CH658131A5 - REAGENT AND METHOD FOR IMMUNOLOGICAL ANALYSIS. - Google Patents

Description

The object of the present invention was to create a reagent for immunological analysis which enables a remarkable reduction in the time required for the analysis without reducing the sensitivity and the accuracy.

The invention therefore relates to a reagent according to claim 1.

Furthermore, the invention relates to a sandwich method according to claim 9, which enables a noticeable reduction in the required analysis time without reducing the sensitivity and the accuracy.

5 Fig. 1 is a schematic illustration of the reaction according to the conventional sandwich method;

Figs. 2 and 3 are graphs showing the presence or absence of competition between antibodies;

Figure 4 is a schematic illustration of the reaction mode of the present invention;

Fig. 5 is a graph showing a comparison between the present invention and the conventional method;

Fig. 6 is a graphical representation of the results of Example 1;

Figure 7 is a graphical representation of the results of Example 2;

Figure 8 is a graphical representation of the results of Example 8.

The objects are achieved by using monoclonal antibodies for the production of the insolubilized antibody and the labeled antibody, which different antigenic determinants on the same antigen differ and combine with different antigenic determinants, while so-called polyclonal antibodies according to the conventional method which were obtained from animals immunized with the same antigen were used to produce the two antibodies so that they compete for the same antigenic determinants of the antigen.

The reagent according to the invention is based on the principle that the insolubilized antibody and the labeled antibody each contain antibodies which recognize different antigenic determinants and can combine with different antigenic determinants of the same antigen to be determined, or they contain antibodies which different antigens Distinguish determinant of the 40 antigen so that the two antibodies can bind to different antigenic sites on the same antibody that are sufficiently spaced from one another not to interfere with the binding.

Monoclonal antibodies are extremely suitable for this purpose.

The monoclonal antibodies are obtained by cell fusion between myeloma cells and antibody-producing cells according to the method of Milstein et al. Nature, 256,495 (1975). Since these antibodies are produced by the cells of a single clone which produce antibodies, they are completely homogeneous and their specificities for antigenic determinants are completely identical. Since the antigens to be determined generally have several antigenic determinants, the use of monoclonal antibodies, which recognize different antigenic determinants and combine with different antigenic determinants of the antigen, are used as insolubilized antibodies and as labeled antibodies, no competition between them Antibodies and a strong specific analysis of the antigen can be achieved. Thus, a mixture of two or more monoclonal antibodies that bind to different antigenic determinants can also be used as insolubilized antibodies and / or labeled antibodies, provided that these antibodies do not compete.

The analysis with the reagents according to the invention can be carried out in the same manner as in the prior art. However, it must be emphasized that the

658 131

Reaction mechanism easier than that known to compete with the labeled antibody, as is the post-method, since the insolubilized antibody is not shown below.

ffÄkl -Ag) + (Ak *)

+ (Ag) + (Ak *)

CoeD + CAg "Ak * D

Ya

Äkl "Ag-Ak *)

, Ag, Ak * and - have the same meaning as above and p, p ', q, q', r, r ', s, and s' each represent equilibrium constants.

Since there is no competition between the insolubilized antibody and the labeled antibody if they have different binding sites on an antigen, the reaction proceeds in such a way that the components in the reaction solution form an “insolubilized antibody-per-antigen-labeled antibody” complex .

This can be demonstrated by the following experiment: In the same reaction system as in the conventional sandwich method, two different monoclonal anti-HCG antibodies that could recognize different antigenic determinants of HCG were used as the insolubilized antibody and as the labeled antibody, and a similar experiment it was accomplished. The result, as shown in Fig. 3, shows that once the antigen is bound to the insolubilized antibody, it will no longer dissociate from the insolubilized antibody (solid phase) if an anti-HCG antibody is antigenic to another Corresponds to determinants, is added. This shows that there is no competition for the same antigen sites between the antibodies. Similar results are obtained with the reagent according to the invention if the insolubilized antibody,

the antigen to be determined and the labeled antibody 20 are reacted simultaneously or when the insolubilized antibody and the antigen are first mixed and then reacted with the labeled antibody or when the insolubilized antibody is reacted with the antigen and after a while the mixture is reacted with the labeled antibody with 2s. There is therefore no restriction on the order of the reactions.

