JPS61110059A - Immunological analysis - Google Patents

Abstract

PURPOSE:To perform a required analysis at a high accuracy, by a method wherein in an antibody labelled by a specified substance is made to react with a carrier which has an antibody in solic phase on the surface thereof and is larger than the labelled antibody and then, the labelled antibodies bonded to the carrier in a reaction liquid and those not are separated by gel chromatography. CONSTITUTION:An antibody 3 labelled by a specified substance 4 and a carrier 1 having an antibody 2 in solid phase larger than the labelled antibody 3 are used to cause a reaction by adding a reaction buffer liquid containing a sample antigen 5. Then, with a chromatograph equipment, an antigen-antibody composite (Bound 6) in the reaction liquid is separated from the labelled antibody 3 (Free 7) having a labelling substance 4 not involved in an antigen-antibody reaction so called B-F separa tion. Then, changes in the quantity of scattering light and transmission light are detected with the chromatograph equipment together with the intensity of fluorescent light. At first, the fluorescent peak of the Bound 6 is obtained and that of the Free 7 one later. Thus, the density of the sample antigen is analyzed at a high accuracy from the calibration curve of the intensity of fluorescence and the density of the antigen obtained from an antigen with the known density based on the initial fluores cent peak.

Description

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

(Conventional technique wi) Proteins such as globulins and enzymes, hormones, bacteria, viruses, etc. contained in blood, body fluids, etc. have similar molecular structures and are extremely small in amount, so it is difficult to identify them using normal analysis methods. Quantification is the inner key. Therefore, immunological analysis methods using antibodies, antigens, lectins, etc. are generally used to analyze these substances.

Immunological analysis methods that utilize such antigen-antibody reactions can be roughly divided into labeled immunoassay methods that use labeled substances, and non-labeled immunoassay methods that directly measure antigen-antibody complexes without using labeled substances. However, although non-labeled immunoassays are simple, they are insufficient for precise measurements in terms of sensitivity, quantification, and reproducibility, so labeled immunoassays have recently become mainstream. This labeled immunoassay is broadly classified into radioimmunoassay, enzyme immunoassay, and fluoroimmunoassay depending on the difference in the labeling substance, and in the measurement system, the antibody (antigen) labeled with the labeling substance and the antigen (antibody) in the sample are used. The antigen-antibody complex (goun) that caused an antigen-antibody reaction
d) and a labeled antibody (antigen) that does not participate in the antigen-antibody reaction
(Free) and is classified into a heterogeneous method which requires so-called B-F separation, and a homojunius method which does not require it. The homogeneous method is easy to operate because it does not require B-F separation, and various devices have been proposed that automatically perform this process, but the heterogeneous method does not require B-F separation. As a result, the analysis procedure is complicated, and there are fewer proposals for devices that can automatically perform this procedure than in the homogeneous method.

As an immunological analysis method that employs this heterogeneous method, an analysis method suitable for automation that uses column chromatography to perform B-F separation has been proposed in Japanese Patent Application Laid-open No. 10495/1983. . In this analysis method, the adsorbent packed in the column is a material that selectively adsorbs free antigens in a solution but does not adsorb antigen-antibody complexes, such as anion or cation ion exchange resins or gel chromatography with a molecular sieving effect. A filler for graphics is used. However, although B-F separation by gel chromatography is effective in the analysis of low-molecular antigens such as tobutene and 7-mihapten using a competitive method, when it comes to antigens with a molecular weight higher than that of the enemy, free antigen and antigen-antibody complexes are separated. B-F separation becomes difficult because the size of the antigen-antibody complex is close to that of the antigen-antibody complex, or because the size and shape of the antigen-antibody complex vary.

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 there is a problem that 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 be able to always reliably perform B-F separation by gel chromatography from low molecules to high molecules without being limited to the analysis items. The purpose of the present invention is to provide an immunological analysis method that allows the required analysis to be carried out 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 a surface, and a small amount of the labeled antigen or antibody is immobilized on the surface. After reacting with a carrier having a larger size or molecular weight than the carrier, the labeled antigen or antibody bound to the carrier in the reaction solution is separated from the unbound labeled antigen or antibody by gel chromatography. .

