JPH1054831A - Immunological analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunological analyzer, which can perform measurement in high accuracy even when there are dispersions in sizes and shapes of immunity complexes and can perform the analysis of many items in the short time. SOLUTION: This immunological analyzer has a sample and the parts performing the functions as follows. A reaction device 27 makes to react the respective antigens or antibodies in correspondence with plural kinds of analysis items with a plurality of carriers, which the label antigens or the label antibodies and a plurality of the different particle diameters are provided and the antigens or the antibodies in correspondence with the different analysis itmes for every particle diameter are made to be the solid phase. Detectors 25 and 26 introduce reacting liquid 23 containing the carrier after the reaction in the reaction device 27 into a flow cell 21 and makes the liquid to flow and sequentially detects the information of the particle diameter of the material in the reaction liquid and the information of the presence or absence of the label material. A detected-information fractionation part 29 fractionates two pieces of detected information detected with the detectors 25 and 26 for every particle diameter so as to provide correspondence. Then, plural kinds of the analysis items are analyzed based on the information, which is fractionated and made to correspond for every particle diameter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological analyzer using a carrier on which an antibody or an antigen reacting with an antigen or an antibody in a sample is immobilized.

[0002]

2. Description of the Related Art Proteins such as globulin and enzymes contained in blood, body fluids, etc., hormones, bacteria, viruses, etc. have similar molecular structures or very small amounts. Identification and quantification are difficult with the device. Therefore, in the analysis of these substances, an immunological analysis method in which an antigen or an antibody is immobilized on a particulate carrier to cause an antigen-antibody reaction is generally used.

[0003] In an immunological analysis method, for example, RIA (radioimmunoassay), EIA (enzyme immunoassay), FIA
(Fluoroimmunoassay). In an analysis method using these labeling substances, in an assay system, for example, an immunocomplex (Bou) in which an antibody (antigen) labeled with a labeling substance and an antigen (antibody) in a sample cause an antigen-antibody reaction.
nd) and an operation of separating a labeled antibody (antigen) as an unreacted component remaining in a free state without being involved in the antigen-antibody reaction, that is, a heterogeneous method requiring so-called BF separation. , Which do not require a homogeneous method.

As an analysis method by the above-mentioned heterogeneous method, Japanese Patent Application Laid-Open No. 53-10495 proposes a method in which BF separation is carried out by using column chromatography. For example, an ion exchange resin that selectively adsorbs free substances (Free) in a solution and does not adsorb an immune complex (Bound) or a packing material for gel chromatography having a molecular sieving effect is used as an adsorbent. Then, after a required reaction time, a free component containing a sample is subjected to BF separation as an unreacted component.

[0005]

However, as described above, B
In the case where a multi-item analysis is performed in a column chromatography using column chromatography for -F separation, a column must be created for each item, and an operation of adsorbing the reaction solution to a different column for each item is required. Become. Therefore, the required amount of the reaction solution increases, and the analysis operation becomes complicated. In addition, if the size or shape of the immune complex varies in each column, or if the size of the immune complex and the size of the free substance are close to each other, the BF separation itself becomes difficult and the accuracy becomes worse. , BF separation prevents the subsequent reaction from occurring, so that the sample cannot be used efficiently. Moreover, it cannot be used to measure the same molecule as an antibody used as a reagent such as immunoglobulin, or a molecule chemically or physically similar, and analysis items are extremely limited.

On the other hand, the technique described in Japanese Patent Application Laid-Open No. 52-15815 discloses a method in which a carrier suspended in a buffer is subjected to a flow cell after two BF separation steps using a plurality of carriers having different particle sizes. And the fluorescence generated by the laser light is detected. However, since this technique requires a centrifuge for BF separation, the structure is complicated and the time required for the whole analysis is very long, and furthermore, before the two BF separation steps, Since it is necessary to react a sufficient and excessive amount of the sample with the labeled antibody, the analysis efficiency is low.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned disadvantages and to carry out high-precision measurement even if the size or shape of the immune complex varies, and to carry out a multi-item analysis in a short time. It is intended to provide a device.

[0008]

According to a first aspect of the present invention, there is provided a reaction section for obtaining and reacting a mixed solution containing a sample and a number of carriers on which an antigen or an antibody corresponding to an analysis item is immobilized. A sampler for collecting the mixed solution mixed in the reaction section from the reaction section and introducing it into the needle of the flow cytometer; and irradiating the mixed solution flowing in the flow cell with laser light. And a data processing unit for separating the received light data into individual data represented by particle diameters and obtaining a total amount of data for each particle diameter. It is characterized by.

Further, the present invention according to claim 2 provides a sample and a labeled antigen or antibody obtained by labeling an antigen or an antibody corresponding to an analysis item with a predetermined labeling substance, and an antigen or an antibody corresponding to the analysis item on a solid phase. A reaction section for simultaneously or sequentially obtaining and reacting a mixed solution containing a large number of carriers that have been converted, and a mixed solution mixed in the reaction section is collected from the reaction section and introduced into a needle of a flow cytometer. A detector for obtaining information on the particle diameter and information on the labeling substance from the mixture flowing in the flow cell, and individual data expressed by the particle diameter. And a data processing unit for determining the amount of labeling substance for each particle size by separating the particles into data.

[0010] The labeling substance of the invention according to claim 2 is a substance which emits fluorescence by laser light, and the detecting section comprises a laser.
And a means for irradiating the light and a means for respectively receiving scattered light and fluorescence from the common laser light.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows an embodiment of a schematic reaction diagram in an analyzer according to the present invention. In the present embodiment, reference numerals 1, 2 and 3 each represent a polystyrene latex used as a carrier, and the particle size is, for example, latex 1
0.5 μm, latex 2 1.0 μm, latex 3
One having a uniform diameter of 1.5 μm is used. Reference numerals 4, 5, and 6 denote solid phase antibodies immobilized on latexes 1, 2, and 3, respectively, having different diameters by physical adsorption.
Reference numerals 7, 8 and 9 denote antigens contained in the sample serum or the like, and reference numerals 10, 11 and 12 denote antibodies specifically binding to the respective antigens 7, 8 and 9 by FI.
It is a labeled antibody labeled with a fluorescent substance such as TC.

Hereinafter, the analysis will be described by taking the specificity analysis of the human immunoglobulin class as an example. Anti-human IgG antibody 4 is applied to latex 1 having a particle size of 0.5 μm, anti-human IgA antibody 5 is applied to latex 2 having a particle size of 1.0 μm, and anti-human IgM antibody 6 is applied to latex 3 having a particle size of 1.5 μm. Become In addition,
It is desirable to use a monoclonal antibody for these solid phase antibodies in order to eliminate non-specific adsorption between latexes and to enhance specificity with the antigen. The reaction was performed by adding 50 μl of these antibody-bound latex solutions to 200 μl of reaction buffer.
And 10 μl of a sample containing antigens such as human IgG7, IgA8 and IgM9, and anti-human Ig each labeled with FITC.
G antibody 10, anti-human IgA antibody 11, anti-human IgM antibody 12
And a mixed solution of 50 μl. Here, it is desirable to use Fab fragments for the labeled antibodies 10, 11, and 12 in order to reduce non-specific adsorption and to increase the reaction rate. In addition, all of these reagents may be added at the same time to obtain a reaction solution, or a reaction solution may be obtained by sequentially adding various solid-phase antibodies and labeled antibodies to a common sample. Is also good.

When the reaction is carried out, for example, at 37 ° C. for 10 minutes, the immunoconjugates (Bounds) 13, 14, 15 of each solid-phase antibody latex-antigen-labeled antibody and the remaining labeled antibody (Freund)
e) 16 is generated. In this embodiment, the reaction solution obtained by mixing and reacting the sample and the reagent in this manner is collected and introduced into a flow cytometer as shown in FIG. Can flow.

As is already known, the flow cytometer is a dedicated machine for analyzing cells, in which a reaction solution 23 is caused to flow through a needle 22 in a flow cell 21 and a laser beam 24 is irradiated on the flow to scatter the light from the cells. Measure light and fluorescence.
Normally, the forward scattered light is detected by the detector 25 positioned substantially horizontally with the laser incident light, and is mainly used for measuring the cell size. The fluorescence is detected by a detector 26 located in a direction perpendicular to the incident angle of the laser light 24, and is used for measuring a fluorescent substance or the like on the cell surface. Since the laser beam 24 has a single wavelength, there are limitations on the fluorescent dyes that can be used, but the fluorescent dye FITC used in the analyzer of the present embodiment has a wavelength of
Since it absorbs light near 489 nm and emits fluorescence with a wavelength of 515 nm, an Ar laser with a wavelength of 488 nm may be used in this case.

As the flow cytometer, for example, the one described in US Pat. No. 4,325,706 can be used. In this manner, the reaction solution 23 in which the immune complexes 13, 14, 15 and the remaining labeled antibody 16 shown in FIG. 1 after the reaction are mixed is introduced from the needle 22 into the flow cell 21, and flows through the needle 22. If the scattered light and fluorescence of each immune complex and the remaining components such as the labeled antibody by the laser light 24 are detected by the detectors 25 and 26, respectively,
The size of each immune complex is measured by the detector 25, and if the size of each latex is about 1 μm, the solid antibody-antigen-labeled antibody binding portion of at most several nm is within the error range. Mostly dependent on latex particle size. At the same time, the amount of fluorescence on each latex / 1 latex is measured by the detector 26, and based on these two parameters, the detection information classification processing unit 2
9 outputs the cytogram shown in FIG. In FIG. 3, the vertical axis represents the amount of fluorescence, and the horizontal axis represents the particle diameter.

Here, since the remaining labeled antibody which has not been involved in the antigen-antibody reaction has a small diameter, it concentrates on the position 31 of the amount of fluorescence per one labeled antibody. In addition, the immune complex 13 of FIG.
In addition, the immune complex 14 of FIG. 1, bound by the second smallest latex,
It is shown at the position 33 which is considerably high also as the fluorescence amount per unit. The immune complex 15 of FIG. 1 having the largest latex diameter is shown at the position 34 because the antigen concentration was low.

When the measured value of the fluorescence amount is obtained in this manner, based on a calibration curve representing the relationship between the fluorescence intensity and the antigen concentration, which is similarly obtained in advance from a known concentration series of each antigen such as IgG, IgA, and IgM. The concentration of each antigen in the sample can be determined.

As described above, the combination of the flow cytometer and the latex immunoassay using the latex in which different antibodies are immobilized according to each antigen is combined, so that a single analyzer can analyze many items at high speed. Can be performed with high accuracy. In addition, since there is no restriction on the reaction amount associated with the BF separation, the size of the immune complex with respect to a large amount of latex reacted with a small amount of a sample of about 10 μl is defined as BF
Since measurement is performed without separation by laser light without separation, accurate data can be obtained efficiently. In addition, if latexes having different particle diameters are used simultaneously for each type of antigen, it is possible to display information that can be easily identified for each antigen.

It should be noted that the present invention is not limited to the above-described embodiment, and that various changes and modifications are possible. 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 having a uniform molecular weight. In addition, a sorting function can be added to the flow cytometer to separate the immune complex and the remaining labeled antibody after measurement.
In this way, the remaining labeled antibody can be separated and taken out, and can be reused. Further, automatic measurement can be easily performed by adding a reaction device 27, an autosampler 28, and the like to the flow cytometer. In this case, since the measurement speed in the flow cytometer is about 500 particles / sec, even if 1 × 10 6 particles are measured for one sample, high-speed analysis can be performed in about 3 minutes. Further, the present invention can be effectively applied to analysis by a competition method.

Further, according to the above-described configuration, it can be set so as to carry out an analysis relating to a reaction in which carriers are bound to each other, such as a cell analysis using a general flow cytometer. As the labeling substance, a substance for chemiluminescence as generally known in immunological analysis may be used.

[0021]

According to the present invention, the immune complex in the mixture flowing in the flow cell is measured without any laser light, and the individual data is displayed as the total amount for each particle diameter. In addition, immunological reaction results can be analyzed in a short time and with high accuracy. In addition, since the mixed solution reacted in the reaction section is collected by a sampler and introduced into the needle of the flow cytometer, it is simply distributed in the flow cell, so that a large number of analyzes can be performed with a single automated device. Can be analyzed efficiently.

[Brief description of the drawings]

FIG. 1 is a schematic reaction diagram of one embodiment of the present invention.

FIG. 2 is a diagram for explaining a flow cytometer.

FIG. 3 is a diagram showing a cytogram of measurement data.

[Explanation of symbols]

 1,2,3 Latex 4,5,6 Solid-phase antibody 7,8,9 Antigen 10,11,12 Labeled antibody 13,14,15 Immune complex 16 Remaining labeled antibody 21 Flow cell 22 Needle 23 Reaction solution 24 Laser light 25, 26 Detector 27 Reactor 28 Autosampler 29 Detection information separation processing unit

Claims (3)

[Claims] 1. A reaction section for obtaining and reacting a mixed solution containing a sample and a large number of carriers on which an antigen or an antibody corresponding to an analysis item is immobilized, and reacting the mixed solution mixed in the reaction section. A sampler for collecting from the reaction section and introducing it into the needle of the flow cytometer; a detection section for irradiating the mixed solution flowing in the flow cell with laser light to obtain the received light data; An immunological analyzer comprising: a data processing unit for separating received light data into individual data represented by a particle size and obtaining a total amount of data for each particle size. 2. A mixture comprising a sample and a labeled antigen or antibody obtained by labeling an antigen or antibody corresponding to an analysis item with a predetermined labeling substance, and a large number of carriers having the antigen or antibody corresponding to the analysis item immobilized thereon. A reaction section for obtaining and reacting liquids simultaneously or sequentially; a sampler for collecting a mixed solution mixed in the reaction section from the reaction section and introducing it into a needle of a flow cytometer; and a flow cell. A detection unit that obtains light-receiving data indicating information on the particle size and information on the labeling substance from the mixed solution flowing through the inside, and separates the light-receiving data into individual data expressed by the particle size to label each particle size An immunological analyzer, comprising: a data processing unit for determining an amount of a substance. 3. The labeling substance is a substance that generates fluorescence by laser light, and the detecting unit receives scattered light and fluorescence from a laser beam common to the means for irradiating the laser light. The immunological analyzer according to claim 2, comprising: