candida albicans (yeast) is used 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 batch method was used in which the antigen-antibody reaction solution was stored in a cell and the measurement was performed, but a flow-type method in which the measurement is performed while the antigen-antibody reaction solution is continuously flowing may also be used. Of course it is possible.

(Effect of the invention) The effects of the present invention described above are summarized as follows.

(1) Since the relaxation frequency of the power spectrum density of light intensity fluctuation can be determined accurately and quickly, analysis accuracy is improved and processing efficiency is also improved.

(2) Since there is no need to use expensive and difficult-to-handle reagents such as labeling reagents such as enzymes and radioisotopes, it can be carried out at low cost and easily.

(3) It has higher accuracy and reproducibility than non-labeled immunoanalytical methods such as immunoelectrophoresis, immunodiffusion, and precipitation, so it is possible to obtain highly reliable measurement results with high precision.

(4) Since the method detects the intensity fluctuation of scattered light based on the Brownian motion of fine particles, highly accurate measurement can be performed with an ultra-trace amount of sample, and the measurement time can be shortened.

(5) Compared to methods that quantify antigens or antibodies by determining the average diffusion constant from changes in the spectral width of scattered light, a spectrometer is not required, so the device is smaller and cheaper, and the measurement results are highly accurate and reliable. is obtained.

[Brief explanation of drawings]

FIG. 1 is a miniature diagram showing the configuration of one embodiment of the apparatus for carrying out the immune reaction measurement method according to the present invention, FIG. 2 is a miniature diagram showing the configuration of the main part of another embodiment of the immune reaction measurement apparatus, and FIG. is a graph showing the change in power spectral density before and after the antigen-antibody reaction, Figures 4A-D are diagrams explaining the sequential operation of determining the relaxation frequency according to the present invention, and Figure 5 is a graph showing the antigen concentration and relaxation before and after the reaction. It is a graph showing the relationship with the ratio of frequencies. 1... Laser light, source 2. 4. 5... Luminous flux 3... Semi-transparent 1 6... Condensing lens 7...
Cell 8... Photodetector 9... Fine particles
10... Collimator 11... Photodetector
13-15...Low sourness #XA garden 14...Data processing device 16...Low pass filter 20...Display device patent applicant Olympus Optical Industry Co., Ltd. Figure 2 Figure 3 Number J (jh) - Figure 4 A B

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, The white levels of the power spectral density before and after the antigen-antibody reaction are determined, and the frequencies that give the power spectral density values that are approximately 3 dB lower than these white levels are determined as the relaxation frequencies. A method for measuring an immune reaction using light intensity fluctuation, characterized by determining a ratio of relaxation frequencies after a reaction, and determining the concentration of an antigen or antibody from this ratio of relaxation frequencies.