JPH03252542A - Immunoassey and device thereof - Google Patents

Abstract

PURPOSE:To detect an antigen-antibody reaction with high accuracy even with a sample in which impurities coexist by adding a specified quantity of an element which does not exist in serum into a particulate material which causes agglutination and converting particles to plasma by a laser beam incident breakdown. CONSTITUTION:A laser beam 10 emitted from a pulse laser 1 is condensed 2 and forms a beam west in a cell 3. The particulate material added with the specific element in the liquid sample is converted to the plasma by the laser beam incident breakdown therein. Stimulating light 11 emitted from a stimulating light source 4 of the wavelength conformed to the wavelength of the specific element is made incident on this plasma. Fluorescence 12 emitted by the specific element is condensed 5 and is subjected to a wavelength dispersion by a spectroscope 6; thereafter, the fluorescent intensity of the specific element is detected by a photodetector 7 and is converted to an electric signal 13. The signal 13 is inputted to a data processor 8 by which the signal is compared with the data previously inputted thereto and the number of the particles of the agglutinated lump is determined. The particle distribution is thus determined.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to the analysis of the aggregation state of particulate matter in a liquid, and in particular to the analysis of antigen-antibody reaction products suitable for detecting trace amounts of antigen-antibody reactions. This invention relates to an analytical method and an apparatus for the same.

[Conventional technology]

Turbidity method, which is a conventional method of detecting agglomeration of particulate matter;
In the light scattering method, as discussed in "New Utilization Examples of Immunoassay Methods and Applications to the Development of Diagnostic Reagents and Therapeutic Drugs" (1986), pages 114 to 129, biological samples,
In particular, accurate measurement is difficult because it is strongly influenced by the color and turbidity of the serum.Also, even with photoacoustic analysis, as described in JP-A No. 63-44149, the color and turbidity of the serum However, sufficient consideration was not necessarily given to avoid being influenced by

[Problem to be solved by the invention]

The above-mentioned conventional techniques do not necessarily give sufficient consideration to the fact that the detection and quantification of antigen-antibody reactions is affected by the color and turbidity of the serum in the sample, and the accuracy of quantifying antigen-antibody reactions is not improved. The problem was that this could happen.

An object of the present invention is to provide a method and apparatus for accurately detecting and quantifying antigen-antibody reactions even in samples containing impurities such as serum.

[Means to solve the problem]

The above purpose is to add a certain amount of an element that does not exist in serum to particulate matter that causes aggregation, turn one agglomerate into plasma by laser beam input breakdown, and convert the added element in the plasma into light emission or fluorescence intensity. This can be achieved by repeatedly determining the number of particles in the aggregate and determining the particle number distribution in the aggregate.

[Effect]

The present invention will be explained in detail below.

FIG. 1 shows an example of the basic configuration of an apparatus for measuring the particle number distribution of agglomerates according to the present invention. A laser beam 10 emitted from a pulse laser 1 is condensed by a converging lens 2 and the beam waist is adjusted in a cell 3. Form.

If particles doped with a specific element or aggregates thereof exist within the beam waist, laser beam incidence breakdown occurs and the particles become plasma. Excitation light 11 emitted from an excitation light source 4 having a wavelength matched to a specific element, such as a hollow cathode lamp, is incident on this plasma. A specific element emits fluorescence 12 due to the excitation light 11 . After this fluorescence 12 is wavelength-dispersed by a condensing system 5 and a spectrometer 6, the fluorescence intensity of a specific element is detected by a photodetector 7 and converted into an electric signal 13. By inputting this electrical signal 13 into the data processing device 8 and comparing it with data input in advance, the number of particles in the aggregate can be determined. By repeating this measurement while the liquid sample is flowing, the particle number distribution of the aggregate can be determined.

Further, since a specific element in the plasma emits atomic luminescence 14, this atomic luminescence 12 is wavelength-dispersed by a condensing system 5 and a condenser 2 spectrometer 6, and detected by a photodetector 7, and the following steps are performed in the same manner as above. It can be measured using the following procedure. In this case, the particle number distribution of the aggregate can be determined without using the excitation light source 4.

〔Example〕

An embodiment of the present invention will be described below with reference to FIGS. 2 to 4.

Uniform latex particles having a grain size of approximately 0.3 μm and containing approximately 5% (weight concentration) of copper are used as the particulate material.

DNP (2,4-dinitrophenyl)-converted bovine serum albumin (DNA-BSA) was adsorbed onto this particulate material to prepare a flux reagent coated with DNP-BSA. The agglutination reaction medium was 0.1M glycine-0.0
5M sodium chloride-sodium hydroxide buffer solution (pH
8,2) with 0.5% BSA and 0.01% non-ionic surfactant diluted in 0 reaction medium and diluted to 0.1% in the same reaction medium. Weigh out 25 μΩ of the latex reagent using a micropipette, react for 6 hours while shaking on a microtimer plate, suck up the reaction solution with a micropipette, and add 10 ml of the buffer that had been dispensed into a small beaker. Q
The diluted solution was used as a sample.

