JPH01136051A - Immunological reaction measurement using fluctuation of light intensity - Google Patents

Abstract

PURPOSE:To obtain a smoothed power spectrum density simply with limited statistic variations from limited measuring data, by performing a spectral window processing of a power spectrum density in fluctuation of intensity of a detection output. CONSTITUTION:Scattered light of light projected into a reaction liquid containing a sample and a reagent is detected with a light receiving element 8. As amplified with a low noise amplifier 11 an output of the light receiving element 8 is passed through a lowpass filter 12 to remove a high frequency noise and then, after amplified with an amplifier 13 again, it is converted into a digital signal with an A/D converter 14 to be supplied to a computer 15. The computer 15 determines a power spectrum density by an FFT based on the digital signal from the A/D converter 14. Moreover, after averaged, the power spectrum density thus obtained undergoes a spectrum window processing and then the subsequent smoothing processing is performed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for measuring an immune reaction using fluctuations in light intensity.

[Conventional technology]

A method for measuring an immune reaction using light intensity fluctuations was proposed in, for example, Japanese Patent Laid-Open No. 6.1-28866, and is well known. In this conventional immune reaction measurement method, the scattered light intensity of light projected into the reaction solution is detected before and after the reaction between the sample and the reagent, and the detected output is A/D converted and then fast Fourier transformed ( The power spectral density is determined by FFT), and the concentration of a given antigen or antibody in a sample is measured by utilizing the fact that, for example, the relaxation frequency of the power spectral density changes due to the antigen-antibody reaction before and after the reaction. .

[Problem that the invention seeks to solve]

However, in the above-mentioned conventional immune reaction measurement method, there is a problem that unless a large number of measurement data are collected, statistical variations in power spectral density become large, making it difficult to perform highly accurate measurement. In addition, if you try to collect a large amount of measurement data to solve this problem, it will take a long time to measure, and the time required for calculation processing will also increase.
This results in a problem that the sample processing capacity is reduced.

This invention was made by focusing on these conventional problems, and it is possible to improve the throughput of specimens without collecting a large amount of measurement data, and to improve the light intensity fluctuation that allows highly accurate measurements. The purpose of the present invention is to provide a method for measuring an immune reaction using the method.

[Means and effects for solving the problem] In order to achieve the above object, the present invention detects the scattered light of the light projected into the reaction solution containing the sample and the reagent, and calculates the power of the intensity fluctuation of the detection output. After determining the spectral density, the power spectral density is subjected to spectral window processing to obtain a smoothed power spectral density with less statistical variation, and the immune response is measured based on this power spectral density.

〔Example〕

FIG. 1 shows the configuration of an example of an immune reaction measuring device implementing the present invention. In this measuring device, He
- A light beam from a light source 1 such as a Ne laser is divided into two by a half mirror 2, and one of the beams is used as a signal light by an NO filter 3.
After the light is attenuated by the lens 4, it is focused on the center of the cell 5,
The other luminous flux is made to enter the photodiode 6 as a reference light to monitor fluctuations in the output light intensity of the light source 1.

The cell 5 accommodates a sample and a reagent containing a substance that specifically binds to a predetermined substance to be measured in the sample, and the scattered light 906 caused by the particles in the cell 5 is divided into a diameter Q, for example. The light is received by the light receiving element 8 through a pair of circular slots 7) 7a and 7b having pinholes of 3 mrn, and the light directly transmitted through the cell 5 is blocked by the optical trap 9. In addition,
A pair of circular picks 7) 7a.

The distance between 7b is set to be the coherence length, and a hollow cylinder IO is provided between these to cassette noise such as stray light.

After the output of the light receiving element 8 is amplified by the low noise amplifier 11,
After cutting high frequency noise through a low-pass filter 12 and then amplifying it again with an amplifier 13, the A/D converter 14
The signal is converted into a digital signal and supplied to the computer 15. The computer 15 performs FFT on the digital signal from the A/D converter 14 to obtain the power spectrum density, and after averaging the obtained power spectrum density several times to suppress statistical fluctuations, The power spectral density is subjected to spectral window processing, and then the power spectral density obtained by this spectral window processing is smoothed in order to improve the S/N.

In this embodiment, Hanning window processing is performed as the spectral window processing. That is, the values of the power spectral densities obtained by FFT are divided into At, A2. A3. .

, AN, perform Hanning window processing using these data, B1=0.58t+o, 5A2 B2=0.25A1+0.5A2+0.25A3B3=
0.25A2+0.5A3+0.25A48N=0.5
A (N-1) +Q, 5AN is determined, and this power spectrum density Bl, B2. Measure the immune response based on B3°, ON.

Figure 2A shows the power spectral density after Hanning window processing, and Figure 2B shows the power spectral density before Hanning window processing, that is, according to the conventional measurement method.
It shows measurement data before the start of the reaction between a 0-6 g/ml sample and a reagent (dashed line) and 15 minutes after the reaction (solid line). As is clear from Figures 2A and B, by performing the houning window process, it is possible to obtain a smoothed power spectral density with small statistical fluctuations from limited measurement data. Based on this, the presence or absence of an immune reaction and its concentration can be measured with high accuracy.

Here, the presence or absence of an immune reaction and the concentration of a predetermined substance can be measured, for example, as follows.

1) Relaxation frequency after the reaction between sample and reagent (frequency that indicates a power spectral density that is 3 dB lower than a flat level (white level) in the low frequency region)
The presence or absence of an immune reaction and the concentration of a predetermined substance in the sample are determined from a calibration curve of the concentration determined in advance and the relaxation frequency.

li) Calculate the average value or integral value of the white level after the reaction between the sample and the reagent, and determine the presence or absence of an immune reaction and the specified substance in the sample from a calibration curve between the concentration determined in advance and the average value or integral value of the white level. Determine the concentration of

iii) The ratio of relaxation frequencies before and after the reaction between the sample and the reagent is determined, and the presence or absence of an immune reaction and the concentration of a predetermined substance in the sample are determined from a calibration curve of the concentration determined in advance and the relaxation frequency ratio.

iv) Calculate the ratio of the average value or integral value of the white level before and after the reaction between the sample and the reagent, and use the calibration curve between the predetermined concentration and the average value or integral value ratio of the white level to determine the presence or absence of an immune reaction and the concentration of the white level in the sample. Determine the concentration of a given substance.

(2) The ratio of relative fluctuation before and after the reaction between the sample and the reagent is determined, and the presence or absence of an immune reaction and the concentration of a predetermined substance in the sample are determined from a calibration curve of the concentration determined in advance and the relative fluctuation ratio.

Note that the present invention is not limited to the above-mentioned embodiments (many changes and modifications are possible). For example, in the above-mentioned embodiments, Hamming window processing is performed as the spectral window processing. Hamming window processing can also be performed.In this case, F
Power spectrum density Al obtained by FT, A2.
A3. - Based on AN 2, C2 = 0.54A1 + 0.5482 C2 = 0.23A1 + 0.54A2 + 0.23A3C3
=0.23A2+0.54A3+0.2384CN=0
．． The power spectral density of 54A (N-1) +0.54AN may be found. Further, as other spectrum windows, for example, a box-shaped window, an Akaike window, etc. can also be applied.

〔Effect of the invention〕

As described above, according to the present invention, the power spectral density of the intensity fluctuation of the detection output is subjected to spectral window processing, so that a smoothed power spectral density with less statistical variation can be obtained from limited measurement data. can be obtained easily. Therefore, the immune reaction can be measured with high accuracy without increasing the amount of measurement data, and the processing capacity of the specimen can also be improved.

[Brief explanation of the drawing]

Fig. 1 is a block diagram showing the configuration of an example of an immune reaction measuring device implementing the present invention, and Figs. 2 A and B are diagrams showing a comparison of the power spectral density according to the present invention and the power spectral density according to the conventional method. be. ■...Light source 2...Half mirror 3.
...N, D filter 4...Lens 5...Cell 6...Photodiode 7a, 7b
... Circular slit ... Photodetector 9 ... Optical trap 10 ... Hollow cylinder 11 ... Low noise amplifier 12 ... Low pass filter 13 ... Amplifier
14... A/D converter 15... Computer Figure 1 Figure 2 Frequency (Hz)

Claims (1)

[Claims] 1. Projecting light into a reaction solution of a sample and a reagent containing a substance that specifically binds to a predetermined substance in the sample,
After detecting the light scattered by the fine particles present in the reaction solution and determining the power spectral density of the intensity fluctuation of the detection output, the power spectral density is subjected to spectral window processing, and the power spectrum obtained by this spectral window processing is An immune reaction measurement method using light intensity fluctuation, characterized in that the immune reaction is measured based on density. 2. The immune reaction measuring method using light intensity fluctuation according to claim 1, wherein the spectral window is a Hanning window. 3. The immune reaction measuring method using light intensity fluctuation according to claim 1, wherein the spectral window is a Hamming window.