The implementation according to the invention is shown in a schematic illustration in FIG. 4. Since the insolubilized antibody 30 and the labeled antibody 1 do not compete with each other, a larger amount of the labeled antibody 1 can be bound to the antigen 3 in a shorter time and not only the reaction time can be shortened, but also the accuracy and 35 the sensitivity of the analysis can be increased.

Table 1 summarizes the advantages of the method according to the invention over the conventional method using polyclonal antibodies in the determination of a-fetoprotein (AFP). Table 40 clearly shows that several advantages can be achieved, such as significantly reducing the analysis time, simplifying the analysis process and increasing the accuracy and sensitivity of the analysis.

Table 1

Method according to the invention

Known procedure

Action

Antibodies used required time sensitivity

'53 Internal error C.V. at

.m 10 times repetition '3

c Internal errors C.V. with repetition 5 times

Washing reaction - »enzyme reaction

Monoclonal antibodies 30 minutes 3 ng / ml 1.2%

3.7%

1st reaction -

2nd reaction - reaction

• washing -> •

• washing - * enzyme

Polyclonal antibodies 270 minutes 10 ng / ml 2.5%

5.6%

The analysis was carried out using a fluoropotometer with a pregnant woman's serum as an analysis sample, a polystyrene test tube as a solid phase, horseradish peroxidase as a labeling enzyme and phloretic acid as a substrate.

It is also possible to use only one of the antibodies, i.e. produce either the insolubilized or the labeled antibody from monoclonal antibody and the other from ordinary polyclonal antibody. In this case, however, there is competition for the antigenic determinant, which is common to both the monoclonal and the polyclonal antibody and which in some cases

658131

The affinity of the antigenic determinant for the jolyclonal antibody can be stronger, which results in reduced accuracy and sensitivity, not only compared to the method in which both antibodies are monoclonal, but even with the known method.

Figure 5 shows standard curves for the determination of CEA 'Carcinoembryones antigen) when the insolubilized antibody and the labeled antibody are combined as shown in Table 2. It is clearly evident that the analysis system, which is based on the combination according to the invention, achieves the best results.

Table 2

Marked insolubilized

Antibody Antibody

Method according to the invention

Comparative procedure 1

Comparative procedure 2

Conventional procedure monoclonal monoclonal polyclonal polyclonal monoclonal polyclonal monoclonal polyclonal

The monoclonal antibodies used were obtained by cell fusion technique using mouse myeloma cells as described below in Example 2 and obtained from clones corresponding to different antigenic sites. The polyclonal antibodies were commercially available products from the Dako Company (Denmark). Polystyrene test tubes were used as insoluble supports; horseradish peroxidase was used as the enzyme and o-phenylene-diamine as the substrate.

Any desired combination of myeloma cells and antibody-producing cells can be used for the production of monoclonal antibodies, and the choice of the animal from which the cells are obtained is not limited, as long as both cells combine to form hybridomas and, during growth, antibodies can.

All insoluble carriers customary for immunoassays can be used for the reagents according to the invention.

Examples of such insoluble carriers are polystyrene, polyethylene, acrylic resins, polytetrafluoroethylene ("Teflon"), paper, glass and agarose. The insoluble carrier can be in the form of a roller, pearl, gear, rod, disc or cuvette. For example, it can be an optical cell, a test tube, etc. However, it can also be in another form.

Labeling agents that are commonly used are, for example, peroxidase, β-D-galactosidase and alkali lipophosphatase for EIA and fluorescein isothiocyanate for FIA.

If the label is an enzyme, a substrate is required to measure its activity. A substrate can be used such that the amount of the product formed or the decrease or the amount of the remaining substrate can easily be measured during the reaction with the corresponding enzyme. For example, as substrates for peroxidase, 5-aminosalicylic acid-H202, o-phenylenediamine-H202, phloretic acid-H202, etc .; For β-D-galactosidase, the following substrates can be mentioned: fluorescein-di- (β-D-galactopyranoside), o-nitrophenol-β-D-galactopyranoside, 4-methyl-umbelliferyl-β-D-galactoside, etc.

Antigens that can be determined with the reagent according to the invention must have at least two antigenic determinants in the molecule. Since substances which have been determined by the sandwich method have two or more antigenic determinants, essentially all substances which can be determined by the known method can also be analyzed by the method according to the invention. Examples of such substances are enzymes such as y-glutamyl transpeptidase (y-GTP), alkali-io phosphatase and transglycosidase; Protein hormones such as thyroid stimulating hormone (TSH), luteinizing hormone (LH), human chorionic gonadotropin (HCG), insulin, secretin and growth hormone (GH); Plasma proteins such as fibrin degradation products (FDP), C-reactive protein (CRP), acidic glycoprotein (ai-AG), arAnti-trypsin (arAT), a2-plasmin inhibitor (a2-PI), ß2-microglobulin (ß2 -MG) and immunoglobulin; carcinoembryonic antigen such as a-fetoprotein (AFP), carcinoembryonic antigen (CFA) and embryonic ferritin; and lymphocytes and bacterial cells, cell surface antigens, cell debris, etc.

Since the order of the reactions between the insolubilized antibody, the antigen to be determined and the labeled antibody is in no way restricted when using the reagent according to the invention, it is also possible to mix the insolubilized antibody and the labeled antibody in advance. In particular, it is possible that the on a carrier, for. B. plastic beads etc., bound insolubilized anti-30 bodies and the labeled antibody are contained in the same vessel, e.g. in a test tube or it is also possible to attach the antibody to the inner wall of a suitable vessel, e.g. a test tube, which itself serves as an insoluble carrier and in which the labeled antibody 35 is contained. The labeled antibody can be in the form of a solution of a lyophilized product of the solution as well as in the form of tablets, granules or dusts.

In addition to the insolubilized antibody and the labeled antibody, the reagent according to the invention can contain various auxiliaries to increase its usefulness. For example, you can use solubilizers, solvents for the labeled antibody, if the latter is used as a lyophilized product, a washing liquid for washing the solid phase after the reaction of the insolubilized antibody and the antigen to be determined and after further reaction with the labeled antibody Substrate for the determination of the enzymatic activity of the labeling agent (if this is an enzyme) and a means for ending the enzymatic reaction see.

Examples of the invention are given below.

example 1

A reagent for the determination of AFP 55 a) Preparation of purified AFP

16.2 g of crude AFP extract were obtained from 51 of the ascites from Hepatoma patients by salting out with ammonium sulfate (the fraction with a degree of saturation between 45 and 70% was collected). The crude extract was purified by 60 affinity chromatography using 50 ml Se-pharose 4 B to which rabbit anti-AFP antibody was bound (1 mg / ml Sepharose) to obtain 924 mg of purified AFP.

65 b) Production of monoclonal anti-AFP antibody A female BALB / C mouse was immunized by subcutaneous injection of 50% of the purified AFP obtained according to a) together with a complete Freund's adjuvant.

658 131

siert. The immunization was repeated 4 times, at one week intervals. The spleen was removed on the 4th day after the last immunization and the spleen cells were collected. The spleen cells were washed with Dulbecco's modified MEM culture medium (hereinafter abbreviated as D-MEM). 1 x 108 of these cells were counted and mixed with 1 x 107 mouse myeloma cells (P3-NSI / l-Ag4.1). They were subjected to cell fusion for 1 minute at 37 ° C in 1 ml of D-MEM containing 42.5% polyethylene glycol No. 1540 and 7.5% dimethyl sulfoxide. 20 ml of HAT medium (RPMI-1640 medium containing hypoxane-thin, aminopterin, thymidine and 10% fetal bovine serum) were added to the cells subjected to cell fusion in this way. After 0.2 ml aliquots of the cell mixture were placed in a 96-well microplate and incubated for two weeks, the antibody titers of the supernatants in the wells were determined.

Then the cells from those wells in which antibodies were observed were each added to 40 ml of RPMI-1640 medium containing 10% fetal bovine serum and Thymocyte from BALB / C mice. Aliquots of the cell suspension obtained were placed in two 96-well microplates and incubated for one week to obtain 9 clones of anti-AFP antibody-producing hybridomas. They were transferred to large-scale cultures and 10 liters of the resulting supernatant liquid was subjected to affinity chromatography using 50 ml Sepharose 4B, which was fixed with purified AFP (0.5 mg AFP / ml Sepharose). 4.2 to 11.6 mg of monoclonal antibodies with the sample numbers 1 to 9 were obtained.

iii) Identification of the antigen-recognizing sites

1.5 ml of PBS, 0.1 ml of the standard solution of 5 AFP obtained under a), which was diluted to 100 ng / ml with PBS, were added to each of the anti-AFP antibody-sensitized test tubes which were obtained according to i), and 0.4 ml of HRPO-labeled anti-AFP antibodies, which were obtained in accordance with ii) and were diluted 100-fold, were added and the mixture was reacted for 30 minutes. After the reaction, the test tube was washed with washing liquid and 3 ml of substrate solution containing 100 mg / dl o-phenylenediamine and 0.01% hydrogen peroxide were added. The enzyme reaction was carried out for 30 minutes and then 0.05 ml of 4M sulfuric acid was added to terminate the enzyme reaction. The absorbance of the reaction mixture at 450 nm was measured. In Table 3 those combinations which caused positive reactions are denoted by 4 and those which did not cause a reaction by -.

20th

Table 3

Antibody labeled with HRPO 1 2 3 4 5 6 7

25th

■ s cd

1

2nd

3rd

- - + - - + - - +

+ + +

+ + + + + +

0) bû

rC

30 .y

& 4 + + + - + + + '-

m 5 - - - + - + - + +

n'6 + + + + -l- - + + +

<7 - - - + - + - + +

8 + + + - + + + - -

9 + + + - + + + - -

c) Identification of Antigen-Recognizing Sites i) Production of Sensitized with Anti-AFP Antibodies

Test tubes

A physiological saline solution, which was buffered with 0.05M phosphate (pH 6.4; hereinafter abbreviated as PBS) and each contained 0.2 mg of the monoclonal anti-AFP antibodies of sample numbers 1 to 9, was added in an amount of 2 ml placed in a test tube washed with PBS. The test tubes were incubated at 56 ° C for 20 minutes and washed with PBS, whereby the sensitized test tubes were obtained.

ii) Preparation of enzyme-labeled anti-AFP antibodies

5 mg of horseradish peroxidase (grade 1 from Boehringer Mannheim GmbH; hereinafter abbreviated as HRPO) were dissolved in 1.0 ml of 0.3M sodium bicarbonate buffer and 0.1 ml of 1% ethanol solution of l-fluoro-2,4 was added to the solution obtained -Dinitrobenzene added and the mixture was reacted for 1 hour.

Subsequently, 1.0 ml of 0.06M sodium peiodate solution was added to the mixture and reacted for 30 minutes. Then 1.0 ml of 0.16M ethylene glycol solution was added and the reaction was continued for 1 hour. The reaction mixture was dialyzed against 0.0IM sodium carbonate solution at pH 9.5. 5 mg each of the new samples of anti-AFP antibodies produced according to b) were added to the solution obtained and the mixture was reacted for 3 hours at room temperature. Then 5 mg of sodium borohydride was added and the reaction was carried out overnight. The reaction mixtures were in each case dialyzed against 0.01M PBS at a pH of 7.2, HRPO-labeled anti-AFP antibodies being obtained.

35 According to the differences in the antigen-recognizing sites, the new samples of monoclonal anti-AFP antibodies obtained according to b) can be divided into three groups, namely the first group consisting of samples 1, 2, 3, 5 and 7, the second group consisting of Sample 6 and the third group consisting of Samples 4, 8 and 9. These groups of antibodies are called anti-AFP antibodies (A), (B) and ( C) designated.

45 d) Preparation of a reagent for the determination of

AFP

Test tubes sensitized with anti-AFP antibody were prepared according to methods c) -i), as described above, using the monoclonal antibody (A).

The monoclonal antibody (C) was labeled with HRPO according to methods c) to ii) described above and diluted 1:10 with PBS. 2 ml of the diluted labeled antibody was placed in the sensitized test tube. After the contents of the test tube were lyophilized, it was sealed and gave a reagent for determining AFP.

e) Determination of AFP 60 1.8 ml of PBS were placed in the test tube sensitized according to d) with anti-AFP antibody. Then 0.1 ml of the standard solution of AFP prepared according to a) was added to the concentrations of 1000, 100, 10.1 and 0 ng / mmiluted with the serum of a healthy person, followed by a further addition of 0.1 ml of one Solution of the HRPO-labeled anti-AFP antibody, which was prepared according to d), in 2 ml of distilled water. The reaction was carried out for 20 minutes. After the

The test tube was then washed with a physiological saline solution containing 0.005% Tween 20 (referred to below as detergent). Then 3 ml of enzyme substrate solution containing 5 mg / ml o-phenylene diamine and 0.01% hydrogen peroxide were added and reacted for 10 minutes. To terminate the enzyme reaction, 0.05 ml of 4N sulfuric acid was added to the reaction mixture. The absorbance of the reaction mixture was measured at 450 nm with a photometer. The resulting calibration curve is shown in Fig. 6.

Example 2

Reagent for the determination of CEA

a) Production of purified CEA

40 g of colon cancer tissue were homogenized in a homogenizer after adding 100 ml of distilled water. The same amount of 1.2M perchloric acid was added to the homogenate and extracted with stirring for 30 minutes. The supernatant liquid after the centrifugation was dialyzed against distilled water to obtain the crude CEA extract.

The crude extract was concentrated to 10 ml and subjected to gel filtration on Sepharose 4B, which was adjusted with physiological saline, and the first fraction was collected. This was subjected to gel filtration on Sepha-dex G-200, which was set in the same manner, and the second fraction was collected. It was concentrated to 2 ml to give 135 (purified CEA).

b) Production of monoclonal anti-CEA antibodies

Eight clones of anti-CEA antibody-producing hybridoma were obtained using the purified CEA obtained under a) according to a method similar to that described in Example 1b.

Mice were immunized using 30 µg purified CEA per immunization. A female BALB / C mouse intraperitoneally administered with 0.5 ml of pristan (2,6,10,14-tetramethylpentadecane from Wako Pure Chemicals Co., Ltd.) was inoculated with 1 x 106 hybridomas intraperitoneally. The ascites was collected two weeks later. The ascites was chromatographed on DEAE cellulose, which was adjusted to pH 7.0 with 0.0 IM phosphate buffer, and an unabsorbed fraction was obtained as a monoclonal anti-CEA antibody.

By means of a test for identifying the antigen-recognizing sites essentially in accordance with Example 1c, the antibodies obtained could be divided into three groups, i.e. 5 samples, 2 samples and one sample, which were designated as anti-CEA antibodies (A), (B) and (C), are divided.

c) Production of test tubes sensitized with anti-CEA antibodies

Polystyrene test tubes were washed with PBS and 2 ml of the anti-CEA antibody (A) (lmg / ml), which was prepared according to b), were added to the test tube and reacted at 56 ° C. for 20 minutes. After the reaction, the test tube was washed with PBS, whereby a test tube sensitized with anti-CEA antibody (A) was obtained.

d) Preparation of a reagent for the determination of

CEA

HRPO-labeled anti-CEA antibody (B) was obtained using anti-CEA antibody (B) obtained according to b) in accordance with Example lc

7 658131

ii) received. The labeled antibody was diluted 1:50 with physiological saline and 0.2 ml of the diluted antibody solution was placed in a test tube sensitized with anti-CEA antibody (A) according to c) 5 and lyophilized to form the reagent for the determination of CEA.

e) Determination of CEA

The purified CEA prepared according to a) was diluted with the serum of a healthy person to concentrations of 100, 30, 10, 3 and 1 ng / ml. 0.2 ml of the diluted CEA and 0.8 ml of distilled water were simultaneously added to the reagent for the CEA determination, which was prepared in accordance with d), and reacted for 20 minutes with stirring. After the reaction, the test tube was washed with detergent, and then 3 ml of a substrate solution containing 300 mg / dl of phloretic acid and 0.01% of hydrogen peroxide was added. The reaction was carried out for 10 minutes and then 0.1 ml of 5% -20% sodium sulfite was added to terminate the reaction. Subsequently, the fluorescence intensity at the excitation wavelength of 323 nm and a fluorescence wavelength of 420 nm was measured with a fluorophotometer. The resulting calibration curve is shown in FIG. 7.

25th

Example 3

Reagent for the determination of HCG-ß

a) Production of the HCG-ß subunit

I g of HCG (2000 iu / mg) was added to 2 ml of a 0.025M

30 phosphate buffer (pH 5.6) was dissolved and the solution was fractionated chromatographically on DEAE-Sephadex A-50 (3 g), which had previously been adjusted with the same buffer. A fraction eluted with 0.05M phosphate buffer (pH 5.6) was collected and dialyzed against distilled water to give a solution containing 308 mg of purified HCG, which was then lyophilized. 300 mg of the lyophilized HCG were dissolved in 10 ml of a 10M (pH 4.5) urea solution and reacted at 40 ° C. for 1 hour. The reaction mixture was fractionated by chromatography 40 on DEAE-Sephadex A-50 (2 g), which was previously adjusted with a solution containing 0.03M glycine and 10M urea. A fraction eluted with a solution containing 0.2M glycine, IM NaCl and 8M urea was dialyzed against physiological saline 45 to give 147 mg of HCG-ß subunit.

b) Production of anti-HCG-ß antibody

II Samples of anti HCG-β antibody were prepared according to the procedure of Example 2b), with the

50 decided that the antigen was administered to rats from the Wi-star strain instead of BALB / C mice. The antigen-recognizing sites of these antibodies were identified essentially in accordance with the method of Example lc). The antibodies obtained were divided into two 55 groups, namely a group of 8 samples of anti-HCG-ß antibodies (A) and another group consisting of 3 samples of anti-HCG-ß antibodies (B).

c) Preparation of beads sensitized with anti-HCG-β antibodies (insoluble carriers)

100 polyethylene beads were added to 100 ml of PBS containing 50 mg of the anti-HCG-β antibody (A) prepared according to b) and reacted at 56 ° C. for 20 minutes. The polyethylene beads were washed to obtain anti-HCG-β anti-65 body (A) sensitized beads.

d) Production of enzyme-labeled anti-HCG-ß antibodies

658 131

The procedure of Example lc-ii) was repeated using anti-HCG-β antibodies (B) obtained according to b). After dilution to 200 times the volume with PBS, HRPO-labeled anti-HCG-β antibodies (B) were obtained.

e) Preparation of the detergent

9 g of NaCl and 50 ml of Tween 20 were weighed exactly and dissolved in deionized water and made up to 100 ml. The solution was poured into a glass bottle and sealed with a spigot. A detergent was prepared in this way.

f) Preparation of the enzyme substrate

26.40 g of 5-aminosalicylic acid, 54.34 g of monopotassium phosphate and 5.72 g of disodium phosphate were mixed and pulverized in a mortar. 550 mg of the mixture was weighed exactly and placed in a glass vial. 1.5 ml of 30% hydrogen peroxide were diluted to 100 ml with deionized water. 1 ml of this solution was placed in an ampoule, which was then sealed. The enzyme substrate was thus obtained.

g) Preparation of an agent for stopping the enzyme reaction

2 g of sodium azide was weighed exactly and dissolved in 100 ml of distilled water. 2 ml of the solution was placed in an ampoule, which was then melted to give the agent to terminate the enzyme reaction.

h) Preparation of a reagent for the determination of HCG A reagent for the determination of HCG was carried out by

8th

Combination of the materials produced according to c) to g) in the following manner.

1. Beads sensitized with anti-CHG-ß antibody (A)

2. HRPO-labeled anti-HCG-ß antibody (B) 5 3. Detergent (10-fold concentrated solution)

4. Enzyme substrate (5-aminosalicylic acid hydrogen peroxide)

5. Means to terminate the enzyme reaction i) Determination of HCG io The following analysis was used using the reagent for the HCG analysis, prepared according to h). 0.4 ml of the HRPO-labeled anti-HCG-ß antibody (B) was placed in a test tube and a bead sensitized with anti-HCG-ß antibody (A) was added. Then 0.1 ml of a standard solution obtained by diluting HCG by the method described in Japanese Pharmaco-poeia with the serum of a healthy person to a concentration of 10,000, 1,000, 100, 10 and 1 miu / ml , added and reacted for 15-20 minutes. After the implementation, that was

Detergent diluted to 10 times its volume with distilled water and the bead was washed with the diluted detergent. Then, 3 ml of the substrate solution obtained by dissolving the whole enzyme substrate 25 in 100 ml of distilled water was added, and the reaction was carried out for 30 minutes. Then 0.025 ml of the reaction completion agent was added and the absorbance of the reaction mixture was measured at 500 nm. The calibration curve 30 obtained is shown in FIG. 8.

B

4 sheets of drawings

Claims (9)

658 131 2nd PATENT CLAIMS 1. Reagent for immunological analysis of antigens based on the sandwich method, characterized in that it contains an insolubilized monoclonal antibody and a labeled monoclonal antibody, both of which differ from each other different antigenic determinants of an antigen to be determined and differ with different antigenic determinants of an antigen to be determined can connect. 2. Reagent according to claim 1, characterized in that it has beads or a vessel made of plastic or glass as a carrier for the insolubilized antibody. 3. Reagent according to claim 1 or 2, characterized in that the labeled antibody is labeled with an enzyme. 4. Reagent according to claim 1 or 2, characterized in that the labeled antibody is labeled with a fluorescent substance. 5. Reagent according to one of claims 1 to 4, characterized in that it contains the labeled antibody and the -insolubilized antibody in one vessel. 6. Reagent according to one of claims 1 to 5, characterized in that it contains the labeled antibody in a plastic or glass vessel, which is also a carrier for the insolubilized antibody. 7. Reagent according to one of claims 1 to 6, characterized in that it contains a lyophilized labeled antibody. 8. Reagent according to one of claims 1 to 7, characterized in that it contains one to four auxiliaries from the group consisting of solubilizer, detergent, substrate and agent for ending the reaction. 9. sandwich method for immunological analysis of antigens by means of a reagent according to claim 1, characterized in that reacting an antigen to be determined simultaneously with insolubilized antibody and labeled antibody. Advances in immunochemistry in recent years have led to extensive studies of antibodies about which little has been known in the past, and the control mechanism for antibody production and the biochemical structures and properties of the antibodies have been studied in detail. In 1975, C. Milstein et al. by cell fusion of mouse myeloma cells with antibody-producing cells from the spleen to form a monoclonal antibody. The monoclonal antibody is expected to make a major contribution to the development of basic research in immunology, and studies in this field have been actively undertaken. Enzyme immunoassay (EIA) has already been used as a highly sensitive and quantitative immunological analysis method. In particular, the sandwich method based on this analysis is often used because of its simplicity and high sensitivity. However, since the sandwich process requires 1 to 4 days for the reaction, it is desirable to minimize the reaction time required. Due to the recent progress in clinical medicine, rapid evaluation of the test results is also required to enable immediate treatment of the patient. From this point of view, too, it is seriously desired to shorten the response time. However, the shortening of the reaction time in the known sandwich method results in a reduction in the sensitivity and the accuracy of the analysis, as will be shown below, so that no significant reduction in the reaction time can be achieved. The conventional sandwich process is described in detail below. Although the method is described throughout the description for EIA's sake of convenience, the explanation can also be applied to the FIA (fluorine immunoassay) based method. The conventional sandwich process, as shown in the schematic diagram in FIG. 1, is carried out in the following order. i) An insolubilized antibody 2 'obtained by binding the antibody for an antigen 3 to be determined to an insoluble support (solid phase) 4 25 reacted with the antigen 3, the antigen 3 being connected to the antibody 2 'on the solid phase (first reaction). ii) In order to remove unreacted substances, the solid phase 4 is washed. Iii) A washed solid phase 4 is reacted with the labeled antibody Y, the labeled antibody Y being bound to the unreacted antigen site on the antigen 3 which was bound to the solid phase (second reaction). 35 The labeled antibody is obtained by binding an enzyme 5 to an antibody for the antigen 3 to be examined. iv) To remove the deficit of labeled antibody 1 ', the solid phase is washed and a substrate 40 for the enzyme is added and subjected to the enzyme reaction. Since the antigen 3 and the labeled antibody Y were bound to the solid phase 4, the enzyme reaction takes place in proportion to the amount of the enzyme present. 45 The amount of antigen 3 to be analyzed is determined from the amount of the reaction product obtained. Since the sandwich process is generally considered a non-competitive analysis, it can be assumed that there is no competitive reaction. However, this consideration does not necessarily express the exact reaction states. Thus, when the sandwich process is carried out, there may actually be an equilibrium state, which is shown schematically below. 55 First reaction a lAk Q + CA * P ^ ~ T ~ C lAk I ~ AgD a " -Ag-Ak *} (Œ3 -Ag) + (am3 Second reaction fi a * 11 + (Ag-Ak *) HEED + l> gj + (Ak *) 658131 In the illustration above, 1 Ak 1 stands for the insolubilized antibody; Ag for the antigen to be determined; Ak * for the labeled antibody; a, a ', b, b', c, c ', d, d', e and e 'for equilibrium constants and - means that the antigen and the antibody are linked. When the complex of the insolubilized antibody and the antigen to be determined is reacted with the labeled antibody in the second reaction, the insolubilized antibody and the labeled antibody competitively combine with the antigen to be determined according to their affinity for the antigen. Since the properties of the insolubilized antibody and the labeled antibody are approximately the same, their affinities for the antigen to be determined are considered to be approximately equivalent. As a result of the competitive reaction, the insolubilized antibody-antigen complex formed in the first reaction dissociates and complexes of various combinations are formed as shown above. The proof is provided by the following experiment. An anti-HCG-ß subunit antibody bound to a plastic test tube is reacted with HCG-ß labeled with peroxidase and then washed. The enzyme-labeled HCG-ß, which was bound to the solid phase, gradually dissociates from the solid phase upon reaction with an anti-HCG-ß antibody and enters the liquid phase. Fig. 2 shows this process. However, if the reaction time for the first reaction is long enough, the binding between the antigen and antibody becomes partially irreversible and the antigen-antibody complex no longer easily dissociates, not even when the labeled antibody is added. On the other hand, it is claimed that if the reaction time of the second reaction is long enough, the reaction proceeds toward the formation of a complex of the (insolubilized antibody, the antigen to be determined and the labeled antibody ». For this reason, the reaction is necessary to be carried out for a sufficiently long time according to the conventional sandwich method, therefore if the sandwich method is carried out without a first reaction or if the reaction time of the first reaction is extremely shortened, for example if the insolubilized antibody and the labeled antibody are reacted simultaneously with the antigen to be determined ; the second reaction has to be extended and the sensitivity and the accuracy of the analysis are nevertheless reduced. If the reaction time of the second reaction is shortened, the sensitivity and the accuracy are reduced even more. For this reason, it was impossible to conventional methods to shorten the response time without reducing the sensitivity and accuracy. In this context Ichi-hara et al. The Japanese Journal of Clinical Pathology, 26, 1027 (1978) that such a competitive reaction exists and only if the reaction is carried out for a sufficiently long time is it partially irreversible. Therefore, the reaction system of the conventional method is complicated, inefficient and requires a long reaction time.