FIG. 2 shows a configuration diagram of an analyzer for antigen-antibody reaction products. The pulse laser is a YAG laser 14 with an output of 300 mJ/pulse and an oscillation period of 20 Hz, and a part of the laser beam 27 from the YAG laser is transmitted through a half mirror 15. and is monitored by a photocell 16. The remaining laser light is focused by a focusing lens 17 and forms a beam waist inside the flow cell 18 . A sample introduced from the sample introduction port 19 flows inside the flow cell 18, and if particulate matter or its agglomerates are present inside the beam waist, one particle causes breakdown and becomes plasma. This plasma is irradiated with excitation light 28 (327, 4 nm) emitted from a Cu hollow cathode lamp 20, and fluorescence 29 emitted by Cu in a plasma state is focused by a condenser lens 21. The collected fluorescence passes through an interference filter 22 to cut off scattered light and stray light other than fluorescence, is detected by a photomultiplier 23, and the fluorescence intensity is converted into an electrical signal 30. This electric signal 3
0, a trigger is applied by the monitor signal 32 from the photocell 16, and the magnitude of the fluorescence intensity is input to the MCA (multi-channel analyzer) 24.

The computer 25 reads the memory contents of the MCA.

This result is printed by the printer 26.

Figure 3 shows the particle number distribution of aggregates in samples to which anti-DNA-BSA dilution ratios of 100 times and 500,000 times to 52,000 times are added.PL, P2° in Figure 3 P3. For P4, the number of particles in the aggregate is one, two, and three.

Corresponds to four.

FIG. 4 shows a calibration curve of the dilution ratio of anti-DNP-BSA serum and the average number of aggregate particles determined by this analytical method.

The concentration of the anti-DNA-BSA serum in the unknown sample can be determined from this calibration curve.

Note that the error was good at CV=0.3 to 3%.

〔Effect of the invention〕

According to the present invention, the particle number distribution of aggregates can be determined without affecting impurities such as serum in the sample, so immunization can be performed with high precision without any special treatment of the sample after antigen-antibody reactions have occurred. It has the effect of quantifying the reaction.

[Brief explanation of drawings]

FIG. 1 is a block diagram showing the basic configuration of the immune substance quantification device according to the present invention, FIG. 2 is a block diagram of the configuration of an embodiment of the present invention, and FIG. 3 is a particle number distribution diagram of aggregates in a sample. Fourth
The figure is a calibration curve diagram of anti-DNA-BSA serum. 1...Pulse laser, 2...Condensing lens, 3...
Cell, 4... Excitation light source, 5... Condensing system, 6... Spectrometer, 7... Photodetector, 8... Data processing device, 9
...Recorder, 10...Laser light, 11...Excitation light, 12...Fluorescence or atomic emission, 13...Signal, 1
4...YAG laser, 15...Half mirror-16.
... Photocell, 17 ... Condensing lens, 18 ... Flow cell, 19 ... Sample introduction port, 20 ... Hollow cathode lamp, 21 ... Condensing lens, 22 ... Interference filter, 23...Photomaru, 24...Multi...
Channel analyzer, 25...computer, 2
6... Plotter, 27... Laser light, 28... Excitation light, 29... Fluorescence, 30... Electric signal, 31...
・# Entry + inlet Fig. 1 24 WJ 2 section 5 6 Fig. MCA + better balance

Claims (1)

[Claims] 1. Particulate matter doped with a specific element in a liquid sample is turned into plasma by laser beam incident breakdown, and the intensity of light emission or fluorescence of the specific element in the plasma state is measured; An immunoassay method that determines the presence or absence of particle aggregation and the particle number distribution of aggregates. 2. The analysis method according to claim 1, wherein the aggregation is a result of an antigen-antibody reaction between an antibody supported on particulate matter added with a specific element and an antigen in a liquid sample. immunoassay method. 3. An immunoassay method according to claim 1, in which the concentration of an antigen that causes an immune reaction in a liquid sample is determined from the presence or absence of agglutination and the particle number distribution of the agglomerates. 4. A laser that breaks down particulate matter, a cell that emits light from the plasma generated by injecting a liquid sample and laser light to the outside, and a cell that collects and spectrally spectra the plasma emission or fluorescence. An immunoassay system comprising an optical system and a photodetector for measuring intensity, a data processing device for processing signals from the photodetector, and a device for displaying output information of the data processing device.