JPH01270643A - Method for examination of specimen - Google Patents

Abstract

PURPOSE:To facilitate window processing and to enhance measuring accuracy, by a method wherein the respective particle sizes and optical characteristics of several kinds of carrier particles are combined so as to be clearly distinguished when a flocculation state and a particle kind are detected. CONSTITUTION:Two or more kinds of latex particles different in light transmissivity and a particle size and a specimen to be examined (serum) are received in a specimen liquid container 15. Herein, when the antibody sensitized with the particles coincides with the antigen in serum, antigen-antibody reaction is generated and the same kind of the particles are flocculated. The flow of this sample liquid is irradiated with the laser beam from a laser 1, and forward scattering beam and lateral scattering beam are measured by light detectors 6, 11 and the outputs thereof are inputted to an operation circuit 16 to analyze the particles. That is, when the hystograms of both scattering beams are put together to obtain a sightgram, a plurality of groups appear thereon. By selecting the particle so that each group appears in a spread state, the sightgram easy to perform window processing can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a specimen testing method, particularly to an immunoassay using carrier particles such as latex particles.

[Prior art] Conventionally, as an immunoassay method, carrier particles such as latex particles are sensitized with a predetermined antibody and a test sample is mixed, and the antigen to be identified by the sensitized antibody is detected in the test sample. When the carrier particles are contained, an antigen-antibody reaction occurs and the carrier particles bind to each other, and a method has been used to measure the presence or absence of the antigen or the amount of the antigen based on the state of aggregation of the carrier particles. At that time, the method of determining the agglomeration state of the carrier particles was carried out by measuring the light transmittance and the degree of light scattering of the suspended fA liquid containing the carrier particles.

In particular, using a flow cytometry method, i.e., the suspension is wrapped in a sheath liquid and hydrodynamically converged to sequentially flow individual carrier particles to the test position, and the carrier particles at the test position are irradiated with a light beam. By determining the size of the carrier particles from the intensity of the scattered light, it is possible to determine the state of aggregation of individual carrier particles, and to calculate the presence or absence of antigen or the amount of antigen, making it possible to perform highly accurate measurements. Fuko.

In the above-mentioned conventional example, only a carrier ladle sensitized with one type of antibody can be used, and therefore only one type of antigen can be tested at a time, which hinders efficiency in dog examinations and the like. Therefore, in Japanese Patent Application No. 63-33482, the applicant of the present application proposed a sample testing method that can simultaneously measure the presence or absence of multiple types of antigens or antibodies, or the amount of antigens or antibodies.

[Object of the Invention] The object of the present invention is to further improve the specimen testing method of the above-mentioned reference and to provide a specimen testing method that improves measurement accuracy.

[Means for Achieving the Object] To achieve the above-mentioned object, the present invention provides a first carrier particle having the same particle size that supports a first antibody or antigen and has a first optical property; A second antibody or antigen is supported by a second antibody or antigen.
second carrier particles having the same particle diameter and having optical properties of are mixed with a test sample, flowing the carrier particles in a set to an inspection position, and irradiating the carrier particles flowing to the inspection position with light, The light scattered in the first direction and the second direction from the carrier particles is measured respectively, and the agglomeration state of the carrier particles is determined by calculating the si [Dullam] from the intensities of the measured first and second lights. and a sample testing method for detecting the type of particles and testing for antigens or antibodies in a sample to be tested, which includes the following steps: Select combinations that are separated on the Zytodarum.

[Example] FIG. 1 is a block diagram of an embodiment of the present invention.

In this example, latex ladle, which is fine particles of an organic polymer material, was used as the carrier particle. Metals, grains, cocci, cell membrane fragments, etc. can also be used.

A sample solution containing a sample liquid made by adding a test sample such as serum to a latex ladle of multiple types sensitized with antibodies? The sheath liquid container 14 containing distilled water, which is the sheath liquid, is pressurized, and is wrapped in sample liquid or sheath liquid and converged into a thin flow, which flows approximately in the center of the flow section in the flow cell 4. pass through the section.