In the present invention, the carrier on which the antibody or antigen is immobilized may be latex particles that are larger in size than the labeled antibody or antigen and have a uniform particle size, or polystyrene that has a molecular weight larger and more uniform than the labeled antigen or antibody. Although macromolecules such as , dextran, and cytochrome 0 can be used, latex particles are preferably effective because they can be easily made uniform in shape and size. Although it varies depending on the thickness of latex particles and conditions such as the analysis items and the packing material used in gel chromatography, the particle size is 0.08 μm to 5 μm.
It is possible to use a material of any size up to about 1.5 m, and various materials and surface chemical properties can be selected as well. The antigen or antibody immobilized on this carrier is, for example, a monoclonal antibody Fab or F as the antibody, when analyzing the antigen in a sample by the so-called sandwich method in which the carrier and a recognition antibody are bonded via this. (abz)
A fraction is preferable, and this is solidified by physical adsorption or hidden bonding such as covalent bonding. Further, the labeled antibody in this case is preferably a Fab7 fragment of a monoclonal or reflonal antibody, which is bound to a predetermined labeling substance such as an enzyme, a fluorescent substance, a radioactive substance, a dye, a metal, or the like. Note that, for example, when labeling an antibody with an enzyme, it is desirable to bond the antibody and enzyme molecules one molecule at a time. In addition, the gel used in gel chromatography for B-F separation can be appropriately selected depending on the analysis item, carrier, etc., and the material, size, molecular fractionation range, etc. of the gel particles can be arbitrarily selected.

(Example) First Example As the first example, (h) IgE analysis will be explained. Figure 1 is a schematic diagram of a reaction when analyzing IgE. In this example, polystyrene latex with a particle size of 0.41 μm is used as carrier 1, and solid phase antibody 2 is used on this carrier 1.
A monoclonal anti-human EggE antibody is immobilized by physical adsorption. Further, a Fab fragment of a goat anti-human IgE antibody, which is smaller in size than the carrier 1, is used as the labeled antibody 8, and a fluorescent substance such as fluoren7ine isothiocyanate (FITO) is bound to this as the labeled substance 4. First, 60 μl of the latex reagent containing the carrier 1 having the solid-phase antibody 2, 60 μt of the labeled reagent containing the labeled antibody 3 labeled with the labeling substance number, and 10 μt of the IgE solution containing the sample antigen 5, 0. OIM phosphate buffer (PBS) PH=7.5/0. I M HaC
Jt/0.1% bovine serum albumin (BSA)10.
It is added to a reaction buffer solution of z00 μt consisting of oz% NaN, J: to cause an anti-antigen reaction at 87° C. for 110 minutes by the Sanderch method. Incidentally, each solution may be added all at the same time, or each solution may be added sequentially so that the solid-phase antibody 2 and the sample antigen 5 are first reacted, and then the sample antigen 5 and the labeled antibody δ are reacted. Good too.

After that, the antigen-antibody complex in the above reaction solution, namely B
ound 6 and the labeled antibody 3 having the II labeling substance 4, which did not participate in the antigen-antibody reaction, ie, 1rree 7, are subjected to B-F separation using the chromatography apparatus shown in FIG. 2, and the sample antigen concentration is measured. The chromatography apparatus shown in FIG. 2 has an inlet of a gel chromatography column 11 connected to an eluate tank 14 via an injector 12 and a semp 13, and an outlet connected to a waste liquid tank 16 via a detector 15. The output of the detector x5 is recorded by the recorder 17, and in this example, 0.01
MPBS pH=7.5/0. I M N&O
Cellulofine GCL-20008f (trade name; manufactured by Seikagaku Corporation) is used as a packing material for the column 11. When the above reaction solution is injected into the column 11 of this chromatography device from the injector 12 and the eluate is supplied by the pump lδ, the Boun
Since d6 has a larger carrier 1 than the labeled antibody 8, it elutes at high speed, and yree 7 elutes later, ensuring B-F separation. Therefore, when the detector 15 detects changes in the amount of scattered light and transmitted light at the same time as the fluorescent light intensity and records them with the recorder 17, the very sharp fluorescent peak a of Bound 6 is first detected as shown in FIG. Since the fluorescence peak b of Free7 is obtained after a delay, we can calculate the result using the standard curve representing the relationship between the fluorescence intensity and the antigen concentration, which was determined in advance from the antigen with a known concentration, based on the initial fluorescence peak a. Sample antigen concentration can be determined with high precision and high speed.

In this example, the sample antigen was analyzed by the Sand-Deutsch method, but by using a labeled antigen and an appropriate column packing material, the sample antigen can also be analyzed by a competitive method applied in the analysis of haptens, etc. .

Second Example As a second example, analysis of human insulin will be explained. In this example, polystyrene latex with a particle size of 0.22 μm is used as a carrier, and a guinea pig anti-IV-toinsulin antibody as a [1m antibody is immobilized on this carrier by physical adsorption. In addition, porcine insulin, which is smaller in size than the carrier, was used as a labeled antigen, and this was
Label with C fluorophore. 60 μt of the latex reagent containing the carrier with the above-mentioned solid-phase antibody, 50 pt of the standard reagent containing the labeled antigen labeled with FITO, and 10 μt of the human insulin solution of the sample antigen), 0.01 M
PBS PH=7.0/0. I N Mail
/ 0.1% BSA / 0.02% NaN3 was added to 200 μt of reaction buffer, and an antigen-antibody reaction was performed at 87°C for 10 minutes by competitive method, followed by the same procedure as in Example 1. B-F separation is performed using a chromatography apparatus having the configuration shown in FIG. 2, and the respective fluorescence intensities of Bound and Free are measured, and the insulin concentration in the sample is determined based on these fluorescence intensities. In addition, in this example, Cellulophy/Go is used as the filling IEI for Kara A.
-200m (trade name; manufactured by Seikagaku Corporation) is used.

In this example as well, Bound has a larger carrier than the labeled antigen, so when the reaction solution is passed through the chromatography device configured as shown in Figure 2, Bound elutes at high speed first, and Free elutes later. B-7 separation is reliably carried out, and a fluorescence intensity similar to that shown in FIG. 8 is obtained. Therefore, the insulin concentration in the sample can be determined with high accuracy and high speed based on the Bound peak value and the Free peak value.

Example δ As the eighth example, we will explain the analysis of human egg G.
Ru. In this example, dextran with a molecular weight of 300,000 is used as a carrier, and by activating this with cyanogen bromide (0NBr) and reacting with an excess amount of 6-aminocaproic acid, a carboxyl group is introduced into the dextran molecule. Carbodiimide is used to crosslink the dextran and the 'ab' fragment of the monoclonal anti-human IgG antibody. Further, as a labeled antibody, a goat anti-human IgG Fab7 fragment having a molecular weight smaller than that of the carrier is used, and this is labeled with the fluorescent substance FITC. 50 μt of dextran reagent containing the carrier with the above solid-phase antibody and FITC.
60 μt of a labeled reagent containing a labeled antibody labeled with 0.01 M P and 6 μt of a sample antigen human IgG solution
BS pH=7.5/0.2M Ha (3t/
0.1%BSA/0.05%Tw-ee
n20/O-02% NaN, 200
After adding it to P& reaction buffer and carrying out an antigen-antibody reaction by the Sand-Deutsch method at 87°C for 5 minutes,
As in the example, BF separation is carried out using a chromatography apparatus having the configuration shown in FIG. 2, and the respective fluorescence intensities of BO2 and 7ree are measured to determine the IgG concentration in the sample. In this example, C-C-7O0 product name; Seikagaku Corporation 11! is used as the column packing material for Cellulofine. Use Iri.

In this example, Bound has a carrier with a larger molecular weight than the labeled antibody, so when the reaction solution is passed through the chromatography device configured as shown in Figure 2, Bound elutes at high speed first, and 1rree elutes later. By elution, B-F separation is performed reliably, and a fluorescence intensity similar to that shown in FIG. 8 is obtained.

Therefore, in this example as well, the IgG concentration in the sample can be determined with high precision and at high speed, as in the above-mentioned example.

The present invention can be effectively applied not only to the above-mentioned analysis of human IgE, human insulin, and IgG, but also to various analyzes of proteins such as enzymes, hormones, bacteria, viruses, etc. In addition 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, a carrier having a larger size or molecular weight than the labeled antigen or antibody is used in a labeled immunoassay/analysis method that performs B-F separation by gel chromatography. Therefore, regardless of the analysis items, you can always reliably perform B-
F can be separated, thereby enabling the required analysis to be performed with high precision and high speed.

[Brief explanation of the drawing]

Fig. 1 is a schematic reaction diagram of an example of the labeled immunoassay method, Fig. 2 is a diagram showing the configuration of an example of a chromatography apparatus for performing B-F separation according to the present invention, and Fig. 8 is a diagram showing the configuration of an example of the chromatography apparatus shown in Fig. 2. It is a figure which shows an example of the output waveform obtained. 1... Carrier 2... Antibody 8... Labeled antibody Age... Labeled substance 5... Sample antigen 6... Bound 7... Free
11... Column 12... Injector
18... Pump 14... Eluate tank 15
...Detector 16...Waste liquid tank 17...
Recorder Figure 1 Figure 2 Figure 3

Claims (1)

[Claims] 1. Reacting a sample with a labeled antigen or antibody labeled with a predetermined substance and a carrier having the antigen or antibody immobilized on the surface and having a size or molecular weight slightly larger than the labeled antigen or antibody. After that, the labeled antigen or antibody bound to the carrier in the reaction solution and the unbound labeled antigen or antibody are separated by gel chromatography.