JPS5954966A - Immunological measuring reagent - Google Patents

Abstract

PURPOSE:To achieve a higher measuring sensitivity and a shorter measuring time in the immunological measuring method by using several kins of monoclonal antibodies. CONSTITUTION:This measuring reagent features the use of an insoluble antibody which comprises one or more kinds of monoclonal antibodies 2b and 2c capable recognizing different antigenic determinants of same antigen 3 to be measured to bond to and a labelled antibody comprises more than one different kinds of monoclonal antibodies 1b and 1c capable of recognizing antigenic determinants different from those mentioned above of the antigen 3 being measured to bond to. This eliminates competition between the monoclonal antibodies 2b and 2c as the insoluble antibody and the monoclonal antibodies 1b and 1c to bond to antigenic determinants 6b and 6c of the antigen 3 being measured for a short space of time thereby achieving a shorter measuring time and a higher measuring accuracy.

Description

[Detailed description of the invention] The present invention relates to immunological measurement reagents.

In recent years, due to advances in immunology, research on antibodies, which was previously an unknown field, has progressed, and the control mechanism of their production and the biochemical composition and properties of the antibodies themselves have been investigated in detail.

In 1975, C. Mllstein et al. succeeded in producing monoclonal antibodies by cell fusion of mouse myeloma epilepsy and antibody-producing cells in the spleen. This monoclonal antibody is expected to greatly contribute to the development of basic research in immunology, and research in this direction is being actively conducted.

Conventionally, highly sensitive and quantitative immunoassay methods include radioimmunoassay (RIA) and enzyme immunoassay (E
In particular, the sandwich method based on the method of these authors is frequently used because it is easy to operate and has high measurement sensitivity, but it takes 1 to 4 days for measurement. Since this is necessary, it is desirable to make this time as short as possible. Furthermore, with recent advances in clinical medicine, it has become necessary to know test results quickly and take necessary measures immediately, so there is a strong desire to shorten test times in this respect as well.

However, the conventional sandwich method has a problem in that measurement sensitivity and accuracy decrease when the reaction time is shortened, and it has not been possible to significantly shorten the reaction time for reasons described below.

As a result of repeated research to solve this problem, the present inventors have conventionally used almost identical antibodies, so-called polyclonal antibodies, obtained by administering the same antigen as insolubilized antibodies and labeled antibodies. However, in the present invention, by using multiple types of antibodies that recognize and bind to different antigen-determining sites of the same antigen, so-called monoclonal antibodies, measurement sensitivity can be widened without reducing measurement accuracy. This has resulted in significant improvements and a significant reduction in measurement time.

Hereinafter, the present invention will be explained in detail in comparison with a conventional method. For convenience of explanation, a sandwich method based on EIA will be described as an example, but the same applies to FIA (fluorescence immunoassay).

The conventional measurement method is performed in the following order as shown in the schematic diagram of FIG. .

i) An antibody against the antigen to be measured 3 is transferred to an insoluble carrier (in phase) 4
The insolubilized pile 2a bound to the antibody 2a is reacted with the antigen 3 to be measured, and the antigen 3 to be measured is bound to the antibody 2a on the same phase.

ii) Washing the solid phase 4 to remove unreacted substances.

iii) The washed solid phase 4 is reacted with an enzyme-labeled antibody 1a (hereinafter simply referred to as labeled antibody), which is an antibody against the antigen to be measured 3 bound to an enzyme 5, to remove the antigen to be measured bound to the solid phase 4. Labeled antibody 1a is bound to the unreacted site of 3.

iV) After washing the solid phase 4 to remove excess labeled antibody 1a, an enzyme substrate is added to perform an enzyme reaction.
,.

Since the antigen to be measured 3 and the labeled Rg body 1a are bound to the solid phase 4, an enzymatic reaction occurs in proportion to the amount of this enzyme present, and the amount of the antigen to be measured 3 is determined from the amount of the resulting reaction product. measure quantity.

Generally, the Sand Inch method is classified as a non-competitive reaction method, so it is easy to think that no competitive reaction occurs at all. However, this is just the classification; in reality, an equilibrium relationship is established. A schematic diagram of this is shown below.

(Here, {Ab} is the insolubilized antibody, Ag is the antigen to be measured,
Ab* is labeled antibody, a, a', b, b', c, c', d
, d', e, and e' each represent an equilibrium constant, and - represents that the antigen and antibody are bound. ) That is, in the second reaction, when the labeled antibody is reacted with the bond between the insolubilized antibody and the antigen to be measured, the insolubilized antibody and the labeled antibody compete for the antigen to be measured according to their respective affinities for the antigen to be measured. (Since the insolubilized antibody and the labeled antibody are almost the same antibody, their affinities for the antigen to be measured are considered to be the same.) Is it dissociated and mentioned above? This results in the formation of conjugates with various bond states.

This is demonstrated by the following experiment. That is, anti-HC
A plastic test tube bound with a G-β subunit antibody is reacted with peroxidase-labeled HCG-β, and then washed. When this is reacted with an anti-HcG-β antibody, the enzyme-labeled HCG-β bound to the solid phase becomes
As time passes, it dissociates from the solid phase and transitions to the liquid phase.

FIG. 2 shows this state.

However, if the first reaction time is sufficiently prolonged, some of the antigen-antibody binding becomes irreversible, so that even if a labeled antibody is added to the bound product, dissociation of the bound product becomes difficult to occur. It is also said that if the second reaction time is made sufficiently long, the reaction proceeds in the direction of producing an insolubilized antibody-analyte antigen-labeled antibody conjugate. Therefore, in conventional therapy, it is essential to make the response time sufficiently long. Therefore, if the first reaction is not performed or is extremely short <L,
For example, if an insolubilized antibody and a labeled antibody are reacted with the antigen to be measured at the same time, the reaction time of the second reaction will be prolonged and the measurement sensitivity and accuracy will be reduced, and if the reaction time of the second reaction is shortened, A significant reduction in measurement sensitivity and accuracy occurs. For these reasons, it has been impossible with conventional methods to shorten one reaction time without reducing measurement sensitivity and accuracy. It should be noted that Ichihara et al. [Clinical Pathology, 26, 1027 (1978
)) was also reported.

As described above, the reason why the reaction mode in conventional measurement methods is complicated, inefficient, and takes a long time is that the antibodies used are polyclonal antibodies, and the insolubilized antibody and labeled antibody are almost identical. This is thought to be due to the fact that both antibodies react competitively to the same antigen-determining site of the same antigen.

The present invention is an antibody in which an insolubilized antibody and a labeled antibody can recognize and bind to different antigen-determining positions of the same antigen to be measured; otherwise, one antibody can bind to the antigen. It is characterized by being composed of a wide array of antibodies that connect to sites where they do not interfere with each other, avoiding adjacent sites that would interfere with the binding of the other antibody.

Monoclonal antibodies are suitable as such antibodies, but in order to make the antigen-antibody reaction more effective, one or more types of insolubilized antibodies and two or more types of labeled antibodies, each with an antigen recognition site, are used. Preferably, different monoclonal antibodies are used.

For monoclonal antibodies, myeloma cells and antibody-producing cells were prepared using the method of Milstein et al. (Nature, 256, 4).
95 (1975)) and produced from antibody-producing cells of the same clone, the antibodies are completely identical and have the same specificity for the antigen. It is thought that. Since the antigen to be measured usually has several antigen-determining sites,
It can be concluded that by using a combination of monoclonal antibodies directed against different Kakehara-determined sites as the insolubilized antibody and the labeled antibody, competition between the antibodies does not occur and a measurement with high specificity 9 can be performed. Furthermore, by using a mixture of two or more types of single antibodies that do not cause competition as an insolubilized antibody and a labeled antibody, a short reaction time and even higher sensitivity and reliability can be obtained.

Since monoclonal antibodies have very high specificity, there is a concern that if these antibodies are used in combination, the overall specificity will decrease. If the specificity of an antibody is sufficiently high, there is no reduction in overall specificity.

It is appropriate to mix 2 to 5 types of monoclonal antibodies to obtain the desired measurement sensitivity and reaction rate; even if more than 5 types are mixed, the measurement sensitivity and reaction rate will improve more than just that. It is not allowed.

Measurements in the present invention can be performed in the same manner as conventional methods. However, the reaction mechanism is considered to be simpler than the conventional method, as the insolubilized antibody and labeled antibody do not compete with each other, as shown below.

p>>p', q>>q', r>>r', s>>s' (where {Ab}, Ag, Ab*, - represent the same meaning as above, p, p', q, q', r, r', s, and s' each represent an equilibrium constant.] In other words, if the antigen-binding sites of the insolubilized antibody and the labeled antibody are different from each other, competition between the two does not occur. The reaction proceeds in a direction in which the components in the reaction solution produce an insolubilized antibody-antigen-labeled antibody conjugate.

This is supported by the following experiment. That is, in the same experimental system as described in the conventional method, experiments were conducted in the same manner using two types of monoclonal anti-HCG antibodies that recognize different antigen-determining sites of HCG as an insolubilized antibody and a labeled antibody. As shown in Figure 3, once the antigen bound to the insolubilized antibody, no dissociation from the insolubilized antibody (solid phase) was observed even when an anti-HCG antibody with a different antigen recognition site was added. , it is thought that there is no competition between antibodies against the antigen. This reaction pattern is the same even when multiple types of monoclonal antibodies having different antigen recognition sites are used simultaneously as an insolubilized antibody and a labeled antibody, and the reaction efficiency is thought to be further improved. Therefore, in the present invention, the insolubilized antibody, the antigen to be measured, and the labeled antibody may be reacted simultaneously, the insolubilized antibody and the antigen to be measured may be mixed, and then the labeled antibody may be reacted. Similar results can be obtained even if the labeled antibody is reacted with the antibody and the antigen to be measured after a certain interval, so the reaction order is not limited at all. Such an effect was not possible with conventional reagents using polyclonal antibodies.

The reaction mode of the present invention is schematically shown in FIG.

Since insolubilized antibodies 2b and 2c do not compete with labeled antibodies 1b and 1c, more labeled antibodies 1b can be produced in a shorter time.
and 1C are the antigen-determining sites 6b and 6 of the antigen to be measured 3, respectively.
It is possible to combine with c, and it is possible to shorten measurement time and increase measurement accuracy and sensitivity.

Table 1 shows the method of the present invention when measuring α-fetoprotein (AFP), the conventional method using a polyclonal antibody, and the method using the insolubilized particles and the labeled antibody, which have different antigen recognition sites.
This is a comparison with a method using one type of monoclonal antibody (comparative method 1). As is obvious, many advantages can be obtained, such as a significant reduction in measurement time, simplification of measurement operations, and improvement in measurement accuracy and sensitivity.

The measurement was carried out using a fluorophotometer using pregnant woman's serum as the specimen, a polystyrene test tube as the solid phase, horseradish peroxidase as the labeled enzyme, and phloretic acid as the substrate.

It is also possible to use either the insolubilized antibody or the labeled antibody as a monoclonal antibody, and the other as a conventionally used polyclonal antibody. However, in this case, competition occurs at the antigen-determining site shared by the monoclonal antibody and polyclonal antibody, and the affinity with the polyclonal antibody is thought to be strong. Of course, if this is the case, the measurement accuracy and sensitivity will be lower than that of the conventional method.

FIG. 5 shows a standard curve in a CEA (carcinoembryonic protein) measurement system in which insolubilized antibodies and labeled antibodies are combined as shown in Table 2. It is clear that the measurement system based on the combination of the present invention is the most excellent.

The monoclonal antibodies used were those derived from clones with different antigen recognition sites obtained by a cell fusion method using mouse myeloma cells according to Example 2 below, and the polyclonal antibodies were obtained from DAKO. (Denmark)
A polystyrene test tube was used as the insoluble carrier, horseradish peroxy 7'-ase was used as the enzyme, and o-phenylenediamine was used as the substrate.

The combination of myeloma cells and antibody-producing cells to obtain monoclonal antibodies can be carried out using the following methods: As long as each cell can fuse and produce antibodies while proliferating, the type of animal each cell is derived from can be determined. There is no limitation, and any combination may be used.

As the insoluble carrier in the present invention, all those used in conventional immunoassays, such as polystyrene, polyethylene, polyacrylic, Teflon, paper, glass, agarose, etc., can be used. Also, its shape is rollet-like,
Spherical, rod-shaped, disc-shaped, or container-shaped shapes such as optical cells, test tubes, etc. can be used, but other shapes are also possible. , ・As a labeling agent, E
In IA, peroxidase, β-D-galagtodide, alkaline phosphatase, etc. are usually used, and in FIA, fluorescein inthiocyate, etc. are usually used, but other labeling agents can be used as long as the activity of the labeling agent can be measured. It may be.

If the labeling agent is an enzyme, a substrate is required to measure its activity. Any substrate may be used as long as it is possible to easily measure the amount of the product produced by reacting with the corresponding rope, or the amount of reduction or remaining amount of the substrate. For example, peroxidase substrates include 5-aminosalicylic acid-H2O2, o-phenylenediamine-H2O2,4
-aminoantibiline-H2O2, phloretic acid-H2
As a substrate for β-D-galactosidase such as O2, fluoresin-di(β-D-galactopyranoside), o
-Nitrophenol-β-D-galactobiranoside, 4
-Methylumperipheryl β-D-galactoside and the like.

Substances that can be measured by the reagent of the present invention must have three or more antigen-determining sites in one molecule, but conventionally,
Since most of the substances measured by the sandwich method have three or more antigen-determining sites, virtually all substances that can be measured by the conventional method can be measured. For example,
γ-glutamyl transbetide (γ-GTP),
Alkaline phosphatase, enzymes such as glycosyltransferase, thyroid stimulating hormone (TSH), luteinizing hormone (LH)
), placental gonadotropin (HCG), insulin,
Secretin, proteinaceous hormones such as growth hormone (GH), fibrin degradation product (FDP), C-reactive protein (C
RP), α1-acidic glycoprodein (α1-AG),
Plasma proteins such as α1-antitrypsin (α1-AT), α2-plasmin inhibitor (α2-PI), β2-microglobulin (β2-MG), and immunoglobulin,
α-Fedoprotein (AFP), carcinoembryonic protein (CE)
A), oncofetal proteins such as fetal ferritin, lymphocytes, cells such as microorganisms, cell surface antigens, cell fractions, etc.

As described above, in the reagent of the present invention, the reaction order of the insolubilized antibody, the antigen to be measured, and the labeled antibody is not limited in any way, so it is possible to mix the insolubilized antibody and the labeled antibody in advance. That is, a suitable container can be used as an insoluble carrier, and the antibody can be bound to the inner wall of the container to make it insoluble, and the labeled antibody can be stored therein. The stored labeled antibody may be in the form of a solution or a lyophilized product of the solution, and may also be in the form of tablets, granules, or powder.

Although the reagent of the present invention is composed of an insolubilized antibody and a labeled antibody, various auxiliary agents can be included to facilitate the use of this reagent. For example, a solubilizing agent for dissolving the labeled antibody when it is in a solid state, a washing solution for washing the solid phase after reacting the insolubilized antibody with the antigen to be measured, and after reacting the labeled antibody with the insolubilized antibody. , a substrate for measuring the enzyme activity when the labeling agent is an enzyme, and a reaction terminator for stopping the enzyme reaction can be included in any combination.

Next, examples of the present invention will be shown.

Example 1 AFP measurement reagent, 'a) Purification A
Production of FP Salting out method using ammonium sulfate from 5 liters of ascites of a liver cancer patient (
AFP crude extract 16
．． 2g was obtained. This was purified by affinity chromatography using 50 ml of rabbit anti-AFP antibody-conjugated Sephalone 4B (1 mg/ml Sepharose) to obtain 924 mg of purified AFP.

b) Production of monoclonal anti-AFP antibody 50 μg of the purified AFP produced in a) above was subcutaneously administered to female BALB/c mice together with complete Freund's adjuvant (CFA). After administration four times every week, the spleen was removed on the fourth day and splenocytes were collected. Dulbecco'
After washing with modified MEM medium (hereinafter abbreviated as D-MEM), 1 x 108 cells were measured and 1 x 1
07 mouse myeloma cells (P3-NSI/1-A
g 4.1) and fused for 1 minute in 1 ml of D-MEM containing 42.5% polyethylene glycol + 1540 and 7.5% dimethyl sulfoxide at 37°C.

The cells were mixed with HAT medium (RPMI-16 containing hypoxanthine, aminopterin, thymidine, and 10% fetal bovine serum).
40 medium) was added to make 20 ml, and 0.2 ml each was dispensed into a 96-well microplate and cultured for 2 weeks. After that, the antibody activity of the culture supernatant in the wells where the cells were grown was measured.

Next, the cells in the wells in which activity was observed were added to RPMI-16 containing 10% fetal bovine serum containing BALB/C mouse thymocytes.
40 medium in 40 ml. 96% of this cell suspension
Dispense into two well microplates. After culturing for one week, 9 strains of anti-AFP antibody-producing hybridomas were obtained.

These were cultured in large quantities, and 10% of each culture supernatant was obtained.
Separose 4B (0.5 m
Purification was performed by affinity chromatography using 50 ml of gAFP/ml Sepharose to obtain 4.2 to 11.6 ml of each monoclonal antibody. Lot No. of each antibody. was set as 1 to 9.

c) Identification of antigen recognition site i) Production of anti-AFP antibody sensitized test tube 0.05M phosphate buffered saline pH 6.4 (hereinafter PB
Place the lot in a polystyrene test tube washed with
No. Add 2 ml of PBS containing 0.2 mg each of monoclonal anti-AFP antibodies 1 to 9 to separate test tubes,
After reacting at °C for 20 minutes, the tubes were washed with PBS to prepare sensitized test tubes.

ii) Production of enzyme-labeled anti-AFP antibody 5 ml of horseradish peroxidase (Boehringer Mannheim Grade 1; hereinafter abbreviated as HRPO) was added to the O.I.
Dissolve in 1.0 ml of 3M sodium bicarbonate buffer, add 0.1 ml of 1% 1-fluoro-2,4-dinitrobenzene in ethanol, and react for 1 hour.

Furthermore, 1.0 ml of 0.06 M sodium periodate solution was added and reacted for 30 minutes, and then 1.0 ml of 0.06 M sodium periodate solution was added and reacted for 30 minutes.
．． After adding a 16M ethylene glycol bath solution and reacting for 1 hour, the mixture was dialyzed against 0.01M sodium carbonate solution, pH 9.5. To this solution, 5 mg of the 9 types of anti-AFP antibodies prepared in b) above were added, and
After reacting for an hour, 5 mg of sodium borohydride was added and reacted overnight. Zarani, 0.01M PBS p
HRPO-labeled anti-AFP antibody was obtained by dialysis against H7.2.

iii) Identification of antigen recognition site Add PBS to each anti-AFP antibody sensitized test tube prepared in 1) above.
1.5 ml, 100 ng/AFP produced in a) above
0.1 ml of the standard solution diluted with PBS to make 10 ml of the HRPO-labeled anti-AFP antibody prepared in ii) above.
0.4 ml of the 0x diluted solution was added, and the reaction was carried out for 30 minutes.

After the reaction is complete, wash with washing solution. 100mg/ml 0-
3 ml of an enzyme substrate solution containing phenylenediamine and 0.01% hydrogen peroxide was added, and the reaction was carried out for 30 minutes. , 4
After stopping the enzyme reaction by adding 0.05 ml of M sulfuric acid,
The absorbance at 50 nm was measured and the resulting combination of reactions was +. Combinations that could not be obtained are shown in Table 3.

Based on the difference in antigen recognition sites, the nine monoclonal anti-AFP antibodies obtained in b) above were Lot No. 1, 2.3, 5.
7 of 5 stocks and Lot No. 6 and 1 strain, Lot No.
It could be divided into three types, 4.8 and 9, and the anti-AFP antibodies [A], (B) and [C] were labeled respectively.

d) Production of AFP measurement reagent [A] of the monoclonal antibodies produced according to b] above.
), an anti-AFP antibody sensitized test tube was produced by the method of c-i] above.

In addition, monoclonal antibody (B) can be obtained by the method of C-ii).
After labeling [C] with HRPO, this was added to PBS.
The solution was diluted 10 times and filled into 2 ml portions. Next, both were freeze-dried and sealed to produce an AFP measurement reagent.

e) Measurement of AFP Add PBS1 to the anti-AFP antibody sensitized test tube prepared in d) above.
．． After adding 8 ml, add 10 ml of AFP prepared in a) above.
0.1 ml of each standard solution diluted with healthy human serum to give 0, 100, 10, 1, 0 ng/ul and the HRPO-labeled anti-AFP antibody prepared in step d) above were dissolved in 2 ml of purified water. 0.1 ml of the solution was added and the reaction was carried out for 10 minutes. After the reaction was completed, it was washed with physiological saline containing 0.005% Tween 20 (hereinafter abbreviated as detergent) and washed for 300 m.
3 ml of an enzyme substrate solution containing g/Ul of phloretic acid and 0.01% hydrogen peroxide was added, and the reaction was carried out for 10 minutes.

After 0.1 ml of 5% sodium sulfite was added to stop the enzyme reaction, the fluorescence intensity was measured using a fluorometer at an excitation wavelength of 325 nm and a fluorescence wavelength of 420 nm. The obtained standard curve is shown in FIG.

Example 2 CEA measurement reagent a) Production of purified CEA 40 g of colon cancer tissue was cut into small pieces, and 100 ml of distilled water was added thereto and crushed using a homogenizer. The same amount of 1.2M perchloric acid was added to this liquid, and extraction was performed for 30 minutes while stirring. A supernatant was obtained by centrifugation, and this was dialyzed against distilled water to obtain a CEA crude extract.

This crude extract was concentrated to 10 ml and subjected to gel filtration using Sepharose 4B equilibrated with physiological saline to obtain a first fraction. Sephade equilibrated in the same way
Gel filtration was performed again using x G-200, and the second fraction was collected and concentrated to 2 ml to obtain 135 μg of purified CEA.

b) Production of monoclonal anti-CEA antibody Example 1-b) using the purified CEA produced in a) above
Twenty-three anti-CEA-producing hybridoma strains were obtained by the same procedure.

The mice were immunized with 30 μg of purified CEA for each administration.
was used. Into the abdominal cavity of a female BALB/C mouse, 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane; Wako Pure Chemical Industries, Ltd.) was administered into the abdominal cavity in advance.
1×106 hybridomas were inoculated, and ascitic fluid was collected 2 weeks later. Each ascites was diluted with 0.01M phosphate buffer p
Chromatography was performed using DEAE-cellulose equilibrated with H7.0, and the unadsorbed fraction was obtained as a monoclonal anti-CEA antibody. As a result of the antigen recognition site identification test according to Example 1-C), each antibody was divided into nine types,
There are 9 Lots, 5 Lots, 3 Lots each, and 1 Lot each, each with anti-CEA antibodies [A], [B], and [C
], [D], [E], [F], [G], [H], and (J).

c) Production of anti-CEA antibody sensitized test tube After washing the polystyrene test tube with PBS,
) anti-CEA antibodies [A], [E] (1 mg/m
l) 1 ml of each was added and reacted at 56°C for 20 minutes. After the reaction, the tubes were washed with PBS to produce anti-CEA antibody (A) and (E) sensitized test tubes. - d) Production of CEA measurement reagent Anti-CEA antibodies [B] and [C) produced in b) above.

HRPO-labeled anti-CEA antibodies [B), (C], [D] according to Example 1-c-ii) using [D] and [G]
, [G] was obtained. This was diluted 50 times with physiological saline, 0.2 ml each was dispensed into the anti-CEA antibody [A) and [E] sensitized test tubes prepared in c) above, and lyophilized.
An EA measurement reagent was manufactured.

e) Measurement of CEA The purified CEA produced in a) above was diluted with healthy human serum to give 100, 30, 10, 3.1, 0.3, 0.1 ng/m.
0.2 ml each and 0.8 ml each of distilled water.
and simultaneously added to the CEA measurement reagent prepared in d) above,
The reaction was allowed to proceed for 15 minutes under stirring. After the reaction was completed, the test tube was washed with a detergent, and then 0.5 ml of a substrate solution containing 500 mg/ul of o-phenylenediamine and 0.02% hydrogen peroxide was added and reacted for 10 minutes. 2N sulfite 0
．． After adding 1 ml to stop the reaction, absorbance at a wavelength of 492 nm was measured using a spectrophotometer.

The obtained standard curve is shown in FIG.

Example 3 HCG-β measurement reagent a) Production of HCG-β subunit 1g of HCG (2000iu/mg) to 2ml0.025
DEAE-Sephadex A-5 dissolved in M phosphate buffer pH 5.6 and equilibrated with the same buffer in advance
Chromatography was performed using 3 g of 0.03 g. 0.0
The 5M phosphate buffer pH 5.6 elution fraction was collected and dialyzed against distilled water to obtain 308 mg of purified HCG, which was lyophilized. Of this, 300mg was added to 10M urea (pH 4,
5) Dissolved in 10 ml and incubated at 40°C for 1 hour. DEAE-Sephadex A-50 pre-equilibrated with a solution containing 0.03M glycine and 10M urea.
Chromatography was performed using 2 g of HCG-β, and the fraction obtained by elution with a solution containing 0.2 M glycine, 1 M NaCl, and 8 M urea was dialyzed against physiological saline.
147 mg of subunit was obtained.

b) Production of anti-HCG-β antibody According to the method of Example 2-b], Wistar rats were used instead of BALB/c mice as the animals to which the antigen was applied.
17 lots of anti-HCG-β antibodies were produced. The antigen recognition sites of these antibodies were identified according to the method of Example 1-c), and 7, 5, 4, and 1 L were obtained, respectively.
The anti-HCG-
The β antibodies were designated as [A], [B], [C) and [D].

c) Production of anti-HCG-β antibody-sensitized beads (insolubilized carrier)
100 containing 25 mg each of CG-β antibodies [A] and [B]
ml of PBS, reacted at 56°C for 20 minutes, washed with PBS, and prepared anti-HCG-β antibodies (A), [B].
Sensitized beads were produced.

d) Production of enzyme-labeled anti-HCG-β antibodies Anti-HCG-β antibodies (C) and [D
] and operated in the same manner as in Example 1-c-ii), diluted 200 times with PBS, and prepared HRPO-labeled anti-HCG.
-β antibodies [C] and [D] were produced.

e) Preparation of detergent 9 g of NaCl and 50 ul of Tween 20 were accurately weighed and made up to 100 ml with purified water. This solution was filled into a vial to produce a 10-fold concentrated detergent solution.

f) Production of enzyme substrate 26.40 g of 5-aminosalicylic acid, and 54.34 g of potassium phosphate and 5.72 g of disodium phosphate.
After mixing and atomizing g in a mortar, 500 mg was accurately weighed and filled into a vial. Hydrogen peroxide (30% solution) 1.5
After diluting the ml with purified water to 100 ml, 1
ml was measured, filled into an amble, and melted. An enzyme substrate was produced in the above manner.

g) Production of enzymatic reaction terminator 2 g of sodium azide was accurately weighed, dissolved in 100 ml of purified water, 2 ml each was filled into ampoules, and the mixture was melted and closed to produce a fibrillation reaction terminator.

h) Preparation of HCG measurement reagent The materials produced in steps c) to g) above were combined as shown below to prepare an HCG measurement reagent.

1. Anti-HCG-β antibody (A), [B) sensitized beads 2, HRPO-labeled anti-HCG-β antibody (C), [D]3.
Cleaning agent (10x diluted solution) 4. Enzyme substrate (5-aminosalicylic acid, hydrogen peroxide) 5. Enzyme Reaction Terminator i) Measurement of HCG The following measurements were performed using the HCG measurement reagent prepared in h) above. HRPO-labeled anti-HCG-β antibody [C], [D
] Transfer 0.4 ml to a test tube, add one anti-HCG-β antibody [A] and one [B] sensitized bead, and then add HCG listed in the Japanese Pharmacopoeia to 5000, 500, 50,
5, 0.1 ml each of standard solutions diluted to 0.5 miu/ml
was added and the reaction was carried out for 10 minutes. After the reaction, the detergent was diluted 10 times with purified water and used to wash the beads. Next, 3 ml of a substrate solution in which the entire amount of enzyme substrate was dissolved in 150 ml of purified water was added, and the reaction was carried out for 30 minutes. Ta. After that, 0.025 ml of enzyme reaction terminator was added to stop the reaction.
The absorbance of the reaction solution at 500 nm was measured. The obtained palm standard curve is shown in FIG.

[Brief explanation of drawings]

Figure 1 is a schematic diagram showing the reaction pattern in the conventional sandwich method, Figures 2 and 3 are graphs showing the experimental results of whether competition exists between antibodies, and Figure 4 is the reaction pattern of the present invention. FIG. 5 is a graph showing a comparison between the present invention and the conventional method, FIG. 6 is a graph showing the results of Example 1, and FIG. 7 is a graph showing the results of Example 1.
The figure is a graph showing the results of Example 2, and FIG. 8 is a graph showing the results of Example 4. 1a, 1b, 1c, ... labeled antibody, 2a, 2b, 2c
... Insolubilized antibody, 3... Antigen to be measured, 4... Insoluble carrier (solid phase), 5... Labeling agent (enzyme, etc.), 6b, 6
c...antigen-determining site

Claims (7)

[Claims] (1) In an immunoassay reagent using a sandwich method, the insolubilized antibody is composed of one or more monoclonal antibodies that can recognize and bind to different antigen-determining sites of the same antigen to be measured, and the labeled antibody is An immunological measurement reagent comprising two or more different monoclonal antibodies capable of recognizing and binding to antigen-determining sites different from the above-mentioned antigen-determining sites of the antigen to be measured. (2) The reagent according to claim 1, wherein the insoluble carrier is a glass or plastic container or plastic beads. (3) The reagent according to claim 1, wherein the labeling agent is an enzyme or a fluorescent substance. (4) The reagent according to claim 1, wherein the labeled antibody is housed in a glass or plastic container that is an insoluble carrier for insoluble the antibody. (5) The reagent according to any one of claims 1 to 4, wherein the insolubilized antibody and the labeled antibody are lyophilized products. (6) The reagent according to claim 1, consisting of the following components: a) an insolubilized antibody, b) a labeled antibody, c) one or more selected from a solubilizer, a detergent, a substrate, and a reaction terminator. 4 types of adjuvants. (7) In an immunological measurement method using a sandwich method, an insolubilized antibody containing one or more monoclonal antibodies capable of recognizing and binding to different antigen-determining sites of the same antigen to be measured; It is characterized by simultaneously reacting the antigen to be measured with a labeled antibody containing two or more different monoclonal antibodies capable of recognizing and binding to antigen-determining sites different from those described above on the antigen to be measured. Immunological measurement method.