JPS6166148A - Immunological-reaction measuring apparatus utilizing fluctuation of light intensity - Google Patents

Abstract

PURPOSE:To measure antigen-antibody reaction highly accurately with good reproducibility by a simple measuring apparatus, by projecting coherent light on an antigen and antibody cell, receiving scattered light by a light detector, and obtaining the power spectrum density of the intensity fluctuation of the output signal. CONSTITUTION:Laser light from a laser light source 1 is split into luminous fluxes 4 and 5 by a semitransparent mirror 3. The luminous flux 4 is inputted to a cell 7 containing antigen and antibody through a condenser lens 6. The scattered light is inputted to a light detector 11 through a collimator 10 having a pair of pinholes. Its output signal is inputted to a data processor 14 through a low noise amplifier 15 and an LPF 16. Meanwhile, the luminous flux 5 is inputted to the a light detector 8. Its output monitoring signal is inputted to the device 14 through a low noise amplifier 13. The input signal are processed through an AD converter 17, an FFT 18 and an operating device 19. Fluctuation of laser light intensity is removed. Then the power spectrum density of the intensity fluctuation of the scattered light is obtained and displayed on a display device 20.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to an apparatus 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, medical crystals, and immunomodulation.
-Labeled immunoassay using antigens as labeling substances and
Two methods of label-free immunoassays that directly measure antibody complexes are well known 42.

C is Fusio Immuno +
2 ((RIA>, M immune analysis ("IA>, m
Photoimmune phase splitting (11△) is a well-known method, and although it is sensitive, there are limitations in the number of seconds due to handling of Tsunoiso 1 heave, waste disposal, and it takes a long time to measure. 4!
There are disadvantages such as the high cost of the test because the test book is expensive.

Immunoelectrophoresis, immunoJ1 for non-labeled immunoassay on the back right
7: There are fiQγ people and sedimentation people 1q, and there is a simple analytical method, but it is a sensorineural, constant EI of 41'l, reproducibility (-viscosity 1-1; measurement and C is insufficient. Regarding the immunoassay method, please refer to [Clinical Inspection Gamma Summary 1 (Kinj #1 Higashihara author, Kin 1111 Hikari Ha, Kin 1i Ha) j da) ya), ``Clinical Inspection'' VOρ, 22゜No, 5 ( 1978), No. 4
It is explained in detail on page 71-/187.
+1 (4m1str■j, V Of!,
, 12° No. 4 (1975), No. 349-3
1) On page 1, anti-14I: I or anti-I on the surface
−1 Mochi7＼[↓What is the response of the rod tr to antigen j, or antibody?
! , iiZ A method for quantitatively analyzing antigens or antibodies is disclosed by determining the average diffusion constant, which is an index of Brownian motion that decreases due to the size of uE powder particles, from changes in the spectral width of scattered light of a laser beam. There is. Although this analysis method has the advantage of not using a labeling reagent, it uses spectroscopy t1 to detect the broadening of the spectrum of the incident light due to the Doppler effect caused by the Brownian motion of the particles, making the equipment large and expensive. There are disadvantages and errors occur when mechanically driving the spectrometer, 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 there is little information.

As mentioned above, conventional immunoassay methods use expensive labeling reagents, which increases the running cost of the analysis, makes handling and processing of liquids troublesome, and increases processing time. It requires an expensive and large spectrometer, has poor accuracy and reproducibility, and the information obtained is difficult to obtain.
1 also had the disadvantage of being too small.

Also, as mentioned above, J is used to detect scattered light! In addition, the scattered light to be detected is extremely weak, and if even a small amount of 9.11 sound light such as stray light is included, the S/N will deteriorate and the measured viscosity will be prevented from deteriorating. In particular, the light emitted from the cell containing the reaction solution is reflected by other parts e and pj.
Bi1? When I put it in the room, it makes a lot of noise.

(Objective of the Invention) The object of the present invention is to measure the antigen-antibody reaction by utilizing the fact that the intensity fluctuation of scattered light on microparticles is closely related to the antigen-antibody reaction. The above-mentioned slave M! It is possible to measure high viscosity and F1 frequency without using expensive labeling reagents or expensive and expensive spectrometers, and to shorten the measurement time. antigen-
It is possible to automate the measurement of antibody reactions, and it is also possible to obtain a large amount of information about antigen-antibody reactions.
Moreover, it is possible to effectively prevent the intrusion of 911 sound and light.
C-Giro-like immunity fshi1. ? , ■ Set the fixed device t to 1
It is something that I would like to offer.

