GB2095831A - Monoclonal antibody reagent and method for immunological assay - Google Patents

Abstract

A reagent for immunological assay of an antigen comprises an insolubilized antibody and a labelled antibody respectively prepared from monoclonal antibodies, the antibodies being bindable to different antigenic determinants of the antigen. The reagent attains a remarkable shortening of reaction time without any adverse effect on the sensitivity and accuracy. In an improved sandwich method the insolubilized antibody and labelled antibody are reacted simultaneously with the antigen to be assayed.

Description

SPECIFICATION Reagent and method for immunological assay In recent years, the advances in immunochemistry have led to extensive investigations of antibodies of which little was known in the past, and the control mechanism for antibody production and the biochemical structures and properties of antibody have been studied in detail.

In 1975, C. Milstein et al. succeeded in producing a monoclonal antibody by cell fusion of mouse myeloma cells with antibody-producing cells from the spleen. The monoclonal antibody is expected to contribute greatly to the development of fundamental researches of immunology, and investigations in this area have been actively undertaken.

Enzymoimmunoassay (EIA) has previously been used as highly sensitive and quantitative immunological assay method. In particular, a sandwich method based on this assay is frequently used because of the simplicity of the procedure and the high sensitivity. However, since the sandwich method as hereinafter described requires from 1 to 4 days for the reaction, it is desired to minimize the required reaction time. Furthermore, due to recent progress in clinical medicine, rapid evaluation of the test results is demanded to give an immediate treatment to the patient. From this standpoint, too, shortening of the reaction time is earnestly desired.However, according to the conventional sandwich method, shortening of the reaction time results in a lowering of the sensitivity and accuracy of the assay as will be explained herein below, so that significant shortening of the reaction time cannot be attained.

Now the conventional sandwich method is described below:-- Although the method is described in relation to EIA throughout the specification it will be understood by those skilled in the art that FIA (fluoroimmunoassay) techniques can be substituted for EIA.

The conventional sandwich method is carried out in the following sequence as shown in the schematic diagram of Figure 1 of the drawings hereinafter referred to.

i) Insolubilized antibody 2' obtained by bonding an antibody for an antigen 3 to be assayed to an insoluble carrier (solid phase) 4 is reacted with the antigen 3 to bind the antigen 3 to the antibody 2' on the solid phase (first reaction).

ii) The solid phase 4 is washed to remove unreacted substances.

iii) The solid phase 4 thus washed is reacted with labelled antibody 1' obtained by bonding an enzyme 5 to an antibody for the antigen 3 to be assayed to bind the labelled antibody 1' with an unreacted antigenic site of the antigen 3 which have been fixed to the solid phase 4 (second reaction).

iv) The solid phase 4 is washed to remove the excess of the labelled antibody 1', and then a substrate for the enzyme is added and subjected to the enzyme reaction.

Since antigen 3 and the labelled antibody 1' have been bound to the solid phase 4, the enzyme reaction takes place in proportion to the amount of the enzyme present. The amount of the antigen 3 to be assayed is determined from the amount of the resulting reaction product.

Since the sandwich method is generally classified as a non-competitive assay, it might be conceived to include no competitive reaction. However, this classification is not necessarily expressing the exact reaction states. Thus, actually, an equilibrium state can exist in sandwich method, which is shown schematically as follows.

First reaction

Second reaction

(In the above scheme, Ab represents the insolubilized antibody; Ag represents the antigen to be assayed; Ab* represents the labelled antibody; a, a', b, b', c, c', d, d', e and e' represent equilibrium constants; and - means that the antigen and the antibody are bound together) When the complex of the insolubilized antibody and the antigen to be assayed is reacted with the labelled antibody in the second reaction, the insolubilized antibody and the labelled antibody competitively bind to the antigen to be assayed according to their affinity levels for the antigen (since properties of the insolubilized antibody and the labelled antibody are nearly the same, their affinity levels for the antigen to be assayed are considered to be nearly equivalent), and due to the competition, the insolubilized antibody-antigen complex formed by the first reaction dissociates to form complexes of various combination as shown above.

This is evidenced by the following experiment.

A plastic test tube with an anti-HCG-P subunit antibody fixed thereto is reacted with HCG-p which has been labelled with peroxidase, and then washed. When it is reacted with an anti-HCG-P antibody, the enzyme-labelled HFG-P which has been fixed to the solid phase dissociates gradually from the solid phase and moves into the liquid phase. Figure 2 shows this state.

If, however, the time of the first reaction is sufficiently long, binding of antigen and antibody becomes partly irreversible, and the antigen-antibody complex does not easily undergo the dissociation even when the labelled antibody is added thereto. It is said on the other hand that if the time of the second reaction is made sufficiently long, the reaction proceeds in a direction to form a complex of "the insolubilized antibody, the antigen to be assayed and the labelled antibody." For these reasons the reaction in the conventional sandwich method has to be conducted over a long period.Accordingly, if the sandwich method is carried out without the first reaction or if the time length of the first reaction is extremely shortened (for example, when the insolubilized antibody and the labelled antibody are simultaneously reacted with the antigen to be assayed), the second reaction must be prolonged and the sensitivity and accuracy of the assay are reduced. If the time length of the second reaction is shortened, the sensitivity and accuracy are reduced still further. For these reasons, it has been impossible in the convention method to shorten the reaction time without causing a reduction in the sensitivity and accuracy.In this regard, Ichihara et al. (The Japanese Journal of Clinical Pathology, 26, 1027 (1978)) reported that such a competitive reaction does exist, and that only if the reaction is conducted over a sufficiently long period does the reaction partly become irreversible.

According to the present invention there is provided a reagent for immunological assay by the sandwich method as hereinbefore described, characterized in that said reagent comprises insolubilized monoclonal antibody and labelled monoclonal antibody, said monoclonal antibodies being capable of distinguishing and binding to different antigen determinants of an antigen to be assayed.

The present invention is to provide a reagent for immunological assay having a shortened time for completion of the assay without reduction of the sensitivity and accuracy.

The use of monoclonal antibodies for preparing insoluble antibody and of a labelled antibody which distinguish and bind to different antigenic determinants on the same antigen contrasts with the conventional method whereby so called polyclonal antibodies (obtained from animals immunized with the same antigen) have been used for preparing those two antibodies so that they compete for the same antigenic determinants of the antigen.

Thus, the present invention is characterised in that the insolubilised antibody and the labelled antibody respectively comprise antibodies capable of recognizing and binding to different antigenic determinants of the same antigen to be assayed, or they comprise antibodies which distinguish the different antigenic determinants of the antigen so that it is possible to make the respective two antibodies bind to different antigenic sites located on the same antigen sufficiently apart not to disturb the binding of each other.

Monoclonal antibodies are most suitable as such antibodies.

The monoclonal antibodies may be obtained by cell fusion between myeloma cells and antibodyproducing cells as described by Milstein et ai. (Nature, 256, 495 (1975)). Since these antibodies are produced from antibody-producing cells of single clone, they are completely homogeneous and their specificities for antigenic determinants is compietely identical. Because the antigen to be assayed usually have several antigenic determinants, if monoclonal antibodie,s capable of recognizing and binding to different antigenic determinants of the antigen are used as the insolubilized antibody and the labelled antibody, no competition between these antibodies occurs and a highly specific assay of the antigen can be attained.Thus, also a mixture of two or more of monoclonal antibodies which bind to different antigenic determinants may be used as the insolubilized antibody and/or the labelled antibody provided that these antibodies do not cause such competition.

The assay in this invention can be conducted in the same way as in the prior art. It must be emphasized, however, that the reaction mechanism is simpler than in the conventional method because the insolubilized antibody does not compete with the labelled antibody as shown below.

( IA61 Ag, Ab* and-have the same meanings as given hereinabove, and p, p', q, q', r, r', s, s' respectively represent equilibrium constants).

Since no competition exists between the insolubilized antibody and the labelled antibody when they have different binding sites to an antigen, the reaction proceeds in a direction such that the components in the reaction solution form an "insolubilized antibody-antigen-labelled antibody" complex.

Following is a description by way of example only and with reference to the accompanying drawings of methods of carrying the invention into effect.

In the drawings: Figure 1 is a schematic diagram showing the reaction mode in a conventional sandwich method.

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

Figure 4 is a schematic diagram showing the reaction mode in accordance with this invention.

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

Figure 6 is a graph showing the results of Example 1.

Figure 7 is a graph showing the results of Example 2; and Figure 8 is a graph showing the results of Example 8.

The lack of competition within the system between the insolubilized antibody and the labelled antibody can be demonstrated by the following experiment: In the same reaction system as the conventional sandwich method, two different monoclonal anti HCG antibodies capable of recognizing different antigenic determinants of HCG were used as the insolubilized antibody and the labelled antibody, and the similar experiment was conducted. The resulting assays are shown in Figure 3, and shows that once the antigen is bound to the insolubilized antibody, it does not dissociate from the insolubilized antibody (solid phase) when an anti-HCG antibody corresponding to a different antigen determinants is added thereto. Thus, it can be inferred that competition between the antibodies for same antigenic sites does not occur.In the present invention, similar results are obtained whether the insolubilised antibody, the antigen to be assayed and the labelled antibody are simultaneously reacted, or the insolubilized antibody and the antigen are first mixed and then reacted with the labelled antibody, or the insolubilized antibody is reacted with the antigen and after a while, the mixture is reacted with the labelled antibody. In the present invention, therefore, there is no restriction on the sequence of the reactions.

A typical reaction in accordance with this invention is shown by a schematic diagram in Figure 4.

Since the insolubilized antibody 2 and the labelled antibody 1 do not compete with each other, a larger amount of the labelled antibody 1 can be bound to the antigen 3 within a shorter period of time, and it is possible not only to shorten the assay time but also to increase the accuracy and sensitivity of the assay.

Table 1 summarizes the advantages of the method of this invention over the conventional method using polyclonal antibodies in the assay of a-fetoprotein (AFP). It is clear from the table that many advantages, such as the marked shortening of the assay time, the simplification of the assay procedure and the increase of the assay accuracy and sensitivity, can be obtained.

TABLE 1

Method of the invention Conventional method Procedure Reaction e washing 1st reaction washing enzyme reaction 2nd reaction washing enzyme reaction Antibodies used Monoclonal antibodies Polyclonal antibodies Time required 30 minutes 270 minutes Sensitivity 3 ng/ml 10 ng/ml intraexperimental error, C.V. in 10 1.2 % 2.5 % repetition a, 0 0 interexperimental error, C.V. in 5 3.7 % 5.6 % repetition The assay was performed using a flurophotometer with the serum of a pregnant woman as an assay sample, a polystyrene test tube as the solid phase, horseradish peroxidase as the labelling enzyme and phloretic acid as a substrate.

It is also possible that only one of the insolubilized antibody or the labelled antibody is prepared from monoclonal antibody, and the other is prepared from ordinary polyclonal antibody. However, in this case, competition occurs at an antigenic determinant common to both of the monoclonal and the polyclonal antibody and sometimes the affinity of the antigenic determinant for the polyclonal antibody may be stronger, which causes a reduced accuracy and sensitivity of the assay as compared even with the conventional method, not only the method where both antibodies are monoclonal.

Figure 5 shows standard curves in the assay of CEA (carcinoembryonic antigen) when the insolubilized antibody and the labelled antibody are combined as shown in Table 2. It is evident that the assay system based on the combination in accordance with this invention gives the best result.

TABLE 2

1 Insolubilized antibody Labelled antibody Method of the invention Monoclonal Monoclonal Comparative method 1 Monoclonal Polyclonal Comparative method 2 Polyclonal Monoclonal Conventional method Polyclonal Polyclonal The monoclonal antibodies used were obtained by the cell fusion technique using mouse myeloma cells in accordance with Example 2 given hereinbelow and derived from clones corresponding to different antigenic sites. The polyclonal antibodies used were the commercial products from Dako Company (Denmark). Polystyrene test tubes were used as the insoluble carrier; horseradish peroxidase was used as the enzyme; and o-phenylenediamine was used as the substrate.

Any desired combination of myeloma cells and antibody-producing cells can be used in obtaining monoclonal antibodies and the kind of the animal from which the cells are derived is not limited, as long as the both cells in the combination can together form hybridoma and produce an antibody while growing.

All insoluble carriers used in conventional immunoassays can be used in this invention. Examples of such insoluble carriers are polystyrene, polyethylene, acrylic resins, polytetrafluoroethylene (Teflon(!)), paper, glass and Agarose. The insoluble carrier may be in the form of a Rollet (bead in a shape of toothed wheel), bead, rod, disc or cuvette, e.g. it may be an optical cell or a test tube.

Labelling agents normally used include, for example, peroxidase, /3-D-galactosidase and alkaline phosphatase for ETA, and fluoroescein isothiocyanate for FIA.

When the labelling agent is an enzyme, a substrate is required in order to measure its activity. The substrate may be such that when it is reacted with the corresponding enzyme, the amount of the resulting product or the amount of the decreased or remaining substrate can be easily measured. For example, 5-aminosalicyclic acid-H202, o-phenylenediamine-H202 and phloretic acid-H202, may be cited as substrates for peroxidase and fluorescein-di-(p-D-galactopyranoside), o-nitrophenol-P-D- galactopyranoside, and 4-methyl-umbelliferyl-,B-D-galactoside as substrates for ,B-D-galactosidase.

Substances which can be assayed by the reagent of this invention should have at least two antigenic determinants in the molecule. Since substances which have been assayed by the sandwich method have two or more antigenic determinants, substantially all of the substances which can be assayed by the conventional method can be assayed in accordance with the present invention.

Examples of such substances include enzymes such as y-glutamyl transpetidase (y-GTP), alkaline 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 product (FDP), C-reactive protein (CRP), a!,-acidic glycoprotein (et1-AG), a,-antitrypsin (a1-AT), a2-plasmin inhibitor (a2-Pl), 2-micrnglobulin (,B2-MG) and immunoglobulin; carcinoembryonic proteins such as cg-fetoprotein (AFP), carcinoembryonic antigen (CEA) and embryonic ferritin; and lymphocytes and bacterial cells, cell surface antigens and cell fractions.

Since the sequences of the reactions among the insolubilized antibody, the antigen to be assayed and the labelled antibody is not limited, it is also possible to mix the insolubilized antibody and the labelled antibody in advance. More specifically, it is possible that insolubilized antibody bound on a carrier, e.g. plastic beads, and labelled antibody are contained in the same vessel, e.g. a test tube, or it is also possible to bind antibody to the inner wall of a suitable vessel, e.g. a test tube which itself serves as an insoluble carrier and then labelled antibody is contained therein. The labelled antibody may be in the form of a solution or a lyophilized product from the solution as well as in the form of tablets, granules or powders.

Besides the insolubilized antibody and the labelled antibody, the reagent of the invention may contain various auxiliaries in order to increase its usefulness. They may be, for example, a dissolving agent for the labelled antibody (when it is served as a lyophilized product), a washing liquid for washing the solid phase after the reaction of the insolubilized antibody and the antigen to be assayed and after further reaction of the labelled antibody a substrate for measuring the enzymatic activity of the labelling agent (when it is an enzyme), and a reaction stopper for the enzymatic reaction.

The following examples further illustrate the invention.

EXAMPLE 1 A reagent for AFP assay a) Preparation of purified AFP 1 6.2 g of crude AFP extract was obtained from 5 litters of the ascites of hepatoma patients by salting out with ammonium sulfate (fraction between 45% and 70% saturation was collected). The crude extract was purified by affinity chromatography using 50 ml of sepharose 4 B on which rabbit anti-AFP antibody had been fixed (1 mg/ml Sepharose) to obtain 924 mg of purified AFP.

b) Preparation of monoclonal anti-AFP antibodies A female BALB/C mouse was immunized by subcutaneous injection of 50 yg of the purified AFP produced in a) above together with complete Freund's adjuvant. The immunization was repeated four times at the intervals of one-week. The spleen was removed on the fourth day after the final immunization, and spleen cells were collected. The spleen cells were washed with Dulbecco's modified MEM culture medium (to be abbreviated hereinbelow as D-MEM). 1 x 108 of these cells were counted and mixed with 1 x 107 mouse myeloma cells (P3-NSl/1-Ag 4.1). They were subjected to cell fusion for 1 minute at 370C in 1 ml of D-MEM containing 42.5% of polyethylene glycol #1540 and 7.5% of dimethyl sulfoxide.To the cells thus subjected to the cell fusion was added 20 ml of HAT medium (RPMI--1640 medium containing hypoxanthine, aminopterin, thymidine, and 10% fetal bovine serum).

After 0.2 ml aliquots of the cell mixture were added into a 96-well microplate and cultured for two weeks the antibody titers of the supernatants in the wells were determined.

Then, the cells in those wells where the antibodies were observed were respectively added to 40 ml of RPMI--1 640 medium containing 10% fetal bovine serum and thymocytes of BALB/c mouse.

Aliquots of the resulting cell suspension were added into two 96-well microplates, and cultured for one week to obtain 9 clones of anti-AFP antibody-producing hybridoma. They were transferred to large scale cultures, and 10 liters each of the resulting supernatants were subjected to affinity chromatography using 50 ml of Sepharose 4B fixed with purified AFP (0.5 mg AFP/ml Sepharose). As a result, 4.2 to 11.6 mg of monoclonal,antibodies were obtained which are designated as lots Nos. 1 to 9.

c) Identification of antigen-recognizing sites i) Preparation of anti-AFP antibody-sensitized test tubes Physiological saline solutions buffered with 0.05M phosphate (pH 6.4; to be abbreviated as PBS hereinbelow) respectively containing 0.2 mg of the monoclonal anti-AFP antibodies Lots Nos. 1 to 9 were added in an amount of 2 ml to polystyrene test tubes washed with PBS. Then the tubes were incubated at 560C for 20 minutes and washed with PBS to give sensitized test tubes.

ii) Preparation of enzyme-labelled anti-AFP antibodies 5 mg of horseradish peroxidase (Grade 1 of Boehringer Mannheim GMBH; to be abbreviated hereinbelow as HRPO) was dissolved in 1.0 ml of 0.3M sodium bicarbonate buffer, and to this solution 0.1 ml of 1% % ethanol solution of 1 -fluoro-2, 4-dinitrobenzene was added and the admixture was reacted for 1 hour.

Subsequently 1.0 ml of 0.06M sodium periodate 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 carried out for 1 hour. The reaction mixture was dialyzed against 0.01 M sodium carbonate solution at pH 9.5.

Each of the nine lots of anti-AFP antibodies produced in b) above was added in an amount of 5 mg to the resulting solution and the mixture was reacted at room temperature for 3 hours.

Then, 5 mg of sodium borohydride was added, and the reaction was carried out overnight. The reaction mixtures were respectively dialyzed against 0.01 M PBS at pH 7.2 to obtain HRPO-labelled anti AFP antibodies.

iii) Identification of antigen-recognizing sites To each of the anti-AFP antibody-sensitized test tubes produced in i) above were added 1.5 ml of -PBS, 0.1 ml of standard solution of the AFP produced in a) above which has been diluted with PBS to 100 ng/ml and 0.4 ml of HRPO-labelled anti-AFP antibodies produced in ii) above and diluted 100 fold, 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 of o-phenylenediamine and 0.01% hydrogen peroxide was added. The enzyme reaction w6s carried out for 30 minutes, and then 0.05 ml of 4M sulfuric acid was added to stop the enzyme reaction. The absorbancy of the reaction mixture at 450 nm was measured.In Table 3, those combinations which induced positive reaction are indicated with +, and those which did not induce reaction are indicated with TABLE 3

HRPO-labelled antibody 1 2 3 4 5 6 7 8 9 1 - - - + - + - + + 2 2 - - ~ + - + - + + 3 3 - - ~ + - + - + + U 4 + + + ~ + + + ~ ~ a, a) 5 - - - + - + - + + N sg ++++t-+++ o X 7 - - - + - + - + + 8 + + + - + + + - 9 + + + - + + + - By the differences in antigen-recognizing sites, the 9 Lots of monoclonal anti-AFP antibodies obtained in b) above can be divided into three groups, namely the first group consisting of Lots Nos. 1, 2, 3, 5 and 7, the second group consisting of Lots No. 6 and the third group consisting of Lots Nos. 4, 8 and 9. These group of antibodies were designated respectively as anti-AFP antibodies [A], [B] and [C].

d) Preparation of a reagent for AFP assay Anti-AFP antibody-sensitized test tubes were prepared by the method of c-i) above using the monoclonal antibody [A].

The monoclonal antibody [C] was labelled with HRPO by the method of c-ii) above, and diluted to 1:10 with PBS. Two milliliters of the diluted labelled antibody were put into the sensitized test tube.

After the content of the test tube was lyophilized, it was sealed to give a reagent for AFP assay.

e) Assay of AFP 1.8 ml of PBS was added to the anti-AFP antibody-sensitized test tube produced in d) above, and then 0.1 ml of the standard solution of AFP produced in a) above and diluted to a concentration of 1000, 100, 10, 1 and 0 ng/ml with the serum of a healthy person added followed by further addition of 0.1 ml of a solution of the HRPO-labelled anti-AFP antibody produced in d) above in 2 ml of distilled water. The reaction was carried out for 20 minutes. After the reaction, the test tube was washed with physiological saline containing 0.005% of Tween 20 (to be referred to as a washing agent hereinbelow). Then, 3 ml of enzyme substrate solution containing 5 mg/ml of o-phenylenediamine and 0.01% hydrogen peroxide was added, and reacted for 10 minutes. 0.05 ml of 4N sulfuric acid was added to the reaction mixture to stop the enzyme reaction. The absorbency at 450 nm of the reaction mixture was measured by a photometer. The resulting standard curve is shown in Figure 6.

EXAMPLE 2 A reagent for CEA assay a) Preparation of purified CEA 40 g of cancer tissues of the large intestine were homogenized by a homogenizer after addition of 1 00 ml of distilled water. The same amount of 1.2M perchloric acid was added to the homogenate, and was extracted for 30 minutes with stirring. The centrifuged supernatant was dialyzed against distilled water to give a crude CEA extract.

The crude extract was concentrated to 10 ml, and applied to a gel filtration using Sepharose 4B equilibrated with physiological saline and the first fraction was collected. It was again applied to a gel filtration on Sephadex G--200 similarly equilibrated and the second fraction was collected. It was concentrated to 2 ml in which 135 g of purified CEA was obtained.

b) Preparation of monoclonal anti-CEA antibodies Eight clones of anti-CEA antibody-producing hybridoma were obtained by using the purified CEA produced in a) above, by a similar method as in Example 1-b).

Mice were immunized using 30 ,ug of the purified CEA in each immunization. 1 x 106 hybridoma were inoculated intraperitonealy in a female BALBIc mouse which had been intraperitoneally given 0.5 ml of pristane (2,6,10,1 4-tetramethylpentadecane; a product of Wako Pure Chemicals, Co., Ltd). Two weeks later, the ascites was collected. The ascites was applied to a chromatography on DEAE-cellulose equilibrated with 0.01 M phosphate buffer at pH 7.0, and an unadsorbed fraction was obtained as a monoclonal anti-CEA antibody.

By a test for identifying antigen-recognizing sites substantially in accordance with Example 1-c), the antibodies obtained could be classified into three groups i.e. 5 lots, 2 lots, and 1 lot which were respectively designated as anti-CEA antibodies [A], [B] and [C].

c) Preparation of anti-CEA antibody-sensitized test tubes Polystyrene test tubes were each washed with PBS, and 2 ml of the anti-CEA antibody [A] (1 mg/ml) produced in b) above was put in it and reacted at 56"C for 20 minutes. After the reaction, the test tube was washed with PBS to produce an anti-CEA antibody [A]-sensitized test tube.

d) Preparation of a reagent for CEA assay HRPO-labelled anti-CEA antibody [B] was obtained by using the anti-CEA antibody [B] produced in b) above, in accordance with Example 1 -c-ii). The labelled antibody was diluted to 1:50 with physiological saline, and 0.2 ml of the diluted antibody was put into each of the anti-CEA antibody asensitized test tubes obtained in c) above, and lyophilized to form a reagent for CEA assay reagent.

e) Assay of CEA The purified CEA prepared in a) above was diluted with the serum of a healthy person to a concentration of 100, 30, 10, 3 and 1 ng/ml, respectively. 0.2 ml of each of the diluted CEA and 0.8 ml of distilled water were added simultaneously to the CEA assay reagent produced in d) above and reacted for 20 minutes with stirring. After the reaction, the test tube was washed with the washing agent, and then 3 ml of a substrate solution containing 300 mg/dl of phloretic acid and 0.01% hydrogen peroxide was added. The reaction was carried out for 10 minutes, and then 0.1 ml of 5% sodium sulfite was added to stop the enzyme reaction. Subsequently, the fluorescence intensity was measured at an excitation wavelength of 323 nm and a fluorescence wavelength of 420 nm by a fluorophotometer.The resulting standard curve is shown in Figure 7.

EXAMPLE 3 A reagent for HCG-ss assay a) Preparation of HCG-P subunit 1 g of HCG (2000 iu/mg) was dissolved in 2 ml of a 0.025M phosphate buffer (pH 5.6), and the solution was fractionated by a chromatography on DEAE-Sephadex A--50 (3 g) previously equilibrated 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 was dissolved in 10 ml of 1 OM urea (pH 4.5) and reacted at 400C for 1 hour. The reaction mixture was fractionated by a chromatography DEAE-Sephadex A--50 (2 g) previously equilibrated with a solution containing 0.03M glycine and 1 OM urea.A fraction eluted with solution containing 0.2M glycine, 1 M NaCI and 8M urea was dialyzed against physiological saline to give 147 mg of HCG-p subunit.

b) Preparation of anti-HCG-P antibodies Eleven lots of anti-HCG-p antibodies were produced in accordance with the method of Example 2b) except that Wistar-strain rats were used as animal for administration of the antigen instead of the BALB/c mice. The antigen-recognizing sites of these antibodies were identified substantially in accordance with the method of Example 1-c). The resulting antibodies were classified into two groups, namely a group consisting of 8 lots of anti-HCG-p antibodies [A] and the other group consisting of 3 lots of anti-HCG-p antibodies [ C) Preparation of anti-HCG-P antibody-sensitized beads (insoluble carrier) One hundred polyethylene beads were added to 100 ml of PBS containing 50 mg of the anti-HCG antibody [A] produced in b) above, and reacted at 560C for 20 minutes. Then, the polyethylene beads were washed to obtain anti-HCG-p antibody [A]-sensitized beads.

d) Preparation of enzyme-labelled anti-HCG-P antibodies The same method as in Example 1-c-ii) was repeated using the anti-HCG-p antibodies [B] produced in b) above. After dilution to 200 times of the volume with PBS, HRPO-labelled anti-HCG-p antibodies [B] were obtained.

e) Preparation of washing agent 9 g of NaCI and 50 yl of Tween 20 were precisely weighed and dissolved in deionized water to form 100 ml of a solution. The solution was filled in a vial which was plugged. Thus, washing agent was produced.

f) Preparation of enzyme substrate 26.40 g of 5-aminosalicyclic acid, 54.34 g of mono-potassium phosphate and 5.72 g of disodium phosphate were mixed and pulverised in a mortar. 550 mg of the mixture was weighed precisely, and filled into a vial. 1.5 ml of hydrogen peroxide (30% solution) was diluted with deionized water to make 100 ml. 1 ml of the solution was filled into an ampoule which was then sealed by fusing. Thus, enzyme substrate was prepared.

g) Preparation of stopper for enzyme reaction 2 g of sodium azide was precisely weighed and dissolved in 100 ml of distilled water. 2 ml of the solution was filled in an ampoule, which was then sealed by fusing to obtain stopper for the enzyme reaction.

h) Preparation of an assay reagent for HCG.

An assay reagent for HCG was prepared by combining the materials produced in c) to g) above in the following manner.

1.Anti-HCG-P antibody [A]-sensitized beads 2. HRPO-labelled anti-HCG-P antibody [B] 3. Washing agent (tenfold concentrated solution) 4. Enzyme substrate (5-aminosalicyclic acid-hydrogen peroxide) 5. Stopperforenzyme reaction i) Assay of HCG The following assay was carried out by using the reagent for HCG assay prepared in h) above. 0.4 ml of the HRPO-labelled anti-HCG-P antibody [B] was taken into a test tube, and a piece of anti-HCG-P antibody [A]-sensitized bead was added. Then, 0.1 ml of a standard solution obtained by diluting HCG, described in the Japanese Pharmacopoeia, with the serum of a healthy person to a concentration of 10,000, 1,000, 100, 10 and 1 miu/ml, respectively was added and reacted for 15 minutes. After the reaction the washing agent was diluted to 10 times its volume with distilled water, and the bead was washed with the diluted washing agent Then, 3 ml of the substrate solution prepared by dissolving all of the enzyme substrate in 1 50 ml of distilled water was added, and the reaction was carried out for 30 minutes. Then, 0.25 ml of the stopper was added to stop the reaction, and the absorbency of the reaction mixture at 500 nm was measured. The resulting standard curve is shown in Figure 8.

Claims (10)

1. A reagent for immunological assay by the sandwich method as hereinbefore described, characterised in that said reagent comprises insolubilized monoclonal antibody and labelled monoclonal antibody, said monoclonal antibodies being capable of distinguishing and binding to different antigen determinants of an antigen to be assayed.

2. A reagent as claimed in claim 1 wherein beads or a vessel which is made of plastic or glass, is used as a carrier for the insolubilized antibody.

3. A reagent as claimed in claim 1 or claim 2 wherein enzyme or fluorescent substance is used as labelling agent in producing the labelled antibody.

4. A reagent as claimed in any preceding claim wherein the labelled antibody and the insolubilized antibody are contained in one vessel.

5. A reagent as claimed in any one of the preceding claims wherein the labelled antibody is contained in a plastic vessel which is used as a carrier of the insolubilized antibody.

6. A reagent as claimed in any preceding claim wherein the labelled antibody is lyophilized.

7. A reagent as claimed in any preceding claim including at least one auxiliary agent selected from dissolving agent, washing agent substrate and reaction stopper.

8. A reagent as claimed in claim 1 and substantially as described with reference to the accompanying drawings and/or as described in any one of the specific examples hereinbefore set forth.

9. An improved sandwich method wherein the improvement resides in the fact that insolubilized antibody and labelled antibody react simultaneously with antigen to be assayed, said insolubilized antibody and the labelled antibody comprising monoclonal antibodies capable of distinguishing and binding to different antigen determinants of said antigen.

10. A method as claimed in claim 9 and substantially as described with reference to the accompanying drawings and/or as described in any one of the specific examples hereinbefore set forth.