At this time, the individual latex particles contained in the sample liquid sequentially flow one after another or one after another. For this flow of latex particles, is it the laser light emitted from the laser light source 1 or the syringe force? , 3 converges into a lazy shape and is projected to 11. The shape of the optical beam irradiated onto the latex particles is an ellipse that is horizontally elongated with respect to the flow. This is to ensure that the flowing latex particles are irradiated with a light beam with a substantially uniform intensity even if the position of the flow of the sample liquid changes.

When the latex particles are irradiated with a beam of light, scattered light is emitted. Further, when a sample liquid sample is fluorescently stained for fluorescence measurement, fluorescence is also generated at the same time. 119 above. Of the scattered light, forward scattered light emitted in the direction of the optical path )1τj is photometered by a condenser lens 5 and a photodetector 6. Note that the irradiated light beam is directly incident on the photodetector 6 [
55, a stopper (not shown) is provided in front of the condensing lens 5 in the optical path to directly remove the light. The output of the photodetector 6 is input to an arithmetic circuit 16 . Also, among the 11 scattered lights,
The side scattered light emitted in the lateral direction perpendicular to the optical path is condensed by a condenser lens 7, reflected by a tychroic mirror 8, and photometered by a photodetector 11. In general, the direction in which side scattered light is photometered is often orthogonal as in this embodiment, but is not limited to orthogonal and may be, for example, a 45 degree direction or an [iO degree direction. In addition, in order to measure the weak fluorescence that is generated together with scattered light when the sample liquid sample is fluorescently dyed, the fluorescence that is focused by the condenser lens 7 and passed through the tichroic mirror 8 is detected by the tichroic mirror 9 and the photodetector. Green fluorescence is detected by a set of 12, and red fluorescence is detected by a set of a total reflection mirror 1o and a photodetector 13. In front of each photodetector, bandpass filters 2+ and 22.23 are installed to allow only 1M of light in each wavelength range to pass through. Signals from the photodetectors 11, 12, and 13 are input to an arithmetic circuit 16, and the arithmetic circuit performs calculations for particle analysis.

The sample liquid container 15 contains a mixture of three types of latex particles having different light transmittances and particle sizes, each sensitized with a specific antibody, to which serum, which is a test sample, is added or the sample liquid is added. ing.

These latex particles of the same type have the same light transmittance and particle size. When the antibodies sensitized to the latex particles match the antigens in the serum, an antigen-antibody reaction occurs and latex particles of the same type stick together and aggregate.

A method of detecting an antigen-antibody reaction from the intensity of forward scattered light and the intensity of side scattered light by irradiating the flow of the sample liquid with a beam of light will be explained with reference to FIGS. 2 to 9.

Figures 2 to 4 and Figure 8 show the analysis results when three types of latex particles having the same particle size but different light transmittances were sensitized with different antibodies.

When a certain type of latex particles are flowed, the histogram of forward scattered light due to the particles aggregated by the antigen-antibody reaction is expressed as shown in FIG. 2(a). In the figure, the horizontal axis represents the intensity of forward scattered light (FS) measured by the photodetector 6, and the vertical axis represents the number of particles (N). Since the forward scattered light intensity depends on the particle size, the apparent size of latex particles changes due to aggregation, and the graph shows I, , I2, 13
are displayed separately as in . These are considered to have aggregation numbers of 1, 2, and 3 latex particles, respectively.

FIG. 2(b) is a histogram of side scattered light, and the horizontal axis is side scattered light (SS) measured by the photodetector 11.
The vertical axis is the number of particles (N). Since the side-scattered light intensity changes depending on the light transmittance of the particles, the light transmittance of the aggregate changes due to aggregation, and the graph shows J, , I2.
It is displayed separately like I3.

Fig. 2 (C) shows both histograms displayed together. In the figure, the horizontal axis is the side scattered light intensity, and the vertical axis is the forward scattered light intensity. ■■ in FIG. 2(a) and Jl in FIG. 2(b) are the same particles, and similarly ■2J2 and I3. Group I3 is also made of the same particle agglomerate. Therefore, on the cytogram, side scattered light (Jl), forward scattered light (11 groups), side scattered light (I2), forward scattered light (12 groups), and side scattered light (I3), forward scattered light. Three groups appear, such as the group I3.

This can be determined to be an agglomeration state of one, two, or three particles. The above is an explanation of the case where an antigen-antibody reaction occurs and the particles agglutinate, or if the target antigen is not present and agglutination does not occur, I
2, I2, 13J3 does not appear on the graph, I,,J.

Only. Furthermore, when four or more large aggregates are present, they appear on the broken line connecting each group in FIG. 2(C). By looking at the groups appearing on the cytogram as described above, the presence of the desired antigen can be detected.

Similarly, using another type of latex particle having the same particle size as the latex particle and having a different light transmittance, - if an antigen is present, a histogram and a cytodrum as shown in FIGS. 3 and 4 are obtained. Can you get it?

FIG. 8 shows the site graphs 12 of FIGS. 2(C), 3(C), and 4(c) combined into one. In the cytodaram of FIG. 8, the groups on each broken line are information regarding the same antigen (antibody). This is a great success when all types of antibodies are present.

Here, it is a commonly used method to perform the window processing shown in W to check the number of particles (count number) within this window. However, in the case of Figure 8, the particle size is the same but the light transmittance is different, that is, the forward scattered light intensity is almost the same, and only the side scattered light intensity is different because the latex particles are selected. The groups are close together, and in some cases they are displayed overlapping each other. This makes window processing difficult.

Therefore, latex particles with different light transmittances and particle sizes are selected so that each group appears broadly on the cytogram. Figure 5 shows that the forward scattered light intensity (FS) is small and the side scattered light intensity (SS) is caused by latex particles that produce a large output. In the figure, FS is thick and SS is small latex particles. By selecting three types of latex particles in such a combination, the resulting cytodarams are widely separated into groups as shown in FIG. 9, and a zutogram that is easy to perform window processing can be obtained.

In this example, three types of latex particles having different light transmittances were used, or four or more types could be measured simultaneously, or two types could be distinguished more clearly.

In this example, a specific antibody is identified by sensitizing a latex ladle with an antibody or, conversely, by sensitizing a latex particle with an antigen and adding a test sample containing the antibody for testing. It is also possible.

[Effects of the Invention] As described above, according to the present invention, it is possible to identify multiple types of antigen-antibody reactions at the same time, and each antigen-antibody reaction appears clearly separated on a tie-breaker. (Kashiyazuku measurement) Particle analysis with high l'+ degree is possible.

[Brief explanation of the drawing]

FIG. 1 is a block diagram of an embodiment of the present invention, and FIGS. 2 to 9 are 111 diagrams of scattered light measurement data. 1... Laser light source, 2.3... Cylindrical lens, 4... Flow cell, 5.7... Condensing lens, 6.
11.12.13...Photodetector, 8.9...Tichroic mirror, 10...Total reflection mirror, 14...
Sheath liquid container, 15...Zambre liquid container, 16...Arithmetic circuit 2〉No. 30

Claims (1)

[Scope of Claims] 1. A first carrier particle having the same particle diameter that supports a first antibody or antigen and has a first optical property, and a second carrier particle that supports a second antibody or antigen and has a second optical property. A second particle of the same particle size
The carrier particles are mixed with a test sample, the carrier particles are sequentially flowed to a test position, the carrier particles flowed to the test position are irradiated with light, and the carrier particles are mixed with a test sample in a first direction and a second direction. A sample for which the antigen or antibody in the test sample is tested by photometrically measuring the light scattered by each of the carrier particles and detecting the aggregation state and particle type of the carrier particles based on the intensity of the measured first and second lights. In the inspection method, the combination of particle diameters and optical properties of the first carrier particles and the second carrier particles is such that the aggregation state and particle type can be clearly distinguished when detecting the aggregation state and particle type. A specimen testing method characterized by: