FR2500166A1 - REAGENT AND METHOD FOR IMMUNOLOGICAL ASSAYING OF ANTIGENS - Google Patents

Abstract

THE INVENTION RELATES TO A REAGENT FOR AN IMMUNOLOGICAL DOSAGE OF ANTIGENS, COMPRISING AN INSOLUBILIZED ANTIBODY AND A TRADEMARK ANTIBODY, RESPECTIVELY PREPARED FROM MONOCLONAL ANTIBODIES. THE REAGENT ALLOWS A CONSIDERABLE REDUCTION OF THE REACTION TIME WITHOUT IMPAIRING SENSITIVITY AND PRECISION. A ADVANCED SANDWICH METHOD USING THE ABOVE REAGENT, IN WHICH THE INSENSITIZED ANTIBODY AND THE BRANDED ANTIBODY REACT SIMULTANEOUSLY WITH THE ANTIGEN TO BE DOSED, IS ALSO PROVIDED.

Description

- 1 - Advances in immunochemistry in recent years have led to

  extensive research on antibodies that have not been known in the past, and a detailed study of

  mechanism governing the production of antibodies, the structures

  biochemical properties and properties of antibodies. In 1975, C. Milstein and ai managed to produce a

  monoclonal antibody by cell fusion of myeloma cells

  mice with spleen cells, producing antibodies. The monoclonal antibody is expected to contribute greatly to the development of basic research in

  immunology, studies in this area have been

companies.

  The enzyme immunoassay (EIA) has already been used

  as a very sensitive and quantitative immunoassay method

  tative. In particular, the sandwich method based

  on this assay is frequently used because of the sim-.

  plicity of the process and its high sensitivity. However, since the "sandwich" method requires 1 to 4 days for the reaction, it is desirable to reduce the necessary reaction time. In addition, due to recent advances in clinical medicine, a rapid assessment of

  test results are required to give an im-

  mediate the patient. From this point of view too, a reduction

  reaction time is seriously desired. But, according to the classic sandwich method, a reduction in

  reaction time results in a decrease in sensi-

  the accuracy and precision of the assay, as will be explained below, so that it is not possible to reduce the

  reaction time significantly.

  The classic sandwich method will now be described in detail. Although the method is explained in connection with the EIA throughout this application, for the sake of convenience, the explanation may apply

  also to the method based on the fluoyoimnunolo-

(FIA).

  The conventional sandwich method is carried out according to the following sequence, iiustrae on the diagram of FIG.

1 of the attached drawings.

  i) An insolubilized antibody 2 'is reacted, obtained

  By attaching an antibody to an antigen 3 at cos = R on an insoluble support (solid phase) 4, with the same 3 to bind the antigen 3 of the antibody 2 to

  the solid object (first rfact cn).

  (ii) The solid ohase is washed to remove the

what! have not reacted.

  iii) The solid phase 4 thus washed is reacted with a labeled antibody 11 ', obtained by forming an enzyme on an antibody opposite to the antigen 3 to be assayed, in order to fix the labeled antibody 1' with a site free anti genic

  antigen 3 which has been fixed on the solid phase 4 (two

th reaction).

  iv) The solid phase 4 is washed to remove the excess of labeled antibody 1 ', then a substrate for

  the enzyme and subjected to an enzymatic reaction.

  Since the antigen 3 and the antibody mark 1 '

  have been fixed on the solid phase 4, the reaction ean-

  zymatic is proportional to the amount of enzyme pr5-

  senta. The quantity of niteriene 3 to be determined is determined

  oartir the nuantit. of the resultant rroduct.

  Given the sandwich method is generally

  categorized as a non-comnetitive dosage, suDDo-

  that it does not involve a competitive reaction. But this classification does not necessarily express the exact state of the reactions. Thus, in fact, a state of equilibrium can exist in the sandwich method, such as

  where it is illustrated schematically below.

  First reaction: Ab [+ (Ag - (@ - Ag) a "> a '3 - 3 rs: (Ab -Ag-Ab-,) c -Ag) + (Ab-Bu) A + (Ag-Ab *) of (Ib) + Ag) + (Abg-A '"In the scheme above, Abl represents the antibody

  insolubilized; Ag represents the antigen to be assayed, Ab + reorients

  feel the labeled antibody; a, a ', b, b', c, c ', d, d', e and e 'represent equilibrium constants; and - indicates

  that the antigen and the antibody are linked together).

  When the complex of the insolubilized antibody and

  the antigen to be assayed reacts with the labeled antibody in the.

  second reaction, the insolubilized antibody and the marbled antibody binds competitively to the antigen to be assayed according to their affinity for the antigen (since the properties of the solubilized antibody and the labeled antibody are almost idunetic, their affinity levels for the antigen to be assayed are considered almost equivalent)

  and the reason for this is that the antibody complex is

  lubilized-antigen formed by the first rection dissociates itself by closing complexes of diverse combination such that

mounted above.

  This is highlighted by the following experience.

  A test tube made of plastic material was reacted, on which was fixed an anti-subunit anti-unit of HCG with HCG-, (HEG = human chorcnic zcnadotropin) marcated with the 2-hydroxycase, then wash. When reacting with an anti-HCG-anticrush, the HCG-2 is annealed with an enzyme or is fixed on the solid phase

  little by little of the solid phase and migrates in the liquid phase.

  This & 3 is illustrated in Figure 2 of the accompanying drawings.

  If, however, the time of the first class is

  sufficiently long, the connection between the antigen and the anti-

  The body becomes irreversibly perceptible, and the antigenic complex does not dissociate easily, even when the labeled anticcrs are added. We know, on the other hand

  if the time of the second reaction becomes sufficiently

  long, the reformation leads to the Formatio-n of a complex

  of the insolubilized antibody, the antigen Z doser and the anti-

  For these reasons, it is essential in the conventional sandwich method to continue the cure for a sufficiently long time, therefore, if the method of the sandwich is carried out without the first reaction.

  or if the duration of time of the first reaction is

  For example, when the insolubilized antibody and the labeled antibody react simultaneously with the antigen to be assayed, the second reaction must be prolonged and the sensitivity and accuracy of the assay are, however, less. If the duration of the second reagent is reduced, the sensitivity and accuracy are even smaller. Therefore, it has been impossible in the conventional method to reduce the reaction time without causing a decrease in sensitivity and accuracy. In this regard, Ichihara et al (The Japanese Journal of Clinical Pathology 26, 1027 (1978)) have reported that such a competitive reaction exists well and the reaction only becomes partially irreversible if the reaction is continued long enough. Thus the reaction system in the classical method is complicated and inefficient and requires a long time.

reaction time.

  The present invention has the object of providing a reagent for an immunological assay which allows a remarkable reduction in the time required for the assay.

sensibility and precision.

  Specifically, it is an object of the present invention to provide a reagent for the sandwich method, which comprises an insolubilized antibody and a labeled antibody.

  prepared from monoclonal antibodies.

  Another object of the invention is to provide a method

  an improved sandwich that allows a remarkable reduction

  the time required for a determination, without reducing the

sibility and precision.

  FIG. 1 of the accompanying drawings represents a diagram illustrating the reaction mode in a conventional sandwich method, FIGS. 2 and 3 are diagrams showing the presence or absence of competition between the antibodies, FIG. 4 is a diagram showing the According to the invention, FIG. 5 represents a diagram illustrating a comparison between the present invention and the conventional method. Fig. 6 is a diagram showing the results of Example 1, Fig. 7 is a diagram showing the results of Example 2, and Fig. S is a diagram showing the results.

of Example 3.

  The above objects of the invention are achieved by using monoclonal antibodies to prepare an insoluble antibody and a labeled antibody which are distinguishable and bind to different antigenic determinants on the same antigen, whereas in the conventional method , so-called polyclonal antibodies (obtained from animals immunized with the same antigen) are used to prepare these two antibodies, so that they compete for the same determinants

antigenic antigens.

  Thus, the invention is characterized in that the insolubilized antibody and the labeled antibody respectively comprise antibodies capable of recognizing and releasing on different antigenic determinants of the same

  antigen to be assayed, or they include antibodies that are

  tingent the different antigenic determinants of the

  Thus, it is possible to obtain that the two respective antibodies bind to different antigenic sites on the same antigen at a sufficient distance from each other so as not to disturb the fixation of the antigen. other.

  As examples of such antibodies, monoclonal antibodies

  clones are the most appropriate.

  The monoclonal antibodies are obtained by cell fusion between myeloma cells and antibody producing cells by Milstein et al., P. (Nature, 256, 495 (1975).) Because these antibodies are produced from cells producing antibodies of a single clone, they are totally homogeneous and their

  specificity for antigenic determinants is total-

  identical. Since the antigen to be dosed is genetically

  several antigenic determinants, if monoclonal antibodies capable of recognizing, and

  to fix on different antigenic determinants of the anti-

  gene, as insolubilized antibody and labeled antibody, there is no competition between these antibodies and one can achieve a very specific assay of the antigen. Thus it is also possible to use a mixture of two or more monoclonal antibodies which bind to different antigenic determinants, such as insolubilized antibodies and / or

  labeled antibody, provided that these antibodies do not

  do not breed a single, compitition.

  The dosage of the invention can be carried out in the same manner as in the prior art. It must be emphasized, however, that the reaction mechanism is then simpler than in the classical method because the insolubilized antibody does not compete with the antibody

  marked, as shown below.

  7- [j: -Ag) + (Ab *) CA * Qrr -Ag -Abt) Ab) + CAg-AL,) pAp, P q ',> r' qs Ab, .q, Ab "and - have the significant amounts that

  above, and p, p ', q, q, r, r', s, being representative of

  respectively constants of éuiibrs).

  As there is no competitian between insolubilis antibody. and labeled anticoros when they have different sites of attachment to an antigen, the reaction proceeds in such a way that the constituents in the reaction solution

  form an "insolubilized," - antigen -

labeled antibody ".

  This can be highlighted by the following experience.

  In the same reaction system as the conventional sandwich method, two different monoclonal anti-HCG antibodies are used, capable of recognizing various HCG in-vivo determinants, as well as an insubmitted antibody.

  labeled antibody and the experiment is performed on the same

  Niere. As shown in FIG. 3, the result is that once the antigen is bound to the antibody

  insolubilized, it is not possible to

  neutralized with a solid anti-HCG antibody: aint; Different antigenic terrinants are thus used, so that it is evident that there is no evidence of antibody binding between the antibodies of the same sites. according to the invention,

  obtains similar results when anticorosine

  the antigen to be assayed and the anticoros

  at the same time, or. when the insolubilized antibody and the antigen are first mixed, then react with the labeled antibody, or when the insolubilized antibody reacts with the antigen and some time later the mixture is reacted with the labeled antibody. . This is how he

  There are no restrictions on the reactionary sequence.

  The embodiment according to the invention is illustrated by a scheme in FIG.

  insolubilized 2 and the marzué 1 antibody do not enter into

  in competition, a larger amount of the anti-

  The labeled body 1 can bind to the antigen 3 in a shorter period of time, and then it becomes possible not only to reduce the duration of the dose but also

  to increase the accuracy and sensitivity of the assay.

  Table 1 summarizes the advantages of the process according to the invention compared with the conventional method using

  polyclonal antibodies in the assay of c -foeto-

  protein (AFP). From the table it is evident that many advantages can be obtained, such as a marked reduction in the duration of the assay, the increase in the assay procedure and the increase in the accuracy and

the sensitivity of the dosago.

- 9-

TABLE 1

  Process of the Invention and the Classical Method of the Invention The assay was carried out by means of a photometer 3

  fluorescence on the serum of a pregnant woman as an

  to be assayed, using a polystyrene test tube as solid nhase, horseradish peroxidase as an enzyme

  labeling and phloretic acid as substrate.

  It is also possible that only one of the

  The insolubilized body or the labeled antibody is prepared from one monoclonal antibody and the other is prepared from an ordinary polyclonal antibody. But,

  in such a case, a competition is

  antigenic agent common to the monoclonal antibody and the polyclonal antibody and sometimes the affinity of the antigenic determinant for the polyclonal antibody may be higher, resulting in a decrease in the accuracy and sensitivity of the assay relative to the classic method and not only to the method o the two

anticoros are monoclonal.

  Figure 5 shows control curves in the

  dosage of CEA (carcinoembryonic antigen) when the

  Reactive procedure Rtact-n_-washing 1st reaction _> washing-reaction - 2nd enzymatic reaction washing -> enzymatic reaction Antibody Monoclonal Antibody polyclonal used Clonal Time required 30 minutes 270 minutes Sensitivity. 3 ng / ml i 1C ng / ml Precision:

Intra-house error

  experimental 2 C.V. in 10 2.5% repeat

Internal error

  Experimental 53.7% 5.6% C.V in 5 repetitions,

- 10 -

  The insolubilized body and the labeled antibody are combined as set forth in Table 2. It is evident that the dosage system is based on the combination according to the invention.

gives the best result.

TABLE 2

  Antibody Insolubilized Antibody Method of the Invention Monoclonal Monoclonal Comparative Method 1 Monoclonal Polyclonal PtfVahode Comparative 2 PRolyclonal Monoclonal Conventional Polyclonal Polyclonal Method The monoclonal antibodies used are obtained by the technique of cell fusion from mouse myeloma cells according to the example 2 given below and come from clones corresponding to different antigenic sites. The polyclonal antibodies used are commercial products from Dako Company (Denmark). Polystyrene test tubes are used as insoluble support; the enzyme is raifott peroxidase; and the

substrate of o-phenylenediamine.

  Any combination of myeloma cells and

  lules producing antibodies can be used to obtain

  monoclonal antibodies-and the species of the animal from which the cells originate is not limited, to the extent that

  o the two types of cells entering in a neutral combination

  together form hybridomas and produce an anti-

body during development.

  All insoluble carriers used in dosages

  classical immunological agents can be used in

  vention. Examples of insoluble carriers are polystyrene, polyethylene, acrylic resins, polytetrafluoroethylene (Teflon), paper, glass and rust. The insoluble support may be in the form of a gear-shaped part, a ball, a bauoette, a disk or a bowl ure. For example, it can be an optical cell, a

- 11 -

  test tube, etc. He may also have other forms.

  Examples of normal labels which are used include, for example, poroxidase, α-alactosidase and phosphatase aiciline for EIA and isothiocyanate in the form of fluoride. When the labeling agent is an enzyme, a substrate is needed to measure its activity. The substrate

  may be as it does with the corrosive enzyme

  As a result, the amount of the resulting product or the amount of the substrate residue can easily be measured. For example,

  mention may be made of 5-arminosalicylic acid-H2C2, o-phenylne-

  diamine-H2O2, phlorotic acid-H2O2, etc. as substrates

  for peroxidase, and fluorescein-di (i -D-galactopyra-

  noside), o-nitrophenol-D-a3alactoDyranoside, 4-methyl-

  ombllifryl-6-D-galactoside, etc. as substrates for -Dalgalactosidase.

  Substances that can be dosed with the xactive ingredient

  must have at least two antigenic determinants in the molecule. As the substances that have been dosed

  by the sandwich method have at least

  Most of the substances which can be assayed by the conventional method can be assayed according to the present invention. As examples of these

  substances, they will be used to induce enzymes such as glu

  tampropeptide ('-GTP), alkaline phosphatase and

  transglycosidase; nrotic hormones such as hor-

  thyrotropic hormone (thyrotropin) (TSH), the luteinizing hormone

  health (gqondostimulin) (LH), human chorionic gonadothine (HCG), insulin, secretin and growth hormone. (GH); plasma proteins such as

  Fibrin Degradation Product (FDP), Protein C-

  reactive (CRP), the,. acid glycoprotein (AG), the

  antitrypsin (α-1-AT), antiplasmin (o.2-PI), 2-microglobulin (2MG) and immunoglobulin; of the

  c-aminoembryonic proteins such as at -foetropro-

- 12 -

  AFP), carcinoembryonic antigen (CEA) and embryonic ferritin; These are lymphocytes and bacterial cells, cell surface antigens, cell fractions, and so on. Given that according to the reagent of the invention, the sequence of reactions between the insolubilized antibody, the anti-4: 4 to be assayed and the labeled antibody is not at all limited, it is ecally possible to mix in advance

  the insolubilized antibody and the labeled antibody. More

  it is possible that an insolubilized antibody fixed on a support, ie plastic beads,

  etc. and a labeled antibody are contained in the emer-

  for example a test tube, or it is also possible to fix an antibody on the inner wall of a suitable element, for example a test tube, which itself serves as an insoluble support, then introduce a labeled antibody. The labeled antibody may be in the form of a solution or freeze-dried product from a solution, or

  present in the form of tablets, granules or powders.

  Apart from the insolubilized antibody and the anti-

  labeled body, the reagent of the invention may contain various auxiliaries to increase its usefulness. It can be for example a dissolution agent for the antibody

  marked (when used as a lyophilic product)

  philisé), a washing liquid for washing the solid phase after the reaction between the insolubilized antibody and the antigen to be assayed and after the other reaction of the labeled antibody, a substrate for measuring the enzymatic activity

  of the labeling agent when it is an enzyme) and a.

  reaction blocking agent for the enzymatic reaction.

  Examples illustrating the invention are described below. EXAMPLE 1 Reagent for AFP Assay a) Purified AFP Prloar3ticn containing 16.2 o of crude AFP extract from 5

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  liters of ascites from patients with hepatoma, by salting out, with arraoniuni sulphate

  between 45% and 70% saturation is collected).

  The crude extract is purified by

  Finished using 50 ml of Sepharose 4 B to which was fixed a rabbit anti-AFP antibody

  (1 mg / ml Sepharose) to obtain 924 mg of purified AFP.

  b) Repair of Monoclonal Anti-AFP Antibodies BALB / C is injected by injection

  subcutaneous 50 μg of the purified AFP produced in a) above

  with Freund's complete adjuvant. Immunization is repeated 4 times at one week intervals. The spleen is taken on the fourth day after the final immunization and the sphenic cells are collected. The sphenene cells are washed with Dulbecco's modified MEM culture medium (abbreviated as D-MEM hereinafter). We count 1 x 108

  of these cells and mixed with 1 x 107

  mouse loci (P3-NSI / 1-Ag 4.1). The mixture is subjected to cell fusion for 1 minute at 37 ° C in 1 ml of D-MEM containing 42.5% polyethylene glycol 1540 and 7.5% dimethyl sulfoxide. To the cells thus subjected to cell fusion, 20 ml of HAT medium (medium of RPMI-1640 containing hypoxanthine, aminopterin, thymidine and serum of beef serum) is added. After transferring 0.2 ml aliquots of the cell mixture into a 96-well microplate and culturing for 2 weeks, the antibody titers of the products are determined.

supernatants in the wells.

  Then, the cells of the wells where the antibodies were observed respectively to 40 ml of RPMI-1640 medium containing 10 'fetal bovine serum and BALB / c mouse thymocytes. We have t-ansvas; Aliquots of this were added to the cell suspension in two 96-well microplates and cultured for one week to obtain 9 hybridoma clones producing anti-AFP antibodies. We transfer to large scale cultures and submit

- 14 -

  liters of each of the supernatants to a chromato-

  affinity map using 50 ml of Sepharose 4 B on

  purified AFP (0.5 mg AFP / ml Sepha-

  pink). As a result, 4.2 to 11.6 mg of monoclonal antibodies designated as lots 1 to 9 are obtained. C) Identification of antigenic recognition sites i) Preparation of test tubes sensitized with the anti-antibody -AFP In polystyrene test tubes washed with PBS (physiological saline solution buffered with 0.05 M phosphate, pH 6.4), 2 ml of PBS solutions containing 0.2 mg of each of the batches are introduced respectively. 1-9 monoclonal anti-AFP antibodies. The tubes are then incubated at 56 ° C. for 20 minutes and washed with PEc for

  obtain sensitized test tubes.

  ii) Preparation of enzyme-labeled anti-AFP antibodies 5 mg of horseradish peroxidase are dissolved (Grade 1

  Boehringer Mannheim 6mbH, abbreviated HRPO

  after) in 1.0 ml of 0.03N sodium bicarbonate buffer

  and to this solution is added 0.1 ml of an ethanol solution.

  1-fluorophenol 2,4-dinitrobenzene 1% and then

  the mixture react for 1 hour.

  1.0 ml of a periodate solution is added to the mixture.

  0.06 M sodium and allowed to react for 30 minutes.

  1.0 ml of a 0.16 M ethylene glycol solution is then added and allowed to react for 1 hour. The reaction mixture is dialyzed against a 0.01 M sodium carbonate solution at pH 9.5. To the isultant solution, each of the nine batches of anti-AFP antibodies produced is added.

  b) above, in an amount of 5 mg and the reaction is

  at room temperature for 3 hours. Then 5 mg of sodium borohydride is added and allowed to react overnight. The reaction mixtures were dialyzed against 0.01 M PBS, pH 7.2, to obtain

  anti-AFP antibodies labeled with HRPO.

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  (iii) Identification of the recognition sites of the

ticène

  In each of the sensitized test tubes have the anti-

  anti-AFP body produced in i) above, 1.5 ml of PBS, 0.1 ml of AFP solution produced in a) above, which was diluted with PES at 11 ° C. / ml and 0.4 ml of HRPO-labeled anti-AFP antibody produced in (ii)

  above and one dilutes 10C times, then one reacts the

  start penceTnt 30 minutes. In the case of washing, the test tubes are washed with washing liquids, and 3 ml of the substrate solution containing

  1 mg / dl of o-phenylenediamine and 0.01% of hydrogen peroxide

  drogen. The enzymatic reaction was carried out for 30 minutes, and 0.05 ml of 4 M sulfuric acid was added to stop the enzymatic reaction. The absorbance of the reaction mixture is measured at 450 nm. In Table 3, the combinations inducing positive reations are marked

  of the sign + and those inducing no reaction of the sign -.

TABLE 3

  Antibody labeled with HRPO Insolubilized at 1 2 3 4 5 6 7 8 9

1 - - - + - + - + +

t

2 - - - + - + - + +

3 3- - - + - + - + +

4 + + + - + + + - -

- - - + - + - + +

6 + + + + + - + + +

7 - - - + - + - + +

I + + + - + + + - _

9 + + + - + + + -

  According to the differences in the recognition sites

  antigen, the 9 batches of mono anti-AFP antibodies.

* clones obtained in b) above can be divided into 3

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  the first group consisting of lots 1, 2, 3,5 and 7, the second group consisting of lot 6 and

  the third group consisting of batches 4, 8 and 9.

  These groups of antibodies are designated respectively as the antibodies AA-, / BI and / Ct1. d) Preparation of an active ingredient for the AFP assay

  in preparation test tubes szrs.bilisés with an anti-

  anti-AFP body according to the above-mentioned procedure using

the antibody / A / monoclonal.

  The monoclonal antibody is labeled with HRJ by the above procedure (c) (ii) and diluted 1:10 with PS5. In the sensitized test tube, 2 ml of diluted marbled antibody is introduced. After lyophilizing the contents of the test tube, seal for

  obtain a reagent for the assay of -AFP.

  e) Assay of AFP In the test tube sensitized with an anti-AFP antibody produced in d) above, 1.8 ml of PBS and 0.1 ml of the reference solution of AFP produced in a) above and diluted to concentrations of 1000 o, iLOG, 10, 1 and 0 ng / ml with the serum of a well-behaved subject, then another 0.1 ml of an antibody solution is added anti-AFP labeled with the HRPO produced in d) above in 2 ml of distilled water. Allowed to react for 20 minutes. After the reaction, the test tube was washed with physiological saline containing 0.005% of Teen 20 (hereinafter referred to as washing agent). Then, 3 ml of a solution of

  substrate for the enzyme containing 5 mg / ml of o-phenylene

  diamine and 0.01% u hydrogen peroxide and allowed to react for 10 minutes. To the reaction mixture, add

  O, G5 ml of 4 N sulfuric acid to stop the enzyme reaction

  matic. The absorbance of the reaction mixture is measured at 450 nm with a photometer. The rforence curve results

-reproduced in Figure 6.

  Exemnole 2 Reagent for the assay of the CEA a) Preparation of purified C E A 40 g of cancerous tissues of the coarse one homogenizes

  intestine with a homogenizer after addition of water

  tillée. The same amount of acid is added to the homogenate

  1.2M perchloric acid and extracted for 30 minutes with stirring. The centrifuged supernatant is dialysed for

  distilled water to obtain a crude CEA extract.

  Concentrate the crude extract at 10 ml, make it

  bir gel filtration using Sepharose 4B equilibrated with physiological saline solution and the first fraction is collected. Gel filtration is again carried out on balanced Sephadex G-200

  in the same way and we collect the second fraction.

  It is concentrated to 2 ml in which 135 Pg are obtained

purified CEA.

  b) Preparation of anti-CEA monoclonal antibodies Eight antibody-producing hybridoma clones

  anti-CEA are obtained using the purified CEA

  in (a) above, by a process similar to that

of Example 1b).

  Mice are immunized using 30 μg of CEA

  purified with each immunization. We inoculate 1 x 106 hybrid

  intraperitoneally to a BALB / c female mice intraperitoneally administered with 0.5 moles of pristane (2,6,10,14 tetramethylpentadecane;

  product of Wako Pure Chemicals, CO., Ltd.). Two weeks

  Later, ascites is collected. We submit

  ascites to a DEAE-cellulose balance chromatography

  bred with 0.01 M phosphate buffer at pH 7.0, and

  obtains an unadsorbed fraction as mono-

clonal anti-CEA.

  By an identification test of the recognition sites

  substantially as described in Example 1-c), the antibodies obtained can be classified in three

  groups, namely 5 lots, 2 lots and 1 lot which are

  respectively as anti-CEA IV, Zb8 and / or c) Preparation of test tubes sensitized with a

- 1a -

Anti CEA antibodies.

  Polystyrene test tubes are washed with PBS

  and introduced into each of them 2 ml of the anti-

  CEA [A] (1 mg / ml) nroduct enb) above, then reacted at 56 C for 20 minutes. After the reaction, the test tube is washed with PBS to produce a test tube

  sensitized with an anti-CEA antibody (A7.

  d) Preparation of a reagent for the assay of CEA An anti-CEA -BJ antibody labeled with HRPO is obtained

  using the anti-CEA antibody JbJ produced in b) above

  sus, according to Example 1-c-ii). The labeled antibody is diluted 1:50 with physiological saline and in each of the anti-CEA antibody-sensitized test tubes (A) obtained in c) above, 0.2 ml of the diluted antibody and lyophilized to form an

dosage of the CEA.

  e) CEA Assay The purified CEA prepared in a) above was diluted with the serum of a healthy subject at concentrations of 100, 30, 10, 3 and 1 ng / ml, respectively. To the reagent for the determination of the CEA produced in d) above, 0.2 ml of the diluted CEA and 0.8 ml of distilled water are added simultaneously and allowed to react for 20 minutes with stirring. After the reaction, the test tube is washed with

  the washing agent and 3 ml of a solution of

  stratum containing 300 mg / dl of phloretic acid and 0.01% of hydrogen peroxide. It is reacted for 10 minutes and then 0.1 ml of 5% sodium

  stop the enzymatic reaction. We then measure the intensity

  fluorescence at an excitation wavelength of 323 nm and a fluorescence wavelength of 420 nm at

  medium of a fluorophotometer. The reference curve results

  aunt obtsnue is shown in Figure 7.

Example 3

Active ingredient for the determination of HCG

  a) Preparation of the HCG subunit p

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  1 g of HCG (2000 IU / mg) is dissolved in 2 ml of a

  0.025 M phosphate (pH 5.6) and the solution is fractured.

  column chromatography of DEAE-Sephadex A-50 (3 g), previously equilibrated with the same buffer. The eluted fraction is collected with 0.05 M phosphate buffer.

  (pH 5.6) and dialysis entrusts distilled water to obtain

  a solution containing 308 mg of HCG purifies that

  then philises. 300 mg of lyophilized HCG are dissolved in 1 ml of 1 M urea (pH 4.5) and reacted for 1 hour.

  C. The reaction mixture is fractured by chromato-

  DEAE-Sephadex A-S0 column (2 g) previously equilibrated with a solution containing 0, U3 M glycine and 10 M1 urn. The eluted fraction is dialyzed with a solution containing 0.2 M glycine, 1 M NaCl and 1 M urea against physiological saline to obtain 146 mg of

subunit e of HCG.

  b) Preparation of anti-HCG-P antibodies 11 batches of anti-HCG-P antibodies are prepared according to the method of Example 2-b), except that WJistar strain rats are used as animals. for antigen administration instead of BALB / c mice. The antigen recognition sites are identified substantially according to the procedure (Example 1c). The resulting antibodies are classified into two groups, namely a group consisting of a lots of anti-HCG-p fA / and another group

  consisting of 3 batches of anti-HCG-fB7 antibodies.

  c) Preparation of beads sensitized with an anti-HCG antibody (insoluble support)

  A 1k .; ml of PBS containing 50 mg of the anti-

  HCG-S [A] produced in (b) above, 1CO

  polyethylene and reacted at 56 ° C. for 20 minutes.

  Then, the polylene bead is washed to obtain

  beads sensitized with an anti-HCG- / A / antibody.

  d) Reagents of anti-HCG-3 antibodies marred with an enzyme The procedure of Example 1-c-ii) was repeated using the following method:

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-2O-

  the anti-HCS- / BJ antibodies produced in b) above.

  After having diluted 200 times the volume with PBS, we obtain

  anti-HCG- / BI antibodies marcated with HRPO.

  e) Preparing the agent. Washing 9 g of NaCl and 50 μl of Tween 20 were accurately weighed and dissolved in deionized water to form 100 ml of solution. The solution is poured into a vial which is then capped;

  the washing agent is thus prepared.

  f) Preparation of the Enzyme Substrate 2.40 g of 5-aminosalicylic acid, 54.34 g of monopotassium phoshate and 5.72 g of disodium phosphate are mixed and sprayed in a mortar. 550 mg of the mixture are accurately weighed and poured into a vial. 1.5 ml of hydrogen peroxide (3G solution) is diluted with deionized water to obtain 100 ml of solution. 1 ml of the solution is introduced into an ampoule which is sealed by melting. The

  substrate for the enzyme is then-ready.

  g) Preparation of a blocking agent for the enzymatic reaction

  2 g of sodium azide are accurately weighed and dissolved in 100 ml of distilled water. 2 ml of the solution are introduced into an ampoule which is sealed by fusion to obtain a

  blocking agent for the enzymatic reaction.

  h) Preparation of a HCG Assay Reagent A CGG assay reagent is prepared by combining

  the materials produced in (c) to (g) as follows.

  1. Anti-HCG antibody-sensitized beads. 2. HRPG-labeled antiHCG-VBJ antibody. 3. Washing agent (10-fold concentrated solution).

  4. Substrate for the enzyme (5-amino-salicylic acid

  Hydrogen Peroxide) 5. Blocking Agent for Enzymatic Reaction i) Assay for HCG The following assay is performed using the reagent for the determination of HCG prepared in h) above. In a p

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  0.4 ml of the anti-HCG- (A) antibody, labeled with HRPO, was added to the test tube and a piece of a ball sensitized with an anti-HCG-1 / antibody was added. Finally add 0.1 ml of a reference solution obtained by diluting HCG, described in Japanese Pharmacopoeia, with the serum of a healthy subject at concentrations of 1EOC, 1000, 1GO, 10 and 1 mIU. ml / ml, and reacted for 15 minutes After the reaction, the washing agent is diluted to 10 times its volume with distilled water and the ball is washed with the diluted washing agent. 3 ml of the substrate solution prepared by dissolving the

  the entire substrate for the enzyme in 150 ml of distilled water

  and reacted for 30 minutes. 0.025 ml of the blocking agent is then added to stop the reaction and the absorbance of the reaction medium is measured at 500 nm. The resulting standard curve is shown in Figure B. -22

Claims (9)

PR E V E N D I C A T I 0 N S   A reagent for an antigen immunoassay based on the sandwich method, characterized in that   comprises an insolubilized monoclonal antibody and an   labeled monoclonal body, said monoclonal antibodies being respectively able to distinguish, and to bind to, the different antigenic determinants of the antigen to be dosed.   2. Reagent according to claim 1, characterized in that the support of the insolubilized antibody is used as support.   balls or a container made of plastic or glass.   Reagent according to claim 1, characterized in that an enzyme is used as a labeling agent for produce the labeled antibody.   4. Reagent according to claim 1, characterized in that a fluorescent substance is used as the agent of   labeling to produce the labeled antibody.   5. Reagent according to claim 1, characterized in that the labeled antibody and the insolubilized antibody are contained in the same container.   6. Reagent according to claim 1 or 5, characterized   in that the labeled antibody is contained in a recipe   piez made of plastic or glass that is used   as a carrier for the insolubilized antibody.   Reagent according to any one of the claims   1, 5 and 6, characterized in that the labeled antibody is lyophilized. 8. Reagent for antigen immunoassay based on the sandwich method, characterized in that   comprises an insolubilized monoclonal antibody and an   labeled monoclonal body, said monoclonal antibodies being respectively capable of distinguishing, and of binding to, the different antigenic determinants of an antigen to be assayed, and from 1 to 4 auxiliary agents selected from a dissolving agent, a washing agent, a   substrate and a blocking agent of the reaction.   -23- 9. An improved sandwich method for the immunological assay of antigens, characterized in that the improvement consists in simultaneously reacting the insolubilized antibody and the labeled antibody with an antigen to be assayed, said antibodies insolubilized and labeled antibody being monoclonal antibodies. capable   to distinguish, and to fix themselves respectively on, different   the antigenic determinants of said antigen.