JPH0552848A - Immunoassay and apparatus - Google Patents

Abstract

PURPOSE:To achieve higher measuring sensitivity with the determining of an agglutination state accurately by detecting fluorescence which is generated as each agglutination of fine particles occurs after an antigen/antibody reaction. CONSTITUTION:A reaction reagent employs a latex particle which has a fluorescent pigment such as xanthine or rhodamine bonded to the surface thereof or latex particles contained inside. The latex particles are bonded to each other by an antigen/antibody reaction to form an agglutination body having a distribution with the size thereof corresponding to reaction conditions. After brought to an agglutination reaction, a reaction liquid is introduced from a sample introduction device 1 to be sent to a flowcell 3 through a tube 2 with a pump 17 and a pulse-shaped fluorescence is generated when the particles pass through a beam from a laser 8. The fluorescence is condensed with a lens 10 to be detected with a photomultiplier 12 and an output thereof is amplified with an amplifier 13 to be inputted into a pulse height analyzer 15. The processing results 14 of the analyzer 15 are sent to a personal computer 16 and undergoes a data processing. Then, the results are displayed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to measurement of trace substances in biological components, and more particularly to immunoassay for measuring specific substances in samples such as blood easily and with high sensitivity.

[0002]

2. Description of the Related Art An immunoassay utilizing an antigen-antibody reaction has been developed in order to measure a trace component in a sample such as blood with high sensitivity. Multidimensional binding occurs between the antigen in the sample and the antibody added as a reagent to form an aggregate. This is measured optically. The latex agglutination method is used to further increase the measurement sensitivity. Using latex particles to which an antigen or antibody is bound as a reagent, the degree of aggregation with the antibody or antigen in the corresponding sample is improved by scattering light intensity measurement, turbidity measurement, electrical resistance detection, photoacoustic detection, etc. It is a thing. Regarding each method, for example, Clinical Pathology Extra Number 53, p. 71, 197.
5 years, Journal of the Japan Society for Clinical Laboratory Automation 9 pages 678 1
984, Analytical Chemistry, Volume 59, 2
519 p. 1987 (Anal. Chem. 59, 2519,
(1987)). However, since the measurement was carried out in the presence of the reacted fine particle reagent and the non-reacted fine particle reagent, the sensitivity was poor in the low antigen concentration region. In order to improve this, a method of measuring the particle reagent after the reaction for each aggregation has been developed. About this, for example, Journal of Japan Society for Clinical Laboratory Automation, Vol. 12, 294
There is a particle size distribution measuring method described in page 1987.

[0003]

With respect to the conventional measuring methods based on the above-mentioned antigen-antibody reaction, there are unavoidable problems such as a treatment method under antigen excess reaction conditions and a method of avoiding interference by coexisting substances. In the in-solution antigen-antibody reaction, an abnormally low value is shown in the case of an extreme excess of antigen, and an incorrect measurement value will be submitted. Further, when hemoglobin or chyle normally present in serum is increased, coexisting substances from the sample are inevitable because light scattering and light absorption occur even if highly diluted.

Further, when the turbidity or the scattered light intensity is measured as it is after the antigen-antibody reaction in solution using latex particles, there is a problem that high-sensitivity detection cannot be performed due to the influence of the unreacted fine particle reagent.

In the case of the method of measuring each agglutination, in the detection by the light scattering method, there is a limit in the size of the particles to be detected (wavelength order), while the reaction efficiency decreases when large particles are used as the reaction reagent.

In the electric resistance detection method, the size of the detection particles is limited to the size of the aperture. If the aperture is made small, it is easy to put it down and it is not realistic. Further, similarly to the light scattering method, there is a restriction that small particles cannot be used due to the influence of foreign dust (particles). In the method using photoacoustic detection, the sensitivity resonates in a region where the particle diameter and the excitation light wavelength match, but the sensitivity is very low in a region below that, and it is difficult to apply it to small particles.

Further, in the above measuring method, since the measurement signal is not linear with respect to the number of particles included in the agglomeration, the accurate size cannot be measured.

[0008]

In order to solve the above-mentioned problems, the present invention has fluorescent dyes such as xanthine and rhodamine bound to the surface as fine particles used in a reaction reagent.
Alternatively, latex particles contained therein are used. The reaction liquid after the reaction is introduced into the flow cell, and the flow cell is irradiated with light. A method of measuring the agglutination state is used by detecting the fluorescence generated from the fine particles in the sample and having different intensities for each agglutination.

[0009]

As described above, in the method of the present invention, since the particles are fluorescent, fine particles can be easily measured. Therefore, small particles convenient for the reaction can be used as reagents.

Since the method of the present invention detects fluorescence, it is unlikely to be affected by serum components, foreign debris, particles or the like.

Since the method of the present invention detects each aggregation, the measurement can be performed without the influence of the unreacted particle reagent.

Further, since the fluorescence intensity is proportional to the dye content in the particles, according to the method of the present invention, a linear response is obtained with respect to the size of aggregation (the number of fine particles), and the accurate aggregation state can be grasped. Is possible.

[0013]

Embodiments of the present invention will be described with reference to the drawings.

An example of the latex agglutination reaction will be described with reference to FIG.

Non-antigenic latex particles containing a fluorescent dye serve as a carrier, and a specific antibody (or antigen) is bound to the surface of the latex particle, and in the presence of the antigen (or antibody) in a sample such as serum. The antigen-antibody reaction causes the latex particles to bind to each other via the antigen to form an aggregate having a size distribution according to the reaction conditions.

[0016] The antigen then absorbance method and could not be detected by turbidimetry concentration regions (e.g., 10- ¹¹ M) as described above,
The sample solution obtained by immunoreaction was introduced into a flow cell, and fluorescence was detected by this method. The measurement results before and after the reaction are shown in FIG. As shown in the figure, one peak reflecting the fluorescent fine particle reagent appeared in the histogram before the reaction, that is, in the blank histogram, whereas in the histogram after the reaction, in addition to the above peaks, it corresponds to aggregation. How many peaks were obtained. From this result, even when the concentration of the antigen was very low, the difference from the blank was clearly recognized, which shows that the present invention has higher sensitivity than the conventional method and has a wide quantitative range, and the unreacted fine particle reagent. I was not affected by.

Further, the fluorescence intensity was plotted against the size of aggregation based on the measurement data of FIG. As a result, as shown in FIG. 3, it was found that the fluorescence intensity increased in proportion to the size of aggregation, that is, the number of fine particles. Therefore, it was found that this method can accurately grasp the state of aggregation without the influence of foreign debris and particles.

Next, an example of an apparatus for carrying out the present invention will be shown.

FIG. 4 illustrates the outline of the counting device. The reaction liquid after the agglutination reaction is introduced from the sample introduction device 1 and is sent by the pump 17 through the tube 2 to the flow cell 3 at a constant flow rate. Laser light emitted from the laser 8 is applied to the sample flow flowing through the diaphragm flow cell 3 by the lens 9. The pulsed fluorescence emitted when the particles pass through the laser beam is condensed by the lens 10, passes through the filter 11, and is detected by the photomultiplier tube 12. The output converted into an electric signal by the photomultiplier tube 12 is an amplifier 13
It is amplified by and input to the wave height analyzer 15. Wave height analyzer 15
Then, only the pulses between two types of thresholds that give the upper and lower limits set in advance are counted. The number of pulses becomes the number of particles. A switch 4 is provided in the sample introduction device 1, a trigger signal 5 is sent to the delay timer 6 at the same time as the sample introduction, and a measurement start instruction signal 7 is input to the wave height analyzer 15 after a delay time to start the measurement operation. The wave height analyzer 15 sends the primary processing result 14 to the personal computer 16 to perform data processing and display the result.

The personal computer 16 issues an instruction signal 18 according to a preset program,
Control the progress of the measurement operation.

As the excitation light, a gas laser such as an argon gas laser or a helium neon laser, a solid-state laser such as a YAG laser or a semiconductor laser, or a second or third harmonic having the oscillation line of these as a fundamental wave can be used. it can.

[0022]

EFFECTS OF THE INVENTION It is possible to perform highly sensitive measurement with a simple operation. The conventional reagent production technology can be used as it is.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of a latex agglutination reaction.

FIG. 2 is a diagram showing histograms of samples before and after reaction.

FIG. 3 is a diagram showing the relationship between fluorescence intensity and the size of aggregation.

FIG. 4 is a configuration diagram showing an example of a measuring device.

[Explanation of symbols]

1 ... Injector, 2 ... Tube, 3 ... Flow cell, 4
… Trigger, 8… Laser, 9… Lens, 10… Lens,
11 ... Filter, 12 ... Photomultiplier tube, 13 ... Amplifier,
15 ... Wave height analyzer, 16 ... PC, 17 ... Pump.

Claims (5)

[Claims] 1. A reagent in which the surface of fine particles is sensitized with an antibody or an antigen is prepared, and the reagent is reacted with a sample to be measured,
In an immunoassay for measuring the antigen concentration or antibody concentration in a sample by measuring the aggregation state of the fine particles generated through the antigen or antibody contained in the sample, the particles are fluorescent and An immunoassay method characterized in that the aggregation state is measured by detecting the fluorescence intensity for each aggregate. 2. An immunoassay method characterized in that the fine particles according to claim 1 are spherical and have a diameter of 0.5 μm or less. 3. The fine particles according to claim 1 containing polystyrene, poly (methyl methacrylate) or poly (hydroxyethyl methacrylate) as a main component and binding a fluorescent dye to the surface of the particle, or to the inside of the particle. An immunoassay characterized by containing it. 4. A component which imparts fluorescence to the fine particles according to claim 1, is rhodamine, Neil blue, Neil red, methylene blue, thionine chloride, fluorescein, Texas red, acridine orange, phycobiliprotein. Immunoassay. 5. A mechanism for dispensing a sample into a reaction container, a mechanism for dispensing a reagent containing fine particles into a reaction container, a mechanism for introducing a reaction solution after reaction into a flow cell, and a flow cell irradiated with light.
An immunoassay device comprising a mechanism for detecting fluorescence generated from fine particles in a sample and a mechanism for processing the obtained signal.