JPS61132868A - Immunological analysis - Google Patents

Abstract

PURPOSE:To make possible easily a desired analysis by bringing a labeling antigen (antibody) and a carrier carrying an antigen (antibody) as a solid phase into reaction and separating the reactive liquid to an immune complex and the remaining labeling antibody in accordance with the information on the carrier detected from the reactive liquid. CONSTITUTION:The reactive liquid 13 in which the immune complex and the remaining antibody are mixed is introduced through a needle 12 into a flow cell 11. The scattered light and fluorescence by laser light 14 of the immune complex and remaining and remaining labeling antibody flowing in the needle 12 are detected by detectors 15, 16, by which a sightgramis obtd. from the parameter thereof. The recognition in the measurement of the immune complex and the remaining labeling antibody from the difference in the size of the particles thereof is made easy if the latex immunoassay is used by using the flow sight meter in the above-mentioned manner. The variance in the size and shape of the immune complex is fairly decreased and the analysis with high accuracy is made possible.

Description

[Detailed description of the invention] (Technical field) The present invention relates to an immunological analysis method.

(Prior art) Proteins such as globulins and enzymes contained in blood, body fluids, etc.
Hormones, bacteria, viruses, etc. have similar molecular structures and are very small in amount, so it is difficult to identify and quantify them using normal 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, RIA (radioimmunoassay), E
Examples include IA (enzyme immunoassay) and FIA (fluoroimmunoassay). In addition, in the analysis method using these labeling substances, in the measurement system, an immune complex (Bound) in which an antibody (antigen) labeled with a labeling substance and an antigen (antibody) in a sample undergoes an antigen-antibody reaction,
An operation to separate the labeled antibody (antigen) that does not participate in the antigen-antibody reaction and remains in a free state, the so-called B
-F separation is classified into a heterogeneous method that requires separation and a homogeneous method that does not require it.

As an analytical method based on the above-mentioned heterogeneous method, JP-A-53-10495 proposes a method in which B-F separation is effectively achieved using column chromatography. This is, for example, an educt f(F
selectively adsorbs immune complexes (Bound).
B-F separation is carried out using an ion exchange resin that does not absorb ions 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, it is possible that the sizes and shapes of immune complexes vary, or that the sizes of immune complexes and free substances are close to each other. B-F separation becomes 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 provide an immunological analysis method that can always perform B-F separation reliably and perform desired analysis easily and with high precision. It is something to do.

(Summary of the Invention) The immunological analysis method of the present invention involves reacting a sample with a labeled antigen or antibody obtained by labeling the antigen or antibody with a predetermined substance, and a carrier on which the antigen or antibody is immobilized. The method is characterized by sequentially detecting the carriers while flowing the reaction solution, and separating labeled antigens or antibodies bound to the carrier from unbound ones based on the detected carrier information.

(Example) FIG. 1 shows an example of a reaction schematic diagram in the analytical method of the present invention. In this example, reference numeral 1 denotes a latex used as a carrier, and the solid-phase antibody 2 is immobilized on a polystyrene material having a uniform diameter of, for example, 5 μm by physical adsorption. Reference numeral 3 is an antigen to be analyzed contained in a sample such as serum, and reference numeral 4 is a labeled antibody obtained by labeling an antibody that specifically binds to antigen 3 with a fluorescent substance such as FITC.

In addition, numeral 5 indicates an immune complex (Boun) after antigen-antibody reaction.
d), and numeral 6 is 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 bind to antigens more specifically. The antigen used as a sample is human IgE.
3, and for labeled antibody 4, FITC [anti-human IgB
Use antibodies. 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, use 200 μl of reaction buffer.
Add 50 μl of solid-phase antibody-bound latex solution to sample 1.
0 μl and 50 μl of labeled antibody solution were added.

Note that these reagents may be added all at the same time, or may be added sequentially such that the antigen is reacted with the solid-phase antibody and then reacted with the labeled antibody.

Here, for example, when reacting at 37°C for 10 minutes, the solid-phase antibody-antigen-labeled antibody immune complex 5 and the remaining labeled antibody 6
is generated. In this example, this is passed through a flow cytometer shown in FIG. 2 to electrically separate B and F and simultaneously measure it.

As is already known, a flow cytometer is a machine dedicated to cell analysis, in which a reaction solution 13 is flowed through a needle 12 in a flow cell 11, and a laser beam 14 is irradiated onto the flow to detect scattered light and fluorescent light emitted from cells. Measure light. Normally, the forward scattered light is detected by the detector 1 located almost horizontally with the laser incident light.
5 and is mainly used to measure cell size. The fluorescent light is detected by a detector 16 located 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. However, the fluorescent dye FITC used in the analysis method of this example has a wavelength of 489no+.
Since it absorbs nearby light and emits fluorescent light with a wavelength of 515 na+, in this case, an Ar laser that emits light with a wavelength of 488 nm may be used.

In this way, the reaction solution 13 in which the immune complex 5 shown in FIG.
If the scattered light and fluorescent light caused by the laser beam 14 of each component such as the immune complex flowing therein and the remaining labeled antibody are detected by the detectors 15 and 16, the results will be as shown in FIGS. 3A and 3B depending on these parameters. A cytogram is obtained. In addition, FIG. 3A shows a cytogram when antigen-antibody testing was performed, and in this example, anti-human IgH [i! li
Anti-human IgE that the antibody binds to IgE in the sample
The fluorescent light emitted by the immune complex bound to the xgg portion of the MCA solid-phase latex is detected at position 21 corresponding to the particle size of the latex particle. On the other hand, the remaining labeled antibody that did not participate in the antigen-antibody reaction is detected around position 22 because its particle size is extremely small. In this way, if the measured value of the fluorescence intensity is obtained, the IBE concentration in the sample can be determined based on a calibration curve representing the relationship between the fluorescence intensity and the antigen concentration, which was previously determined from antigen concentrations of known concentration. .

In addition, if there is little IgB in the sample, the remaining labeled antibody will concentrate at position 22, as shown in Figure 3B.
No fluorescence is detected from the antibody-conjugated latex.

As described above, when using latex immunoassay, which is measured using a flow cytometer, since the particle size of the immune complex and the remaining labeled antibody differs greatly,
Easy to identify in measurements.

Furthermore, since the size of latex particles is considerably larger than the size of antigens and antibodies, variations in the size and shape of immune complexes are considerably reduced, allowing highly accurate analysis. Furthermore, the reaction solution is heated electrically rather than mechanically.
Since it separates -F, it can be measured as is, and therefore high speed and multi-analyte measurement 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. Alternatively, measurement can be performed after adding a sorting function to a flow side meter to perform B-F separation of the immune complex and the remaining labeled antibody. In this way, various labeling substances other than fluorescent substances can be used. Furthermore, by adding a reaction device, an autosampler, etc. to the flow cytometer, automatic measurements can be easily performed. In this case, since the measurement speed in a flow cytometer is about 5,000 particles/sec, even if one sample is measured for lXl0'' particles, the analysis can be performed at a high speed in about 3 minutes. can also be effectively applied to competitive analysis.

(Effects of the Invention) As described above, according to the present invention, since a carrier is used, the size of the immune complex and the remaining labeled antibody is greatly different, and therefore measurement separation can be easily performed.
Since variations in the size and shape of immune complexes can be extremely reduced, 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 reaction solution can be electrically separated from B-F and measured at the same time, automatic ratioing for the purpose of high-speed, multi-analyte measurement is possible.

[Brief explanation of the drawing]

FIG. 1 is a schematic reaction diagram of an example of the present invention, FIG. 2 is a diagram for explaining a flow cytometer, and FIG. 3 A and B 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 beam 15.16 - Detector Fig. 1 Fig. 2 Fig. 3 Diameter killing □ aa→

Claims (1)

[Claims] 1. After reacting a sample, a labeled antigen or antibody obtained by labeling an antigen or antibody with a predetermined substance, and a carrier on which the antigen or antibody is immobilized, the 1. An immunological analysis method comprising sequentially detecting carriers and separating labeled antigens or antibodies bound to the carrier from unbound labeled antigens or antibodies based on the detected carrier information. 2. The reaction solution is passed through a flow cytometer to electrically separate the labeled antigen or antibody bound to the carrier from that which is not bound, and at the same time, the labeled antigen or antibody bound to the carrier is measured. An immunological analysis method according to claim 1. 3. The immunological method according to claim 1, wherein the labeled antigen or antibody bound to the carrier is separated from the unbound labeled antigen or antibody, and then the labeled antigen or antibody bound to the carrier is measured. Analysis method.