JPH0588422B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to an immunological analysis method.

(Prior art) Proteins such as globulins, enzymes, hormones, bacteria, viruses, etc. contained in blood, body fluids, etc. have similar molecular structures or are present in very small amounts.
It is difficult to fix and quantify using conventional analytical methods. Therefore, immunological analysis methods that utilize antigen-antibody reactions are generally used to analyze these substances.

Such immunological analysis methods include, for example, those using labeling substances such as RIA (radioimmunoassay), EIA (enzyme immunoassay),
There are FIA (fluoroimmunoassay), etc. Also,
In analytical methods using these labeling substances, for example, an antibody (antigen) labeled with a labeling substance is used in the measurement system.
Separate the immune complex (bound) in which an antigen (antibody) in the sample has caused an antigen-body reaction, and the labeled antibody (antigen) that does not participate in the antigen-antibody reaction and remains in a free state. Operation, so-called B-F
It is classified into heterogeneous methods, which require separation, and homogeneous methods, which do not.

As an analysis method based on the above-mentioned heterogeneous method, a method in which B-F separation is performed using column chromatography is proposed in Japanese Patent Application Laid-Open No. 10495/1983. For example, it selectively adsorbs free substances in a solution,
B-F separation is performed using an ion exchange resin that does not absorb immune complexes (Bound) or a packing material for gel chromatography that has a molecular sieving effect as an adsorbent.

However, when B-F separation is performed using column chromatography, the sizes and shapes of immune complexes vary, and the sizes of immune complexes and free substances are close to each other. This makes B-F separation difficult and accuracy deteriorates. For this reason, it cannot be used to measure molecules that are the same as antibodies used as reagents such as immunoglobulin, or molecules that are chemically or physically similar, and the analytical items are extremely limited.

(Objective of the Invention) The object of the present invention is to solve the above-mentioned problems and to remove the labeling substance bound to the antigen (antibody) in the sample.
The object of the present invention is to provide an immunological analysis method that can be effectively separated and detected from unbound labeling substances, allows desired analysis to be performed easily and with high precision, can be performed rapidly, and is extremely advantageous for large-scale processing.

(Summary of the Invention) The immunological analysis method of the present invention comprises a sample, a labeling reagent in which an antigen or antibody is labeled with a predetermined substance, and a carrier in which the antigen or antibody is immobilized on a carrier that is sufficiently larger than the labeling reagent. The labeled amount of the labeled reagent that binds to the carrier reagent through this reaction is detected separately from the labeled reagent that does not bind to the carrier reagent by detecting the size of the carrier reagent. , the sample is immunologically analyzed.

(Example) FIG. 1 shows an example of a reaction schematic diagram in the analysis method of the present invention. In this example, the code 1
is a latex used as a carrier, for example, one made of polystyrene having a uniform diameter of 5 μm, on which the solid-phase antibody 2 is immobilized by physical adsorption. Code 3 is the antigen to be analyzed contained in the sample, such as serum, and code 4 is the antibody that specifically binds to antigen 3.
It is a labeled antibody labeled with a fluorescent substance such as FITC.
Further, reference numeral 5 indicates the immune complex (Bound) after the antigen-antibody reaction, and reference numeral 6 indicates the remaining labeled antibody (Free).

The analysis of human IgE will be explained below as an example.
In this case, anti-human IgE monoclonal antibody 2 is adsorbed onto latex 1. The purpose of using monoclonal antibodies is to prevent latex particles from aggregating with each other and to bind to antigens more specifically. The antigen as a sample is human IgE3, and the labeled antibody 4 is
Use FITC-labeled anti-human IgE antibody. Note that it is preferable to use a Fab fragment for the labeled antibody 4 in order to reduce nonspecific adsorption and increase the reaction rate. For the reaction, add 200μ of reaction buffer, 50μ of solid-phase antibody-bound latex solution, and 10μ of sample.
Add 50μ of labeled antibody solution. Note that even if all of these reagents are added at the same time,
Alternatively, the antigen may be reacted with a solid-phase antibody and then added sequentially so as to be reacted with a labeled antibody.

Here, when the reaction is carried out at 37° C. for 10 minutes, for example, an immune complex 5 of solid-phase antibody-antigen-labeled antibody and the remaining labeled antibody 6 are produced. In this example, this is passed through a flow cytometer shown in FIG. 2 for measurement.

As is already known, a flow cytometer is a machine dedicated to cell analysis, in which a reaction solution 13 is passed through a needle 12 in a flow cell 11, and a laser beam 1 is applied to the flow cytometer.
4 to the flow and measure the scattered light and fluorescence emitted from the cells. Normally, the forward scattered light is detected by a detector 15 located approximately parallel to the laser incident light, and is mainly used for measuring cell size. The fluorescent light is detected by a detector 16 located in a direction perpendicular to the incident angle of the laser beam 14, and is used to measure fluorescent substances on the cell surface. Since the laser beam 14 has a single wavelength, there are restrictions on the fluorescent dyes that can be used, but the fluorescent dyes used in the analysis method of this example
FITC absorbs light near the wavelength of 489nm and
It emits fluorescent light at 515 nm, so in this case the wavelength
An Ar laser that emits light at 488 nm may be used.

In this way, the reaction solution 13 in which the immune complex 5 shown in FIG. When the scattered light and fluorescent light of each component such as the body and the remaining labeled antibody are detected by the detectors 15 and 16, a sidegram as shown in FIGS. 3A and 3B is generated depending on these parameters. can get. FIG. 3A shows a cytogram when antigen-antibody testing was performed, and in this example, anti-human IgE
The labeled antibody binds to the IgE in the sample.
IgE MCA binds to the IgE part of the solid phase latex, and the fluorescence emitted by the immune complex is detected at position 21 corresponding to the particle size of the latex particles. on the other hand,
The remaining labeled antibody that has not participated in the antigen-antibody reaction is detected around position 22 because its particle size is extremely small. Once the measured value of fluorescence intensity is obtained in this way, the IgE concentration in the sample can be determined based on a calibration curve representing the relationship between fluorescence intensity and antigen concentration, which was previously determined from known antigen concentrations. .

Furthermore, when there is no IgE in the sample, the labeled antibody concentrates at position 22, as shown in FIG. 3B, and no fluorescence is detected from the antibody-bound latex.

As described above, when a latex immunoassay using a flow cytometer is used, the immune complex and the remaining labeled antibody have significantly different particle sizes, making it easy to distinguish them in the measurement. Furthermore, since the size of the latex particles is considerably larger than the size of the antigen or antibody, variations in the size and shape of the immune complexes are considerably reduced, allowing highly accurate analysis. Furthermore, since the immune complex in the reaction solution and the remaining labeled antibody are separated on the measurement rather than physically, the measurement can be performed as is, making it possible to perform high-speed multi-analyte measurements. It is possible.

Note that the present invention is not limited to the above-mentioned example, and can be modified or modified in many ways. For example, the carrier is not limited to latex, but may be of any shape or size depending on the object to be measured, such as artificial cells with uniform molecular weight. Furthermore,
Automatic measurements can also be easily performed by adding a reaction device, an autosampler, etc. to the flow cytometer. In this case, since the measurement speed in the flow cytometer is approximately 5000 particles/sec,
Even if 1×10 ⁶ particles are measured for one sample, the analysis can be performed quickly in about 3 minutes. Furthermore, the present invention can be effectively applied to analysis using a competitive method.

(Effects of the Invention) As described above, according to the present invention, since a carrier is used, the sizes of the immune complex and the remaining labeled antibody are greatly different, and therefore measurement separation can be easily performed. Since the variation in size and shape of immune complexes can be extremely small, highly accurate analysis can be performed. Further, since the carrier can be selected to have any size and shape, it is not limited by the type or size of the sample molecule. Furthermore, according to the above-mentioned embodiment, since the immune complex and the remaining labeled antibody can be separated during measurement,
Automation is possible for high-speed, multi-sample measurements.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an example of a reaction in the present invention, FIG. 2 is a diagram for explaining a flow cytometer,
FIGS. 3A and 3B are diagrams showing cytograms of measurement data. 1... Latex, 2... Solid-phase antibody, 3... Antigen, 4... Labeled antibody, 5... Immune complex, 6...
Remaining labeled antibody, 11...Flow cell, 12...
Needle, 13... Reaction liquid, 14... Laser light,
15, 16...detector.

Claims (1)

[Claims] 1. React the sample with a labeling reagent in which an antigen or antibody is labeled with a predetermined substance, and a carrier reagent in which the antigen or antibody is immobilized on a carrier that is sufficiently larger than the labeling reagent, and through this reaction, it binds to the carrier reagent. The sample is immunologically analyzed by detecting the labeled amount of the labeled reagent to be separated from the labeled reagent that does not bind to the carrier reagent by detecting the size of the carrier reagent. immunological analysis method.