JPH0610678B2 - Immunological analysis method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunological analysis method.

(Prior art) Since globulin contained in blood, body fluid, etc., proteins such as enzymes, etc., hormones, bacteria, viruses etc. have similar molecular structures or are very small in quantity, they can be identified and quantified by ordinary analysis methods. Is difficult. Therefore, for the analysis of these substances, an immunological analysis method utilizing an antibody, an antigen, a lectin or the like is generally used.

Immunological analysis methods utilizing such an antigen-antibody reaction are roughly classified into a labeled immunoassay method that uses a labeling substance and a non-labeling immunoassay method that directly measures an antigen-antibody complex without using a labeling substance. Although the unlabeled immunoassay method is simple, it is insufficient as a precise measurement in terms of sensitivity, quantification, and reproducibility. Therefore, the labeled immunoassay method is predominant in bacteria. This labeled immunoassay is roughly classified into a radioimmunoassay, an enzyme immunoassay, and a fluoroimmunoassay depending on the difference in the labeling substance, and the antibody (antigen) labeled with the labeling substance in the measurement system and the antigen (antibody) in the sample are antigen antibodies. Reacted antigen-antibody complex (Boun
d) and labeled antibody (antigen) (F
ree) and the so-called BF separation, which is a heterogeneous method and a homozygous method, which does not require BF separation. The homogeneous method is simple in operation because it does not require BF separation. Therefore, various apparatuses have been proposed so far for automatically performing the BF separation, but the heterogeneous method requires BF separation. Therefore, the analysis procedure is complicated, and therefore, there are few proposals for an apparatus that automatically performs the analysis procedure as compared with the homogeneous method.

As an immunological analysis method adopting this heterogeneous method, Japanese Patent Application Laid-Open No. 53-10495 proposes an analysis method suitable for automation in which BF separation is performed by utilizing column chromatography. . In this analytical method, the adsorbent packed in the column selectively adsorbs the free antigen in the solution and does not adsorb the antigen-antibody complex, such as anion or cation ion exchange resin or gel chromatography with a molecular sieving effect. It uses a filler for graphy. However, although BF separation by gel chromatography is effective in the analysis of low-molecular-weight antigens such as haptens and semi-haptens by the competitive method, when it becomes an antigen having a molecular weight of tens of thousands or more, the size of free antigen and antigen-antibody complex increases. Are close to each other and the size and shape of the antigen-antibody complex vary, which makes it difficult to separate the B-F. Therefore, for example, it cannot be used for measuring the same molecule as an antibody used as a reagent such as immunoglobulin, or a chemically or physically similar molecule, and there is a problem that analysis items are extremely limited.

(Object of the invention) The object of the present invention is to solve the above-mentioned problems, and to perform BF separation by gel chromatography without fail, regardless of the analysis items, from low molecular weight to high molecular weight. The present invention is intended to provide an immunological analysis method capable of performing required analysis with high accuracy and speed.

(Summary of the Invention) The immunological analysis method of the present invention comprises a sample, a labeled antigen or antibody labeled with a predetermined substance, and an antigen or antibody immobilized on the surface, and at least a small amount of the labeled antigen or antibody described above. After reacting with a carrier having a larger size or a larger molecular weight, the labeled antigen or antibody bound to the carrier in the reaction solution and the unbound one are separated by gel chromatography. .

In the present invention, as a carrier for immobilizing an antibody or an antigen, a latex particle having a size larger than that of the labeled antibody or antigen and a uniform particle size, or a larger molecular weight than the labeled antigen or antibody, and a uniform polystyrene,
Although macromolecules such as dextran and cytochrome c can be used, latex particles that are easy to make uniform in shape and size are effective. The size of latex particles varies depending on the analysis items and the conditions such as packing materials in gel chromatography, but the particle size is 0.08 μm to 5 μm.
Any size up to a level can be used, and various materials and chemical properties of the surface can be similarly selected. The antigen or antibody immobilized on this carrier is, for example, when the antigen in the sample is analyzed by the so-called sandwich method in which the carrier and the labeled antibody are bound via this, _Fab or F of the monoclonal antibody is used as the antibody. _{The (ab ′) 2} fragment is desirable, and it is immobilized by physical adsorption such as physical adsorption or covalent bonding. The labeled antibody in this case may be a monoclonal or polyclonal antibody.
_Fab fragments are preferred, which are enzymes, fluorophores,
It is bound to a predetermined labeling substance such as a radioactive substance, a dye or a metal. When the antibody is labeled with an enzyme, for example, it is desirable to bond one molecule of the antibody and one molecule of the enzyme. Further, the gel in the gel chromatography for performing BF separation can be appropriately selected depending on the analysis item, carrier, etc., and the material, size, molecular fractionation range, etc. of the gel particle can be arbitrarily controlled.

(Example) First Example As a first example, the analysis of human IgE will be described. FIG. 1 is a schematic diagram of the reaction when analyzing human IgE. In this example, a polystyrene latex having a particle size of 0.41 μm is used as the carrier 1, and a monoclonal anti-human IgE antibody as the solid phase antibody 2 is immobilized on the carrier 1 by physical adsorption. Further, as a labeled antibody 3, a _Fab fragment of a goat anti-human IgE antibody having a size smaller than that of the carrier 1 is used, and a fluorescent substance of fluorescein isothiocyanate (FITC) is bound as a labeled substance 4 thereto. First, 50 μm of a latex reagent containing the carrier 1 having the above solid-phase antibody 2,
Labeling reagent 50 containing labeled antibody 3 labeled with labeling substance 4
μ and human IgE solution 10 μ containing sample antigen 5 were added to 0.01M phosphate buffer (PBS) PH = 7.5 / 0.1M
NaCl / 0.1% Bovine Serum Albumin (BSA) /0.02% NaN
_The mixture is added to 200 μl of the reaction buffer consisting of _{3 and the} antigen-antibody reaction is caused by the sandwich method at 37 ° C. for 10 minutes. It should be noted that all of the solutions may be added at the same time, or first, the solid phase antibody 2 and the sample antigen 5 are reacted first, and then the solutions are sequentially added so that the sample antigen 5 and the labeled antibody 3 are reacted. Good.

Then, the antigen-antibody complex in the above reaction solution, namely Boun
The d6 and the labeled antibody 3 having the labeling substance 4 that is not involved in the antigen-antibody reaction, that is, Free7, are subjected to BF separation with the chromatograph shown in FIG. 2 to measure the sample antigen concentration. In the chromatographic apparatus shown in FIG. 2, the inlet of the gel chromatography column 11 is connected to the eluent tank 14 via the injector 12 and the pump 13, and the outlet is connected to the waste liquid tank 16 via the detector 15, Detector 15
The output of is recorded by the recorder 17, and in this example, 0.01 M PBS pH = 7.5 / 0.1 M N is stored in the eluent tank 14.
Cellulofine GCL-2000sf (trade name; manufactured by Seikagaku Co., Ltd.) is used as a packing material for the column 11 which contains an eluate containing aCl. When the above reaction solution is injected from the injector 12 into the column 11 of this chromatographic apparatus and the eluate is supplied by the pump 13, the Bound
Since 6 has the carrier 1 larger than the labeled antibody 3, it elutes at a high speed, and Free 7 elutes after a while, so that BF separation is surely performed. Therefore, the detector 15 detects changes in the amount of scattered light and transmitted light at the same time as the fluorescence intensity, and the recorder 1
When recorded at 7, a very sharp fluorescent peak a of Bound 6 was first obtained as shown in Fig. 3, and was delayed with Free
Since the fluorescent peak b of 7 is obtained, the first fluorescent peak a
Based on the above, the sample antigen concentration can be determined with high accuracy and high speed from the calibration curve showing the relationship between the fluorescence intensity and the antigen concentration obtained from the previously known concentration antigen.

In this Example, the sample antigen was analyzed by the sandwich method, but by using the labeled antigen and an appropriate column packing material, it is possible to analyze by the competitive method applied in the analysis of hapten and the like. .

Second Example As a second example, the analysis of human insulin will be described. In this example, polystyrene latex having a particle size of 0.22 μm is used as a carrier, and a guinea pig anti-human insulin antibody as a solid phase antibody is immobilized on this carrier by physical adsorption. In addition, porcine insulin, which is smaller in size than the carrier, is used as a labeled antigen, and this is labeled with a fluorescent substance of FITC. 50 μm of a latex reagent containing a carrier having the above solid-phase antibody, 50 μm of a labeling reagent containing a labeled antigen labeled with FITC, and 10 μm of a human insulin solution of a sample antigen were mixed with 0.01M PBSpH = 7.0 / 0.1M NaCl /
Add to the reaction buffer of 200μ consisting of 0.1% BSA / 0.02% NaN ₃ and perform the antigen-antibody reaction by the competitive method at 37 ° C.
After 10 minutes at room temperature, BF separation is performed by the chromatographic apparatus having the configuration shown in FIG. 2 as in the first embodiment.
The fluorescence intensity of each of Bound and Free is measured, and the insulin concentration in the sample is determined based on those fluorescence intensities. In this example, Cellulofine GC-200m (trade name; manufactured by Seikagaku Corporation) is used as a column packing material.

Also in this example, since Bound has a carrier larger than the labeled antigen, when the reaction solution is passed through the chromatographic apparatus having the configuration shown in FIG. 2, Bound elutes first at high speed and Free elutes later. As a result, BF separation is surely performed, and the fluorescence intensity similar to that in FIG. 3 is obtained. Therefore, the insulin concentration in the sample can be obtained with high accuracy and high speed based on the peak value of Bound and the peak value of Free.

Third Example A human IgG analysis will be described as a third example. In this example, dextran having a molecular weight of 300,000 was used as a carrier, which was activated with cyanogen bromide (CNBr) to produce an excess of 6
・ Introducing a carboxyl group into the dextran molecule by reacting with aminocaproic acid, and then using carbodiimide, dextran and monoclonal anti-human IgG
Crosslinks the _Fab fragment of the antibody. As a labeled antibody, a _Fab fragment of goat anti-human IgG having a smaller molecular weight than the carrier is used, and this is labeled with a FITC fluorescent substance. Dextran reagent 50 containing a carrier having the above solid-phase antibody
50 μ of a labeling reagent containing μ and a labeled antibody labeled with FITC
And a sample IgG of human IgG solution 5μ, 0.01M
PBSpH = 7.5 / 0.2M NaCl / 0.1% BSA / 0.05% Tw-
een20 / 0.02% NaN ₃ was added to 200 μl of a reaction buffer, and the antigen-antibody reaction by the sandwich method was carried out at 37 ° C. for 5 minutes.
BF separation is performed by a chromatographic apparatus having the configuration shown in the figure, and the fluorescence intensities of Bound and Free are measured to determine the IgG concentration in the sample. In this example,
Cellulofine GC-700m as column packing material
(Trade name; manufactured by Seikagaku Corporation) is used.

In this example, Bound has a carrier having a larger molecular weight than the labeled antibody. Therefore, when the reaction solution is passed through a chromatographic apparatus having the configuration shown in FIG. 2, Bound elutes first at a high speed and Free is delayed. Elution is performed and BF separation is reliably performed, and a fluorescence intensity similar to that in FIG. 3 is obtained. Therefore,
Also in this example, the IgG concentration in the sample can be determined with high accuracy and high speed, as in the above-described examples.

The present invention is the above-mentioned human IgE, human insulin,
Not only analysis of human IgG, it can be effectively applied to various analysis of proteins such as enzymes, hormones, bacteria, viruses, etc., and labeling substances are not limited to fluorescent substances,
Various substances such as enzymes, radioactive substances, dyes and metals can be used.

(Effects of the Invention) As described above, according to the present invention, in the labeled immunoassay method for performing BF separation by gel chromatography, a carrier having a size or molecular weight larger than that of the labeled antigen or antibody is used. Therefore, regardless of the analysis items, B-
It is possible to perform F separation, so that the required analysis can be performed with high accuracy and at high speed.

[Brief description of drawings]

FIG. 1 is a schematic reaction diagram of an example of a labeled immunoassay method, FIG. 2 is a diagram showing a configuration of an example of a chromatographic apparatus for carrying out BF separation according to the present invention, and FIG. 3 is a diagram showing the chromatographic apparatus shown in FIG. It is a figure which shows an example of the output waveform obtained. 1 ... Carrier, 2 ... Antibody, 3 ... Labeled antibody, 4 ... Labeled substance, 5 ... Sample antigen, 6 ... Bound, 7 ... Free, 11 ... Column, 12 ... Injector, 13 ... Pump, 14 ... … Eluent tank, 15… Detector 16… Waste tank, 17… Recorder

Claims (1)

[Claims] 1. A carrier having a sample, a labeled antigen or antibody labeled with a predetermined substance, and an antigen or antibody immobilized on the surface, and at least a carrier having a larger size or molecular weight than the labeled antigen or antibody. And a labeled antigen or antibody bound to the carrier and a non-bound labeled antigen in the reaction solution are separated by gel chromatography.