(Summary of the Invention) The present invention applies light to a reaction solution containing at least an antigen, an antibody, and an antibody or an antigen added to the reaction solution. The scattered light generated by fine particles is detected, and the antigen is detected based on the power spectrum density of the intensity fluctuation of this detection output.
An apparatus for measuring an antibody reaction includes a cell that houses and covers the antigen-antibody reaction solution; a light source device that emits coherent light; and a light source device that makes the coherent light enter the cell; Alternatively, a photodetector that receives light together with Hikaru Okawa receives the output signal from this photodetector, calculates the power spectrum of the intensity fluctuation, and based on that, detects the antigen.
A means for measuring an antibody reaction is provided between the optical path of the input light and the light receiving path of the photodetector, and has a small area only in the path of the incident light and the scattered light emitted to the photodetector. The device is characterized in that it has a cell box that accommodates the cell, which has a through hole, and all other parts are continuously illuminated.

In the present invention, the intensity of scattered light generated by microparticles generated 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. Therefore, we are aware 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 the presence or absence of an antigen-antibody reaction, and the presence or absence of an antigen or antibody reaction. It is possible to obtain a lot of useful information such as the state of aggregation (particle size distribution) of microparticles due to antigen-antibody reactions. In this way, in the present invention, scattered light is received by a photodetector and fluctuations in the output signal intensity are detected, so there is no need to use a labeling reagent, and since spectral analysis of scattered light is not performed, spectroscopic There is no need to use a meter.

In an embodiment of the present invention described later, scattered light is detected in a homodyne manner, and the relaxation frequency of the power spectral density of the intensity fluctuation depends on the particle size. The ratio of relaxation frequencies is determined, and the antigen-antibody reaction is measured from the value of this ratio. Furthermore, in another embodiment, by utilizing the fact that the integral value of the power spectrum density of the power spectral density of the intensity fluctuation of the scattered light depends on the size of the particle, the The ratio of the integral values is determined, and the antigen-antibody reaction is measured from this ratio.

In the present invention, changes in the intensity fluctuations of light scattered by particles due to particle aggregation are detected based on power spectral density, so there is no need to use expensive labeling reagents or spectrometers. A large amount of useful data regarding antigen-antibody reactions can be obtained with high sensitivity and high reproducibility in a short time.

Furthermore, by providing a cell box with the above-described configuration, the present invention can efficiently eliminate the intrusion of noise light, and improve measurement accuracy.

(Example) FIG. 1 is a diagram showing the configuration of an example of the immune reaction measuring device according to the present invention. In this example, the light source that emits coherent light is @e-p4e with a wavelength of 632.8 nm.
A gas laser 1 is provided. As a light source that emits coherent light, in addition to such a gas laser, a solid state laser such as a semiconductor laser or laser can also be used. Light it! 1
A laser beam 2 emitted from the laser beam is separated into a beam 4 and a beam 5 by a semi-transparent mirror 3. One of the light beams 4 is condensed by a condensing lens 6 and projected onto a transparent 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, 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. Scattered light scattered by the fine particles 9 in the self is made to enter a photodetector 11 consisting of a photomultiplier tube J through a collimator 10 having a pair of pinholes. 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 transmitted to the low noise amplifier 1.
5 and a low-pass filter 16 to the data processing device 1.
Supply to 4. The data processing device 14 includes an A/D converter 1
7. 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 18 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 the intensity of the scattered light from the light source is normalized by the short-time average output of the light source intensity monitor signal from the photodetector 8. After removing the A degree variation,
The power spectrum density of the intensity fluctuation of the scattered light is determined, and based on this, the state of aggregation of the fine particles 9 in the self, and therefore the progress state of the antigen-antibody reaction, is measured.

The optical paths of the light beam 2 generated from the laser light source 1 and the light beam 4 reflected by the mirror 21' and directed toward the self are all covered by a light guide tube 12. This light guide tube 12 is, for example,
It is a cylinder made of synthetic resin such as Bakelite to block external light, and its inner surface is painted black to prevent reflection. In addition, the end face of the light guide tube 12 is similarly sealed with a cover painted black.
In the center of this lid, there is a luminous flux 2. Through holes 12a and 12c are provided to allow only the light beam 4 to pass through. Similarly, on the opposite side of the through hole 12a, a through hole 12b is provided which is large enough to allow only the light beam 5 to pass through.

Further, the self is configured to be housed in a cell box 21, and this cell box 21 has an entrance port 22 through which the light flux 4 enters the self, and a photodetector for detecting scattered light generated within the self. The self can be completely covered and shielded from light except for the exit port 23 for emitting radiation to the light source 11.

FIG. 2 is a diagram showing the configuration of this cell box 21. As shown in FIG.

The entire cell box 21 is a box made of synthetic resin such as Bakelite, and is painted black to prevent light from entering the inside.

The inner circumferential surface shape is formed in a shape that is in close contact with the outer circumferential surface of the self, and the above-mentioned entrance port 22 and exit port 23 are formed as small-area through holes on two of the side surfaces that are perpendicular to each other. It is formed.

In addition, a sliding type 125 that can seal the opening 24 is attached to the partial opening 24 for removing the selfie.This lid 25 is also made of synthetic resin such as Bakelite and coated with black paint. In general, a lever 26 is provided protruding from the surface of the lid 25-F so that the manual r25 can be opened and closed.

By providing the cell box 21 with such a structure, the present embodiment can eliminate any room for noise light to enter the cell. In addition, since the optical path from the laser light source 1 to the self is also completely blocked by the above-mentioned light transmitting tube 12, it is possible to prevent noise light from being mixed in between.

Furthermore, although the cell box 21 shown in FIG. 2 can accommodate only one cell, it can also be structured to accommodate multiple cells at the same time, as shown in FIG. 3. .

This is a second box frame 31 made of synthetic resin.
A plurality of cell boxes 21 shown in the figure are incorporated, and in this case, an entrance port 22 is provided on the bottom surface of the cell box 21. Therefore, the optical system is configured so that the light from the laser light source 1 also enters the entrance port 22.

Then, the measurement is performed on one cell box,
By moving the box frame 31 in the direction of the arrow every time a measurement is completed, measurements are continuously performed on the next cell.

Note that the box frame 31 may be moved manually or automatically. Further, the lid 25 may be opened and closed automatically using a power source.

Furthermore, by replacing the light guide tube with an optical fiber, it is also possible to prevent noise light from entering.

Next, using the above-mentioned device, the output signal of the photodetector 11 is supplied to the data processing device 14 via the low-pass filter 16, where it is processed together with the monitor signal from the photodetector 8 to determine the intensity fluctuation of the scattered light. The results of determining the power spectrum density will be explained next. Here, the power spectral density S (f ) of the steady state establishment process x (t) can be expressed as follows.

Based on this equation (1), the power spectral density is calculated using fast Fourier transform. That is, photodetector 1
The output signal from 1 is amplified by the noise amplifier 15 to widen the quantization level of the A/D conversion in the data processing device 14 as much as possible, and the m-digitized data is was calculated by a microprocessor to determine the power spectrum density.The progress status of the immune reaction was numerically displayed on the display device 20 from the power spectrum density thus determined.

In Figures 4 and 5, the particle size is 0.18871, respectively.
This shows the power spectrum density obtained when Ill and 0.3f) Jlm's 7x Net molecule and liquid were placed in IZ Silua 32, which is [
, 1-Lenz-type Patou spectral density is -19, and the power spectral density of the intensity fluctuation of the scattered light is due to the interference effect. One relaxation cycle in these power specs 1 to 1? It can be seen that the IR number is inversely proportional to the target diameter of the fine particles. In other words, the intensity fluctuation of the scattered light depends on the movement of fine particles as described above, and the component due to light interference and the variation in the number of particles within the scattering volume.
In this example, the interference component is detected as J-, and the reciprocal of the time it takes to travel the distance of the power spectrum density wavelength is C', the particle size. As the value increases, the travel time becomes <27, and the relaxation frequency decreases.

In Figure 6, the horizontal axis shows the grain size in μm (1) tli (il), and the vertical axis shows the relaxation frequency, which is expressed as logarithm [b]. In other words, the grain size is 0,0915 μm.
The relaxation frequency of the particle is about 400117. , 0.18
At 8f1m, it is approximately 20011z, 0.305 μm
Then it roars at about 100 Hz. As is clear from the graph in Figure 6, the relaxation frequency of the power spectral density is inversely proportional to the particle size (3), so the presence or absence of antigen-antibody aggregation and the degree of aggregation can be detected from changes in this relaxation frequency. .

FIG. 7 and FIG. 8 are graphs showing power spectral densities when latex particles with a particle size of 0.3 μm are dispersed in a buffer solution at concentrations of 0.1% by weight and 0.09% by weight, It can be seen that a power spectral density of L]-Lentz is obtained. As mentioned above, the intensity fluctuation of scattered light is the sum of the coherent component due to the Brownian motion of particles and the non-interfering component t#, which is caused by changes in the number of particles in the scattering volume. The number of particles decreases,
When the amount of interfering components decreases to the same level as non-interfering components, it is no longer possible to detect components other than changes in the intensity of scattered light due to Brownian motion of particles, making it impossible to accurately detect antigen-antibody reactions. It disappears. Next, the concentration of particles must be selected in a range that is sufficiently low to obtain a sufficient intensity of incident light within the scattering volume, and in which the coherent component is larger than the incoherent component.

The relative fluctuation remains constant over time.

Figures 10 and 11 show that immunoglobulin G antibodies immobilized on the surface of latex particles with a diameter of 0.3 μm were dispersed in a buffer solution adjusted to pH 7 with Tris-1-ICJ2. An antigen-antibody reaction solution containing immunoglobulin G at a concentration of 10 → g/rail and 10-'o/mβ is housed in a cell, and the power spectrum density is shown before and after the start of the antigen-antibody reaction. It is. In the case of an antigen concentration of 10'g 7m 52 shown in FIG. 10, the relaxation frequency before the reaction is approximately 50 Hz, whereas the relaxation frequency after the reaction has changed to 10 Hz.

When the antigen concentration is 1O-9 (J/Aβ), the relaxation frequency before the reaction starts is about 95Hz, and the relaxation frequency after the reaction is about 4011z. Therefore, the antigen-antibody reaction The ratio of the before and after relaxation frequencies is defined as , and this value is calculated for several antigen concentrations and plotted in a graph as shown in Figure 12. In other words, in Figure 12, the horizontal axis represents the antigen concentration. The vertical axis shows the value of the relaxation frequency ratio F, and the antigen concentration can be detected by determining the relaxation frequency ratio [:].

On the other hand, as shown in a3 in FIGS. 10 and 11, it can also be seen that the ratio (R) of relative fluctuation before and after the antigen-antibody reaction has a certain relationship with the antigen concentration. This will be explained next. In FIG. 1, an electric signal obtained by converting scattered light by a photodetector 11 is passed through a low-pass filter having a transfer function as shown below.

f here. is the cutoff frequency of the low-pass filter, which is a frequency sufficiently lower than the relaxation frequency fr.

At this time, the parity of the fluctuation of the current (2) obtained as the output of the low-pass filter is <δ>2-2+2<N>2fo/fr11. (4) becomes. However, K is a constant, and N〉 is the average number of particles in the scattering volume. Therefore, the following equation (5) holds true as the relative fluctuation of the output current of the low-pass filter.

Here γ is a proportionality constant. Here, assuming that the number of particles in the scattering volume is sufficiently large, equation (5) can be rewritten as follows.

Therefore, the relative fluctuation can be calculated by finding the relaxation frequency "1" from the graph of the power spectral density.The relative fluctuation ratio 1< can now be expressed by the following equation.

FIG. 13 is a graph showing the relationship between J and relative fluctuation ratio R using this equation (7) and the antigen concentration. As is clear from this graph, the unknown antigen concentration can be determined by determining the ratio R of the relative fluctuation before and after the antigen-antibody reaction. That is, prior to measurement, the -C relative fluctuation ratio R is determined for standard samples with different known antigen concentrations to obtain a calibration curve as shown in Figure 13, and the relative fluctuation ratio R is determined for the sample with unknown antigen concentration.
The antigen concentration can be determined based on the previously determined calibration curve.

On the other hand, the relative fluctuation ratio R according to equation (7) can also be obtained by comparing the changes in the integral value in the low frequency band of the power spectral density shown in FIGS. 10 and 11. That is, the relative fluctuation ratio R can be determined based on the following.

Here, the integral value A of the power spectrum density before the antigen-pile body reaction and the integral value B after the reaction are 10-1-10'H2
is the integral value in the low frequency band. Therefore, the low-pass filter is assumed to pass frequencies below 10'Hz.

In the above example, as shown in FIGS. 10 and 11, the integral values A and J in the low frequency region of the power spectral density are
Although the relative fluctuation ratio R is determined as the ratio of
The relative fluctuation ratio may be determined from the ratio of the power spectral density levels at . When specifying the frequency in this way, a digital filter can be used instead of a fast Fourier transformer, which simplifies the configuration and improves processing efficiency. The time will also be shorter.

When the particle size is constant, the power spectral density is Lorentzian and decreases inversely as the square of the frequency at frequencies greater than the relaxation frequency.

However, if the particle sizes are distributed, a superposition of Lorentzian spectra with relaxation frequencies corresponding to each particle size will be observed, so the power spectral density in the high frequency region will no longer be the same as the frequency. It is no longer inversely proportional to the square. Therefore, from the shape of this part, the particle size distribution of the particles aggregated by the reaction can be determined.

This kind of data has not been available in the past,
This is useful information in analyzing the state of antigen-antibody reactions.

The present invention is not limited to the devices used only for the above-mentioned measurements, but includes immunoglobulin A (ToA), I (IM),
l! It can be applied to the measurement of all substances that cause agglutination due to antigen-antibody reactions, such as JD, IE, Australian antigen, syphilis antigen, and insulin.

(Effects of the Invention) The above-mentioned effects of the present invention can be summarized as follows.

(1) 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.

(2) 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.

(3) Since it detects the intensity fluctuation of scattered light based on the Brownian motion of fine particles, it is possible to detect high lii
The measurement time can be shortened.

(4) Compared to the method of quantifying antigens or antibodies by determining the average diffusion constant from changes in the spectral width of scattered light, spectral reduction is not required, so the device is smaller and cheaper, and the measurement results are highly accurate and reliable. is obtained.

(5) Since measurements are performed based on the power spectral density of optical fluctuations, a lot of useful information about antigen-antibody reactions in °C can be obtained.

(6) It can effectively prevent stray light from the outside and light from a light source from being reflected by the cell, entering the cell again, and mixing in the measurement light as sound light, and detecting weak scattered light. It is particularly effective for This makes it possible to improve measurement accuracy.

[Brief explanation of the drawing]

Fig. 1 is a diagram showing the configuration of an embodiment of the immune reaction measuring device according to the present invention, Fig. 2 is a perspective view showing an example of a cell box constituting the same embodiment, and Fig. 3 is a diagram showing the structure of the lever box portion. Perspective views showing other configuration examples, Figures 4 and 5 each have grains i\ of 0.188 μm.
The graph shows the power spectrum density for fine particles of 0.305 μm. Figure 6 shows the particle size and the relaxation of power spectrum density -2,3
- Graphs showing the relationship with frequency, Figures 7 and 8 are graphs showing the power spectrum density of cans with particle concentrations of 0.1 type can %a3J and 0.09 weight %, respectively. Figure 9 shows the relationship between particle concentration and relaxation frequency. A degree is 1
A graph showing the power spectrum density before and after the antigen-antibody reaction for O-4a/mρ and 10-9g,/m°C. Figure 12 is a graph showing the relationship between the anti-cavity degree and the relaxation frequency ratio. , FIG. 13 is a graph showing the relationship between antigen 8 degrees and relative fluctuation ratio. 1... Laser light source 2. 4. 5... Luminous flux 3
... Semi-transparent mirror 6 ... Condensing lens 7 ... Cell 8 ... Photodetector 9 ... Fine particles
10... Collimator 11... Photodetector
12...Light guide tubes 13, 15...Low noise amplifier 4-14...Data processing device 16...Low pass filter 20...Display station interval 21...Cell box 22...・
・Incidence port 23... Output [124... Opening 25... Indigo 26... Lever
31...Box frame. Patent applicant: Olympus Optical Industry Co., Ltd. Figure 4 Figure 5 Figure 7 Figure 6 #L (μfn) Figure 9 Particle concentration (I~fn3) Figure 12 Wave number (Hn)

Claims (1)

[Claims] 1. Light is projected onto a reaction solution containing an antigen and an antibody, and the antigen-
Detects the scattered light caused by fine particles generated by antibody reaction or the scattered light generated by fine particles immobilized with antibody or antigen added to the reaction solution, and measures the antigen-antibody reaction based on the power spectrum density of the intensity fluctuation of this detection output. A device comprising: a cell containing a reaction solution for performing the antigen-antibody reaction; a light source device that emits coherent light and makes it incident on the cell; and receiving scattered light from the cell alone or together with the incident light. a photodetection device for receiving an output signal from the photodetection device, determining the power spectrum density of the intensity fluctuation, and measuring an antigen-antibody reaction based on the optical path of the incident light and a light receiving path of the photodetection device; a cell box for accommodating the cell, which is provided between the cell box and the cell box, and has a through hole of a minute area only in a passage portion for the incident light and the scattered light emitted to the photodetecting device, and the other portions are all shielded from light. An immune reaction measuring device using light intensity fluctuation, comprising: