DK158644B - PROCEDURE FOR MANUFACTURING AN ANTIBODY OR ANTISMAR, USING THIS PROCEDURE FOR THE OPERATION OF PATTERN CELLS WHICH ARE ABLE TO PRODUCT ANY ANTIBODY, OR ANTELMALLY ANTELMETELY - Google Patents

Description

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The invention relates to a method for producing an antibody or antiserum of high specificity to a first antigen having a desired antigenic determinant at low cross-reactivity with at least one other antigen containing at least one antigenic determinant structurally related to the desired antigen. antigenic determinant of the first antigen, by immunizing a mammal or culturing appropriate mammalian cells from that mammal after its immunization, and the invention further relates to the use of this method to obtain mammalian cells capable of producing such an antibody, and the use of this antibody or antiserum for immunoassay.

Various methods for immuno-assay of a variety of substances present in humans and animals are known, in which methods utilizing the competitive antigen-antibody reaction using a given amount of an antibody and different amounts of antigens. However, the known immunoassay methods generally have the disadvantage that even in cases where the reagent (antibody) used for the immunoassay is believed to be highly specific for the test substance (antigen), the assay specificity tends to be disturbed by the cross-reactants structurally related to the substance under study.

To overcome such a difficulty, it is advantageous to use an antibody having a higher specificity to the substance to be tested. For this purpose, e.g. has been proposed prior to its use to bind an antigen to a carrier protein at a suitable site in its chemical structure, thereby exposing the recognition site on the antigen to be recognized by the antibody on the surface of the carrier molecule.

Accordingly, such an exposure stimulates the formation of an antibody with low cross-reactivity. Thus, e.g. known to produce a specific antibody at 2

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use of polymers or copolymers of glutamic acid or lysine (DE Publication No. 2,608,096;

Kabat, E.A., Introduction to Immunochemistry and Immunology, Springer Verlag, Berlin 1971, pages 16-19; Beer, 5 O.G. et al., Experimental and Clinical Immunology, Springer Verlag, Berlin 1979, page 68). However, the known methods of this type are still unsatisfactory, as complicated procedures are necessary and, in addition, in some cases it is still difficult to avoid the production of cross-reacting antibodies.

The present invention is based on the realization that it is unexpectedly possible to reduce the amount of cross-reactive antibodies and, in addition, to obtain a desired antibody with a very high specificity against a e.g. for testing the present substance using a copolymer of D-glutamic acid and D-lysine (hereinafter referred to as D-GL) coupled with the cross-reactive antigen.

The object of the invention is to provide an improved antibody with a high specificity and poor cross-reactivity, an antiserum containing such an antibody, for producing such antibodies suitable cells (hereinafter referred to as strains) and a method for producing them. In addition, the invention will provide an immunoassay method using such antibodies or antisera.

The process of the invention is characterized by administering to the mammal a copolymer of D-glutamic acid and D-lysine coupled with the second antigen, and upon induction of a clear immunological tolerance, the mammal immunizes with the first antigen.

Various convenient embodiments of the method of the invention 35 may be used as set forth in claims 2-7.

In the method according to the invention it is possible to reduce the amount of cross-reactive antibody to a minimum and in addition to produce the desired antibody.

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3 or the antiserum with improved properties, as D-GL induces a substantially effective immunological tolerance in B cells, which serves as a precursor for the production of antibodies. When a mammal is treated by the method of the invention, the D-amino acids in the living body are not metabolized readily, and in addition, D-GL does not induce substantially any immunological response in the T cells of the living body. Therefore, when a living creature is treated by the method of the invention, such a hapten-D-GL conjugate is specifically bound to surface immunoglobulin receptors on B lymphocytes and renders these cells irreversibly tolerant.

The desired antibody may be obtained in the form of a pure antibody or in the form of an antiserum containing the desired antibody. The method of the invention leads to the production of cells of the same genetic constitution (hereinafter referred to as strains) in the living body, which cells can produce the desired antibodies. It is therefore possible to obtain the desired antibody by growing the strain thus obtained in the usual manner.

In this connection, it should be noted that the strains taken from the living body's living body to produce the desired antibody can be used even in the absence of the initiating living being, as it is known to fuse a suitable strain with a suitable tumor cell to obtaining a hybridoma, e.g. in that such a strain is fused with a myeloma cell, such as are reported in Nature, vol. 256, 495-497 (1975), and European J. of Immunol., vol. 6, 511-519 (1976) by Kohier et al., Nature, vol. 266, 550-552. (1977) by Milstein et al .; Nature, Vol. 266, 495 (1977) by Walsh). Such a hybridoma is transferred to another living creature, and the hybridoma cells proliferate to produce a large amount of the desired antibody. Thus, it is possible to use such a hybridoma as a new source of the desired antibody.

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According to the invention there is provided a method for immunoassaying a substance which serves as an antigen, wherein the first antigen, i.e. the test substance is reacted with an antibody or antibody prepared according to the invention, the first antigen is separated from the immune complex thus obtained and the activity of the first antigen determined.

The immunoassay may be performed in the usual manner, e.g. by radioimmunoassay or using non-isotopic labels, e.g. enzymes, free radicals, cells, viruses, metal ions as well as fluorescent and chemiluminescent groups. In the radioimmunoassay, the assay can be performed even in the presence of a cross-reactive antigen.

According to the invention, it is preferred to use D-GL having a molecular weight of from about 27000 to approx. The molar ratio of D-glutamic acid to D-lysine is preferably in the range of 70:30 to 30:70, e.g.

20 60:40. Copolymers of this type are commercially available, but it is also possible to produce such copolymers in the usual manner, e.g. by copolymerization of N-carboxyl anhydride of γ-alkyl-D-glutamate and N-carboxyl anhydride of ε-N-carbobenzyloxy-L-lysine in the presence of a suitable amine followed by removal of the protecting groups.

Various naturally occurring substances can e.g. is used in the preparation of the antibody for immunoassay. Examples of preferred materials for this purpose include steroids, their glucuronates and sulfates thereof, catecholamines, peptides, their subunits and related fragments thereof, and various pharmaceutical agents.

Particularly preferred examples of steroids include testosterone (hereinafter referred to as T), 5α-dihydrotestosterone (hereinafter referred to as DHT), androsterone, etiocholanolone, progesterone, 17α-hydroxyprogesterone, pregnenolone, dehydroepiandrosterone, estradiol, estradiol , cortisol, cortisone, corticosterone, 11-deoxycortisol, cholic acid, deoxycholic acid, lithocholic acid and conjugated compounds thereof.

Catecholamines are e.g. dopamine, norepinephrine, epinephrine and the like.

Peptide hormones are e.g. gastrin, cholecystokin pancreozymine, insulin, proinsulin, glucagon, follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (HCG), somatostatin, thyroid stimulating hormone (TSH) and their related units and thus .

Pharmaceutical agents are e.g. 1-propranolol, * which is a β-blocking agent, and 1- or d-cyclazocin, which are analgesics.

Eg. when an anti-T antibody or antiserum 15 is produced by conventional immunization procedures, DHT acts as the cross-reactive antigen since their structures are very similar. Similarly, T acts as a cross-reactive antigen when preparing an anti-DHT antibody or antiserum. In this way, 20 different steroids act as cross-reactive antigens on other steroids.

The aforementioned substances are generally haptenes or the like (i.e., substances capable of being combined with an antibody but unable to induce an immune response, or capable of inducing a weak immune response only if they are not coupled with a carrier prior to their administration to a living being, thus it is necessary to couple them to a suitable carrier to induce the formation of an antibody, for example, to induce an antibody specific to a particular antigen such as anti-testosterone (hereafter referred to as anti-T antibody), this antigen must be coupled with a suitable carrier and used for immunization, however, the anti-T strains and antibodies thus obtained generally have a strong cross-reactivity with substances having an analogous structure, such as DHT. According to the invention, it is possible to specifically inhibit the formation of such cross-reactive anti-DHT stains and antibodies by treating

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6 shows the living being with a substance which is the product of coupling a copolymer of D-GL with a cross-reactive antigen, in this case DHT.

Furthermore, it has also been found that an antibody capable of responding with the specific determinant to the desired antigen was obtained when a living creature is treated with a conjugate of D-GL and a peptide analogous to with said antigen, or a peptide fragment corresponding to the desired antigen.

An example showing such an advantage of the invention includes the case of antibodies to an octa peptide, which is a C-terminal peptide containing eight amino acids from cholecystokinin pancreozyme (hereinafter referred to as CCK), which is a gastrointestinal hormone. the structure (amino acid sequence) of gastrin (human), CCK as well as their fragments is shown in Table 1.

Table 1

Gastrin (human): (Pyro) Glu-Gly-Pro-Trp-Leu-Glu-Glu-20

Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met- (so3h)

Asp-Phe-NH2 CCK-33: Lys-Ala-Pro-Ser-Gly-Arg-Val-Ser-Msb- ^ Ile-Lys-Asn-Leu-Gln-Ser-Leu-Asp-Pro

Ser-His-Arg-Ile-Ser-Asp-Arg-Asp-

Tyr-Met-Gly-Trp-Met-Asp-Phe-NH9 -3h CCK-8-P: Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH-

SO H

30 3

Pentagastrin: Gly-Trp-Met-Asp-Phe-NH2

It has recently been found that CCK octapeptide (hereinafter referred to as CCK-8-P) and its reactive receptor 35 are also present in the brain, and the study of this hormone's function as a neurotransmitter as well as the secretions it promotes, and its effect on various gastrointestinal disorders is of interest. So it's-

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7 it is important to provide an antibody capable of specifically reacting with CCK-8-P.

Antibody-forming strains and antibodies capable of specifically reacting with CCK-8-P can be obtained according to the invention by providing an animal conjugates of D-GL with pentagastrin (i.e., the five amino acids found at the C-terminus). on gastrin and CCK-8-P) to inactivate strains producing antibodies that cross-react with pentagastrin and / or gastrin-like compounds, and then immunize said animal with CCK-8-P.

Thus, according to the invention, it is also possible to prepare gastrin-specific antibody by inhibiting the formation of cross-reacting strains with CCK-33 by treating an animal with a conjugate of D-GL and pentagastrin, which is the C-terminal fragment of the desired antigen and then immunize said animal with gastrin.

As will be apparent from the following examples, it may also be possible to obtain a desired specific antibody by giving an immune animal a conjugate 20 of D-GL with a cross-reactive peptide, even in the case where specific antigenic determinants are not without further can be removed from the entire peptide by peptide fragmentation (cf., e.g., Attasi MZ, Immunochemistry, vol. 15, pages 909-936 (1978).

Such a conjugate of D-GL and a cross-reactive antigen can be obtained either (1) by coupling the antigen directly with D-GL or (2) by coupling the antigen indirectly with D-GL, forming a bridge between antigen and D -GL. For this purpose, various derivatives of the antigen, especially steroid antigens, have been prepared.

These derivatives are e.g. described as oxime derivatives, succinyl derivatives, and chlorocarboxylic acid derivatives by Erlanger et al. [I.e. B.F. Erlanger et al., J. Biol. Chem., 228, 713 (1957)], as carboxylmethylthioether derivatives of 35 a. Weinstein et al. [Steroid, 2Q, 789 (1972)], as carboxyl methyl ether derivatives of P.N. Rao et al. [J.

Steroid Biochem., 9, 539 (1978)], etc. Preferred coupling methods are e.g. the carbodiimide method, the mixed

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8 anhydride method, the Schotten-Baumann method and the isoxazolium method.

When an N1 + group is introduced into such a compound, the coupling reaction is carried out by the glutaraldehyde method and when an N1 + or SH group is introduced into such a compound, the reaction can be carried out using an m-maaleinimidobenzoyl-N-hydroxysuccinimide ester. , succinimidyl 4- (N-Faleimodomethylcyclohexane) -1-carboxylate, succinimidyl-4- (p-maleinimidophenyl) butyrate and the like or N-succinimidyl-3- (2-pyridyldithio) propionate, N-succinimidyl (4-azidophenyldithio) propionate and the like.

In addition, various other methods conventionally used to link peptides with other substances within the peptide chemistry can also be used to link D-GL with an antigen.

The antigens which can be used for the purposes of the invention are generally haptens or the like, and thus it is necessary to couple the antigen with a suitable carrier used in conventional immunization methods before use. Preferred carriers for this purpose are e.g. "keyhole limpet" haemocyanin (KLH), γ-globulin and serum albumin from various animal species used for immunization, such as e.g. humans, goats, calves and the like.

The antigens, their subunits, or related fragments, should be coupled with a carrier protein in a manner similar to that used to couple a cross-reactive antigen, its subunits, or related fragment with D-GL. Thus, the coupling can be performed directly or indirectly. In the latter case, the same intermediate used for coupling D-GL with the cross-reactive antigen should be used for coupling the desired antigen with a carrier protein.

The immunization treatment may be performed in the usual manner. However, it is preferred to immunize small animals, such as mice, with antigen in single doses of 1-100 µg or larger animals, e.g. rabbits, with single doses of 0.1-1 mg, which can be repeated, e.g. 2-5 times with 2-4 weeks in-

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9 intervals. Usually, 2-3 days before the primary immunization, the animal may be given a conjugate of D-GL and a cross-reactive antigen, although in some cases the D-GL conjugate may also be given after the primary or secondary immunization depending on the type of cross-reactive antigen. The dose of the D-GL conjugate with cross-reactive antigen may vary depending on the combined ratio of the cross-reactive antigen (hapten or the like) to D-GL, coupling sites, intermediate types and the like, but it is preferably in the range 100-500 µg / day. smaller animals, e.g. mice, or 2-10 mg / larger animals such as e.g. rabbit. The D-G1 conjugate can be dissolved in a saline solution and administered intraperitoneally.

Since it is advantageous to couple the cross-reactive antigen with D-GL to take the same form as that used for the immunizing antigen with carrier, it is preferred for this purpose to use the same intermediate for the formation of respective combinations.

As described above, it is possible to induce a substantially effective immunological tolerance specific to a particular cross-reactive antigen by giving an animal a conjugate of D-GL and the cross-reactive antigen. Subsequently, the animal is immunized with the desired antigen containing the specific desired antigenic determinants. In this way, it is possible to obtain a strain capable of producing an antibody having low cross-reactivity and excellent specificity to the relevant specific antigenic determinants, and an antiserum containing said antibody.

It should be noted that the above results have not only been tested on smaller animals, such as mice, but also on larger animals such as rabbits. It is significant that the same results have been obtained in both 35 smaller and larger animals, despite the differences that could be expected for the animal species. In this connection, a substantially greater amount of antibody is obtained by use

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10 of larger animals such as rabbit than using relatively smaller animals such as mice.

The invention is particularly advantageous since it was previously difficult to prepare an antibody capable of distinguishing analogous structures such as T and DHT.

As a result of increased productivity of the specific antibody producing strain, it may be practically possible to separate and isolate a specific strain.

10 It was previously known in the literature that the probability of separating and isolating a strain with the ability

C

to recognize a specific antigen was approx. 1/10 to 7 1/10. This means that such separation and isolation was in practice impossible.

Thus, by the method of the present invention, it is possible to obtain without a doubt a specific antibody-producing strain capable of specifically distinguishing a desired antigenic determinant from structurally related antigenic determinants, even by immunization with an antigen which contains cross-reactive determinants. The method of the invention makes it possible to obtain such specific antibodies with greater probability, and as a result it is also possible to increase the probability of production of such specific antibody producing strains.

The method of the invention can be used in any antibody producing method by modifying the method of coupling D-GL with a cross-reacting antigen, the types of the coupling product, and the like.

According to a further feature of the invention, the invention provides a simple method for determining a given substance, the amount of which is unknown in the sample, using an antibody or antiserum obtained by the method described below.

The reagents and assay method used in the Examples below are explained as follows.

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11 (1) Synthesis of DHT-3- (o-carboxymethyl) oxime [hereinafter referred to as DHT-3-CMO]: (A) Scheme of reaction:

OH

\ rh ^ -1 + H2N - o - ch2 - c

OH

10

H OH

r ^ fS

Baking valine ^ I

NaOH / ethanol

C - CH_ - O - N / 2 H

HAY

(B) Materials used: 3 H-DHT (Radiochemical Center, Amersham) 6 µl (0.015 µg as DHT) DHT (Sigma Chemical Co., USA) 0.3 g (1 mmol) of O-Carboxymethyl hydroxylamine hemihydrochloride (Tokyo Kasei Kogyo KK, Japan) 0.3 g (2.7 mmol) 2N NaOH 1.25 ml

Ethanol 15 ml (C) Procedure: The materials were placed in a round bottom flask equipped with reflux apparatus and refluxed under heating for 3 hours. The reaction mixture was added with distilled water (45 ml) and the pH of the solution was adjusted to 8 with a dilute NaOH solution. The solution was transferred to a separatory funnel and added ethyl acetate (about 30 ml) and shaken. The ethyl acetate layer was then removed to remove the unreacted DHT. The aqueous layer was cooled by the addition of ice and acidified

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to obtain a pH of approx. 4 with dilute hydrochloric acid.

The DHT-3- (o-carboxymethyl) oxime thus prepared was extracted by the addition of cold ethyl acetate.

Anhydrous sulfate was used to dry the ethyl acetate layer, which was further removed by evaporation.

The resulting residue was recrystallized in hot methanol to give the desired product.

(2) Synthesis of testosterone-3- (o-carboxymethyl) oxime (hereinafter referred to as T-3-CMO):

The procedure of (1) was repeated to obtain the desired product except that H-T and T were used instead of H-DHT and DHT in the same molar amounts, respectively.

(3) Synthesis of hapten-D-GL:

(A) Synthesis of DHT-3- (o-carboxymethyl) oxime-D-GL

[hereinafter referred to as DHT-3-D-GL]: In accordance with the mixed acid anhydride method by Erlangen et al.

Scheme:

20 OH

I I + ClCOO-C 4 Hg-i HOOC-CH 2 -O-N 2 -

^ H

OH

tC4H3> 3N. / \ Λ / - ^

30 -ϊ O.

Dioxane ^ X. J

C-CH - 0-N / 2 H

i-C4H9-C020 oh ”. rVr1

D-GL II 1 1 J

____. D-GL-NH-C-N-CHo0 T ^^ \ ^

* 2 H

Dioxane / water

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13

Materials used: DHT-3-CM0 containing DMT-3-CM0 labeled with 3 H tracer 20 mg

Dioxane (dried) 6 ml 5 Tri-n-butylamine 20 yl

Isobutyl chloroformate 10 μΐ

Distilled water 6 ml D-GL (Mw = 49,000) 30 mg IN NaOH 150 μΐ 10 Procedure: DHT-3-CMO (20 mg) was dissolved in dry, straight dioxane (1 ml). The mixture was stirred with the addition of tri-n-butylamine (20 μΐ) and then stirred at 11 ° C for 5 minutes. Then, isobutyl chloroforma (10 µΐ) was added to the solution, which was subjected to reaction at 11 ° C for 1 hour with stirring. Separately, D-GjL (30 mg) was dissolved in distilled water (6 ml) and the mixture was added with 1 N NaOH (150 μΐ). The mixture was added dioxin. (5 ml) gradually. This D-GL solution was added to the said reaction mixture at one time with stirring. 10 minutes thereafter the pH of the mixture was adjusted to 6.5-7.0 by adding drops of 1 N NaOH solution and the solution was stirred at ca. 10 ° C for approx. 2 hours. Then, the reaction solution was dialyzed against water at 4 ° C overnight.

The next morning the pH of the solution was adjusted to 4.5 with a dilute hydrochloric acid to form precipitates. The precipitation was terminated by allowing the solution to stand at 4 ° C for 7 hours. The reaction mixture was centrifuged at 4 ° C for 20 minutes (3,000 rpm) and the supernatant removed. The precipitated substance was added with distilled water (10 ml) and the pH adjusted to approx. 7.0 with 1 N NaOH to dissolve the precipitated substance. Then, the solution was dialyzed against distilled water at 4 ° C overnight, followed by lyophilization.

In accordance with the radioactivity per 1 mg of 3 H-DHT-3-CMO used as a tracer and the radioactivity per 1 mg of reaction product was determined the number of

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14 DHT combined with 1 mole of D-GL. The molar ratio of DHT to D-GL in the reaction product was 30: 1.

When the amounts of water and dioxane used were equal to the corresponding amounts calculated on the basis of the 5 mixed anhydride method by Erlangen et al. [J. Biol.

Cheiru, 228, 713 (1957)] relative to BSA, the mixture was gelled. Thus, large amounts of water and dioxane were added to the mixture to avoid the gelation of D-GL therein and further to carry out the reaction appropriately.

Hereby the conjugation of D-GL with the oxime was made possible by the mixed anhydride method, so that a sufficient amount of oxime was combined with D-GL.

(B) Preparation of testosterone-3- (o-carboxymethyl) oxime-D-GL [hereinafter T-3-D-GL]: The same procedure as above was performed except for the use of T-3 oxime containing H-labeled T-3 oxime as tracer to obtain T-3-D-GL (molar ratio of T: D-GL = 30: 1).

(4) Synthesis of hapten-KLH: (A) Preparation of DHT-3- (o-carboxymethyl) oxime-KLH [hereinafter referred to as DHT-3-KLH]:

Materials used: 3 DHT-3-CMO {containing H-labeled oxym as tracer 12 mg 25 dried dioxane 3.0 ml tri-n-butylamine 10 yl isobutyl chloroformate 5 yl distilled water 4 ml KLH (Mw = 100,000) 150 mg N NaOH 75 yl

Procedure:

The same procedure as described above gave DHT-3-KLH (mole ratio DHT: KLH = 10: 1).

(B) Preparation of testosterone-3- (o-carboxymethyl-35-yl) oxime-KLH [hereinafter T-3-KLH]:

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15

Materials used: 3 T-3-CMO (containing H-labeled oxime as trace element) 40 mg dried dioxane 7 ml 5 tri-n-butylamine 40 yl isobutyl chloroformate 20 yl distilled water 10 ml KLH 300 mg 1 N NaOH 300 yl 10 Procedure:

The same procedure as above gave T-3-KLH (molar ratio T: KLH = 8: 1).

(5) Radioimmunoassay procedure:

Reagents: 3 1) H-T standard solution: 3 1,2-H-T (58 Ci / mmol) was diluted with ethanol to give an ethanol solution containing 20,000 dpm / 10 µl (45 µg as T).

2) "H-DHT standard solution: 5α-Dihydro-1,2,4,5,6,7- HT (114 Ci / mmol) was diluted with ethanol to give an ethanol solution containing 40,000 ppm / 10 yl (45 pg as DHT).

3) 0.05 M tris buffer solution (pH 8.0) containing 0.05% calf serum albumin (hereinafter referred to as BSA) 25 and 0.1% calf serum γ-globulin (hereafter referred to as BGG).

4) Saturated ammonium sulfate solution.

5) Unlabeled T standard solution.

6) Unlabeled DHT standard solution.

Procedure 1: 30 Determination of the anti-testosterone antibody titer: 3

A standard H-T solution (10 µl) was placed in a glass tube and dried by evaporation. To this was added an antiserum (0.2 ml, diluted stepwise with a tris buffer solution) and mixed well. The mixture was allowed to stand at room temperature for 2 hours and then a saturated ammonium sulfate solution (0.2 ml) was added. The solution was stirred well and centrifuged (3000 rpm).

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16 minutes) for 20 minutes to obtain the above liquid, 0.2 ml of which was placed in a counter glass. The radioactivity of the sample was determined after the addition of a scintillator [2 ml, prepared by dissolving PPO (2.0 g) in 5 toluene (1 liter)].

Procedure 2:

Determination of cross-reactivity of anti-testosterone antibody with DHT: 3 10 μΐ of a H-T standard solution was placed in 10 glass tubes and the tubes were divided into two groups. Unlabelled standard solutions of T and DHT, respectively, were added to 3 of the two groups of tubes containing H-T standard solutions. However, the added amount of unlabelled steroids was incrementally increased each time. All the mixed solutions were dried by evaporation. To each of them, 0.2 ml of antiserum was added and mixed well. Antiserum was used at the dilution which bound 60% of 3H-T (45 µg).

Procedure 3: 20 Determination of the anti-DHT antibody titer:

The same procedure as described in procedure 1 was used using H-DHT instead of 3 H-T.

Procedure 4: 25 Determination of anti-DHT antibody cross-reactivity with T:

The same procedure as described in procedure 2 was used using H-DHT instead of 3H-T.

Example 1

Preparation of anti-testosterone specific antibody with low cross-reactivity (mouse):

Three groups of mice (each group consisting of 4-5 female mice, the C57BL / 6 strain, 8-10 weeks old) were used in this example. Each mouse from the first group (control group) was given exclusively a saline solution and no DHT-3-D-GL was given. 3 days before a primary

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In immunization with T-3-KLH, DHT-3-D-GL (500 µg) was given to each mouse in the other group by i.p. injection.

All mice were administered (ip) T-3-KLH (100 µg / mouse) in saline with Freund's complete adjuvant 5 and 3 weeks thereafter, further administered the same amount of T-3-KLH in saline along with Freud's incomplete adjuvant. They were also administered the same amount of T-3-KLH in saline after 5, 7, 9 and 17 weeks.

3 days before the secondary immunization and 3 days 10 before the tertiary immunization (5 weeks after the primary immunization) with T-3-KLH, in each case, DHT-3-D-GL (500 µg) was administered to the mouse the third group.

Serum was collected at the following 2-week 15-interval intervals from the mice's retroorbital plexus to determine antibody titers and cross-reactivity. The cross reactivity was determined by Abraham's method. The results are shown in Table 1.

Table 1

20 (A) Titre of anti-testosterone antibody and (B) its cross-reactivity with DHT

Weeks Group 1_Group 2_Group 3_ (not pre-treated (not pre-treated) with DHT-3-D- treated 25 _GL (500 µg / ip)) _ 9 A 3331 1004 *** Formation of antibody + B 30.3 +4.9 7.1 + 9 * 11 A 4320 1711 30 B 54.1 + 24.1 11.2 + 6.2 13 A 2886 819 B 43.0 + 12.5 7.9 + 4.5 19 A 1494 461 B 30.8 + 10.1 '7.4 + 4.1 35 Notes: - 1 - The titer is expressed by the reciprocal number for the dilution of serum capable of combining 50% of 3 H testosterone (45 pg).

18

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* 2 - Cross-reactivity (%) is expressed by [the amount (ng) of testosterone needed to inhibit binding by 50% / amount (ng) of DHT required to inhibit binding by 50%] x 100%.

3 - Mean value of the antibody titer is expressed as the geometric mean and the cross reactivity is expressed as the arithmetic mean + standard release.

4 - DHT-3-D-GL was not used for pretreatment, but 10 was administered after the primary immunization.

As can be seen from this table, the antibody titer in the second group was slightly lower than the titer of the first (control) group and the kinetics of antibody formation in the second group corresponded to the kinetics of the control group. On the other hand, no significant antibody formation was observed in the third group throughout the period. The cross reactivity with DHT for the second group pretreated with DHT-D-GL was significantly less than the cross reactivity for the control group.

The relationship between the antibody titer and the cross-reactivity of the antibody obtained for each of the test mice was plotted.

However, although the data are not shown here, no significant association was found between the antibody titer and the cross-reactivity. In general, treatment with DHT-3-D-GL was found to result in a decrease in cross-reactivity with DHT, and this did not correlate with the decrease in antibody titer. In other words, a decrease in cross-reactivity does not result in a significant decrease in the titer.

From these facts, it is postulated that when the clones having a cross-reactivity with DHT are removed by pretreatment with DHT-3-D-GL, the subsequent immunization with T-3-KLH can selectively enhance the formation of an - 3 b Substance-forming clones with a higher specificity to T. Such an ability for specific formation of the antibody having a lower cross-reactivity in

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The method according to the invention is important for practical purposes.

Example 2

Preparation of specific anti-DHT antibody with 5 small cross-reactivity (mice):

Five groups of mice (each group consisting of 5-7 female mice, the C57BL / 6 strain, 8 weeks old) were used for this example. Mice from the first group (control group) were not administered T-3-D-GL and a primary immunization was performed using DHT-3-KLH. 3 and 5 'Weeks thereafter, a secondary and a tertiary immunization was performed followed by further immunization performed at 2 week intervals. Mice from other groups were also immunized in this way simultaneously with the mice 15 in the control group.

Three days before the primary immunization with DHT-3-KLH, the mice in the second group were administered T-3-D-GL (500 µg / mouse) by ip. injection. As a second control group, mice of the third group were administered DHT-3-D-20 GL (500 µg / mouse) by i.p. injection 3 days prior to primary immunization with DHT-3-KLH.

Three days before the secondary immunization performed 3 weeks after the primary immunization with DHT-3-KLH, the mice in the fourth and fifth groups were administered T-3-D-GL and DHT-3-D-GL, respectively ( every 500 µg / mouse (ip.)).

From 7 weeks after the primary immunization, serum was collected from each mouse in the five groups at 2-week intervals to determine the antibody titer and cross-reactivity in the serum. The results obtained using the serum collected 13 weeks after primary immunization are shown in Table 2.

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20

Table 2

Group Antibody Titer Cross Reactivity 1 3457 90.3 + 12.9 2 1286 38.2 + 14.6 5 3 1079 86.3 + 23.7 4 0 5 0 * 1 and '* 2: Cf. footnote to Table 1.

Table 2 suggests that anti-DHT antibody with low cross-reactivity was obtained by pretreatment with T-3-D-GL (the second group) - In this example no significant formation of anti-DHT antibody in the fourth and fifth groups was observed. that had been treated with T-3-D-GL or DHT-3-D-GL, after the primary immunization.

In the third group, the mice were pretreated with DHT-3-D-GL and then immunized with DHT-3-KLH, and the resulting anti-DHT antibody showed a high cross-reactivity with T as in the case of the control mice, although (Nearly the same order of magnitude 2.0 was observed on antibody t in the second and third groups. From these results, it can be verified that an anti-DHT antibody with low cross-reactivity can be effectively obtained. D-GL.

Example 3 Preparation of anti-T or anti-DHT antibody (rabbit):

Six groups of female rabbits (each group consisting of 2 rabbits, weight approximately 3 kg) were used.

Three days before the primary immunization with 100 µg of 30 DHT-3-KLH emulsified in Freund's complete adjuvant, the rabbits in the first group (ip.) Were administered T-3-D-GL (10 mg / rabbit) and 3 weeks thereafter the rabbits subcutaneously administered a secondary immunization with 200 µg of DHT-3-KLH in Freund's incomplete adjuvant.

They were then booster-injected subcutaneously four

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21 times in a row with 200 µg DHT-3-KLH in alternate Freund's complete adjuvant and Freund's incomplete adjuvant at monthly intervals.

The control rabbits (second group) were also immunized with DHT-3-KLH in a manner similar to that used for the first group. However, these were not administered T-3-D-GL. The rabbits in the third group were administered (ip.) T-3-D-GL (10 mg / rabbit) 3 days before the secondary and tertiary immunization with DHT-3-KLH, performed 3 and 7 weeks, respectively. primary immunization.

The rabbits in the fourth group were administered (ip.) DHT-3-D-GL (10 mg / rabbit) 3 days prior to the primary immunization with T-3-KLH. Immunization with T-3-KLH was performed in a manner similar to that used for the first group.

In the fifth group (control group), the rabbits were immunized with T-3-KLH in a manner similar to that used for the fourth group. However, these were not administered any DHT-3-D-GL.

In the sixth group, the rabbits were administered (ip.) DHT-3-D-GL (10 mg / rabbit) 3 days prior to the secondary and tertiary immunization with T-3-KLH performed 3 and 7 weeks, respectively, after the primary immunization. .

25 Days after each of the above booster injections, serum was collected from each rabbit and determined to obtain the following results: In the third and sixth groups no significant antibody formation was observed throughout the period. An-30 ti-DHT and anti-T antibodies obtained from the second and fifth groups (control groups) showed essentially the same reactivity with the haptenes used for immunization and cross-reactive hapten. Ie anti-DHT antibodies from the rabbits in the second group showed a 100% cross-reactivity with T, and anti-T antibodies from the rabbits in the fifth group showed a 95.2% cross-reactivity with DHT. On the other hand, the rabbits from the first and fourth groups gave significantly low cross-reactivity, and

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The 22 rabbits anti-DHT antibodies in the first group showed an 11/9% cross-reactivity with T, and the rabbits anti-T antibodies in the fourth group showed a 22.0% cross-reactivity with DHT.

Example 4

Determination of T and DHT in human blood using specific anti-T and anti-DHT antibodies from rabbits: 1) Anti-T and Anti-DHT antibodies with low cross-reactivity were obtained from Group 1 rabbits and 4 in the above example. The following studies were performed to investigate the practical utility of these antibodies for the determination of T and DHT present in human serum. To this, given amounts of T and DHT, respectively, were added to human serum samples / and the association between the amount added and the amount of T or DHT determined by radioimmunoassay using said antiserum was investigated. To determine the level of T present in human serum, T (1/2 20 and 4 ng) or DHT (0.1, 0/2 and 0.3 ng) was added to total serum (1 ml, collected from females and with low T value) then added ^ HT (2000 ppm) or ^ H-DHT (2000 ppm) to determine the yield in the extraction step. In each case, the mixture was mixed well and a mixture of hexane / ether (3: 2/3 ml) was added followed by shaking for 1 minute with a vortex mixer. Then, the tube was left in a dry ice / acetone bath for 10 seconds and the organic solvent layer was transferred to another tube. The organic solvent was evaporated and the residue was added with ethanol (2 ml) with shaking. Part of the ethanol solution was placed in a small test tube. The amount of solution transferred to the test tube was adjusted to allow the determination of T using an inhibition curve 35 calibrated with the above antiserum. The solution was dried by evaporation and subjected to radioimmunoassay using the low cross-reacting anti-oxidant.

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23 T antibody obtained by DHT-3-D-GL treatment.

On the other hand, an ethanol solution (0.5 ml) was placed in a glass and dried by evaporation. The residue was added to a scintillator and counted to determine the yield in the extraction step. This yield ratio was used to correct the value obtained by the radioimmunoassay.

To determine the level of serum DHT present, total female serum (1 ml) was added to 10 DHT (0.2 and 0.5 ng) or T (0.2 and 0.5 ng) and then added to HT (2000 ppm) or ^ H-DHT (2000 ppm) to determine the yield. The mixture was added hexane / ether (3: 2, 3 ml) and extracted in a similar manner as described above. The extraction residue was added to ethanol 15 (2 ml) and part of the ethanol solution was placed in a small test tube. The amount of solution in the tube was adjusted to be sufficient to allow the determination of DHT using a DHT inhibition curve obtained using the above antiserum.

The solution was dried by evaporation and subjected to radioimmunoassay using the low cross reactivity antiserum obtained by T-D-GL treatment. On the other hand, the ethanol solution (0.5 ml) was placed in a glass and ethanol was removed by evaporation. The residue was added to the scintillator and counted to determine the yield in the extraction step. This yield ratio was used to correct the value obtained by the radioimmunoassay.

As a result, it was found that when using a small cross-reactivity anti-T antibody to measure T (1, 2 and 4 ng) added to a normal female serum with an initial T value of 0.30 ng, the measured T-value was 1.15, 2.30 and 4.60 ng, respectively.

On the other hand, when DHT (0.1, 0.2 35 and 0.3 ng) was added to female serum instead of T to examine the effect of DHT on the amount of assay-determined T, no significant influence was found. and the T level in normal female serum was almost unchanged.

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In a separate experiment, DHT (0.2 and 0.5 ng) was added to normal female serum and the amount of DHT in the serum was determined using a low cross reactivity anti-DHT antibody. It was found that the amount of DHT 5 in the normal female serum prior to addition was 0.21 ng and then after addition changed to 0.41 and 0.72 ng in each case. These values were sufficiently consistent with the added amounts of DHT.

On the other hand, when T (0.2 and 0.5 ng) 10 was added to normal female serum instead of DHT to examine the influence of Τ on the assay-determined DHT level, no significant change in DHT level in normal female serum by the addition of T.

It is seen that T and DHT levels in the serum can be accurately determined using the low cross-reactivity antibodies described in Example 3.

2) Assay of a sample containing a mixture of specific antigen and cross-reactive antigen: The DHT level in human blood was again determined using a low rabbit cross-reactivity antibody as described above. In each case, DHT and T (5 and 10 ng, 10 and 5 ng, or 10 and 10 ng, respectively) were simultaneously added to female total serum (0.1 ml), and the assay was performed in a manner similar to that of is described by (1) above. As a result, as shown in Table 3, it was found that even a large amount of T, i.e. a cross-reactive antigen present in the sample, the amounts of DHT were 5.67, 10.17 and 9.20 ng in each case. Thus, it can be said that DHT can be accurately determined using the low cross-reactive anti-DHT antibody of Example 3 when even a large amount of T, which is the cross-reactive antigen, is present in the sample.

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25

Table 3

Added amount of DHT Added amount of T DHT found _ (ng) _ (ncf) _ (ng) _ 5 10 5.67 5 10 5 10.17 10 10 9.20 Similarly, T and DHT (5 and 10 ng, 10 and 5 ng or 10 and 10 ng) to the serum (0.1 ml), and T levels were determined using the low cross-reactive anti-T antibody. In each case, the amounts of T found were 6.80, 10.3 and 10.5 ng, respectively, as shown in Table 4, which shows that when the above-mentioned low cross-reactivity anti-T antibody is used, T is accurately determined in close proximity. -15 of DHT, a cross-reactive antigen.

Table 4

Amount added T Amount added DHT T found _ (ng) _ (ng) _ (ng) _ 5 10 6.80 20 10 5 10.3 10 10 10.59 3) Human sample assay (direct method):

The term "direct method" denotes the assay method which does not require prior separation of T 25 and DHT. In comparison, values obtained by a conventional method are also stated. In the latter method, T and DHT are separated by paper chromatography before determination.

3.1) Assay of T in human serum: 30 (A) Sample preparation: Rural serum (each 0.1 ml) and female serum (each 0.5 ml) were placed in respective test tubes and HT (every 3000 ppm) was added. determining the yield in the extraction step or the extraction chromatography step. The male serum was added with distilled water (0.5 ml each). A hexane / ether mixture (3: 2, each 3 ml) was added to the serum samples and mixed well

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26 using a vortex mixer for 1 minute. The tubes were then placed in a dry ice / acetone bath for 10 seconds to freeze the serum fractions. The organic solvent layer was transferred to another test tube and the organic solvent was removed by evaporation.

(B) Direct method:

Ethanol (0.4 ml) was added to each residue of the male serum samples and mixed well, and the solution 10 was transferred to a radioimmunoassay tube. The amount of solution in the test tube was adjusted to range from 50 to 400 µg, i. 100 μΐ in the case of a normal range and 50 μΐ in the case of high T level serum, such as samples Nos. 1, 2 and 3 of Table 5, described below. To determine the yield in the extraction step, the solution (100 μΐ) was transferred to a glass and dried by evaporation. After addition of a scintillator, the remainder is counted.

In the case of female serum, ethanol 20 (0.7 ml) was added to each residue and the ethanol solution (0.5 ml) was transferred to a radioimmunoassay tube. To determine the yield in the extraction step, the solution (100 μΐ) was placed in a glass and dried by evaporation. The remainder was counted after the addition of a scintillator.

In both of the above cases, ethanol was removed by evaporation under a stream of nitrogen and the obtained dry extract was used for radioimmunoassay.

(C) Paper chromatography:

The dried extract, prepared in a similar manner used by the direct method, was placed on a piece of paper with three 50 μ 50 chloroform flushes. On an identical piece of paper, T (10 μg) was placed as a reference and carried out with each set of determinations. The paper strips were placed in a chromatography vessel containing Bush A (a solvent system of cyclohexane / methanol / water = 10: 8: 2). After 2 hours for equilibrium adjustment, they were developed in the upper layer of Bush A. After development for 16 hours,

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The 27 spot used for reference by ultraviolet absorption at 254 nm and a 3 cm long section of the sample paper corresponding to the location of said reference spot / was cut from the paper and extracted with 5 ethanol (3 ml). Ethanol was removed by evaporation under nitrogen flow and the residue was used for radioimmunoassay in a manner similar to that described in the direct method.

(D) Radioimmunoassay: To prepare a standard T-curve, ethanol solutions containing 0, 20, 50, 100, 200 and 400 µg T were placed in test tubes in duplicate. To these test tubes containing test samples was added an ethanol solution (each 10 μ 10) containing 20,000 ppm H-T / 10vl, 15 and then the ethanol was removed by evaporation. A rabbit anti-T antibody was diluted with a 0.05M Tris buffer solution [pH = 8.0 containing 0.05% BSA and 0.1% BGG] to the concentration (B_ = 60%) that is able to

V

Binding 60% of 20,000 ppm H-T (45 µg as T) and 0.2 ml of this antiserum solution were placed in each tube followed by stirring using a vortex mixer. Separately, this tris buffer solution (each 0.2 ml) was transferred to two test tubes, each containing 20,000 ppm H-T alone and mixed well to obtain total ppm (Bq = 100%). After standing at room temperature for 2 hours, the solution was added to saturated ammonium sulfate (0.2 ml) and mixed well. After centrifugation at 3000 rpm. 0.2 ml of the above liquid was transferred to a glass.

After addition of a scintillator (0.2 ml), the activity was counted and calculated to a B / BQ (%).

3.2) Female serum DHT assay: Serum sample preparation, extraction and separation of DHT and T by paper chromatography was the same as 35 performed by assay of T using female serum sample. The location of a reference DHT spot was determined by spraying with a solution of equal volume

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28 1% m-dinitrobenzene in absolute ethanol and 2.5 N NaOH in absolute ethanol. Radioimmunoassay of DHT was performed using the standard ethanol solution of DHT and one

ISLAND

ethanol solution of H-DHT (40,000 dpm / 10 µl) in a manner similar to that used for radioimmunoassay of T. In each case, the result of the radioimmunoassay was corrected with the yield ratio.

In the direct method, the yield ratio of T or DHT was within a range of 95 to 100%, and the yield correction by addition of H-T or H-DHT is therefore not necessarily necessary for routine work. The results are shown in Tables 5 and 6.

(E) Results and analysis:

Table 5 -15 Sample No. J Serum used * excluding T <n (t / ml) (* male; -p -—- I Xk female) Direct method Paper chromato-_i ___ graph 1 * 10.35 9.60 2 * 13.34 15.36 20 3 * 22.84 19.08 4 * 8.92 9.16 5 * 4.40 4.38 6 * 5.00 4.90 7 * 8.20 7.95 25 8 * 7.43 7.10 9 %% 0.88 0.740 10. ** 0.310 0.292 11 ** 0.180 0.191 12 ** 0.160 0.158 30 13 XX 0.340 0.356 14 ** 0.500 0.490 i 15 **! 0.250 0.240 16 ** 0.154 0.218

The table shows that the results of the two 35 assay methods are consistent.

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29

Table 6 (Serum used - female serum)

Detected DHT (ng / ml)

Sample # -

Direct method Paper chromatography

5 1 0.175 0.154 2 0.210 0.201 3 0.198 0.200 4 0.195 0.190 5 0.280 0.280 10 6 0.250 0.260 7 0.221 0.232 8 0.201 0.198 I___ In this table, the results of the two methods are consistent.

As can be seen from these experimental results, by using the direct method of the invention using a low cross-reactive antibody, it is possible to assay and accurately assay the desired substance even in the presence of cross-reactive antigen.

In this case, it is no longer necessary to separate the cross-reactive antigen prior to paper chromatography, which has been used in conventional methods.

The method of the invention in which a specific antibody as well as a clone capable of producing such antibody was obtained by pretreating an animal with a cross-reactive antigen and D-GL to induce an immunological passivity to a cross-reactive antigen. This principle is not limited to the above particular embodiments using T-3-D-GL and DHT-3-D-GL and may be used in other cases as will be apparent from the following Example 5, substantially the same results, even when T and DHT were coupled to the carriers at different locations. In the following example, T and DHT were again used as model systems and their coupling positions changed from the third to the

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30, so that the structure of the hapten freely available on the surface of the carrier molecule changed.

Example 5 Properties of an antiserum obtained using a conjugate of 153-carboxyethyl mercaptotestosterone (hereinafter referred to as 153-CEM-T) and 153-carboxyethyl mercap-to-5a-dihydrotestosterone (hereinafter referred to as 153-CEM-5a-DHT) with KLH and D-GL: 10 In this example, 3-CEM-T was synthesized by the method of Rao, PN et al: [Steroid, 28, p. 101 (1976)) and 153-CEM-5a-DHT were synthesized according to the method of Rao, PN, et al: [Steroid, 29 [, p. 171 (1977)] .

The conjugation of 153-CEM-T-to D-GL and KLH as well as the conjugation of 158-CEM-5a-DHT to D-GL and KLH were performed in a manner similar to that used for the above conjugation of DHT. -3-CM0 or T-3-CM0 to D-GL or KLH. The conjugate thus obtained is called T-15-D-GL, T-15-KLH, DHT-15-D-GL and DHT-15-KLH, respectively.

In this example, 6 groups of mice (each group consisting of 7 mice; C57BL / 6 strain; 8-10 weeks old) were used.

The first group was control group and the mice were given saline (without DHT-15-D-GL). The second group was given DHT-15-D-GL (every 500 µg / mouse) 3 days prior to primary immunization; a second control group was the third group and the mice were given (ip.) T-15-30 D-GL (each 500 µg / mouse) 3 days before the first immunization with T-15-KLH. T-15-KLH (100 µg / mouse) was used to immunize the first, second and third groups. After 3 weeks, the secondary immunization of these mice was performed, and then booster immunizations 35 were performed every 2 weeks after the secondary immunization. The fourth, fifth and sixth group of mice were immunized with DHT-15-KLH. 3 Days before primary immunization with

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31 DHT-15-KLH, the mice in the fifth group were given (ip.) T-15-D-GL (500 µg / mouse). Simultaneously with the fifth group, the mice in the fourth group acting as a control group were given (ip.) Saline instead of T-15-5 D-GL.

The mice in the sixth group (as another control group) were given (ip.) DHT-15-D-GL (500 µg / mouse) 3 days prior to the primary immunization with DHT-15-KLH.

7 Weeks after the primary immunization, 10 serum was collected from each mouse at 2-week intervals to measure the antibody titer and cross-reactivity. The antiserum obtained 13 weeks after primary immunization had the highest antibody titer. Of these, the following results were obtained. The properties of anti-T antisera from the first, second and third groups were as follows:

Expressed as the reciprocal of the antiserum dilution capable of binding 50% HT (45 µg), the anti-T antibody titers of the mice in the first (control) and second groups are within the range of about 20%. 1600 to 9000 and 1000 to 3500, respectively, and the mice in the third group given T-15-D-GL showed no antibody formation.

Determined by Abraham's method, antiserum from the mice in the first group showed cross-reactivity within a range of 4.1 to 8.2, whereas in the antiserum from the mice in the second group, cross-reactivities were obtained from 0.27 to 0.94. The results of the second group are superior in terms of the cross-reactivities with DHT compared to those obtained with all other known anti-T antisera reported by others. Thus, it can be said that the cross-reactivity with DHT for the obtained antiserum was in practice negligible in the invention. It is known that 1.81% is the lowest cross-reactivity with DHT of any anti-T serum reported in the known literature and that Rao et al obtained this lowest value using an anti-T serum prepared by immunization of rabbit with Ιδβ-CEM-T-BSA [PNRao et al and PH Moore Jr.: Steroid, 28 (1976)], which also discloses

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32 is reversed in the above example. The cross-reactivity of the mice in the first group of this example is substantially equal to the lowest value.

Properties of anti-DHT antisera from the fourth, fifth, and sixth groups:

Expressed as the reciprocal of the dilution capable of binding 50% H-DHT (45 µg), the anti-DHT antibody titers in the fourth group (control group) were within a range of 1500 to 5600, and those in the 10th group given T-15-D-GL ranged from 1000 to 7600. The mice in the sixth group given DHT-15-D-GL showed no antibody formation.

When the antisera obtained from the mice of the fifth group treated with T-15-D-GL were tested by Abrahams method, allow the cross-reactivities with T of the antisera thus obtained within a range of 6 , 5 to 19%, in contrast to cross-reactivity values from 48.3 to 68.5 in antisera from the mice of the fourth group (control group). These facts suggest that administration of T-15-D-GL significantly reduces cross-reactivity with T.

Antibody formation was not found in the mice of the sixth group given DHT-15-D-GL. The reason for this is believed to be the specific inactivation of anti-25 DHT antibody-producing clones by administration of DHT-15-D-GL.

The reagents and assay methods used in the following Examples 6-9 are explained as follows. In these examples, the above principle of the invention was applied to peptide determinants.

Preparation of pentagastrin-D-GL conjugate.

Reference: Liu et al., Biochemistry, Vol. 18, 690 (1979).

(6-A) Synthesis of S-acetylmercaptosuccinyl-D-GL (hereinafter referred to as Ac-SD-GL): D-GL (mole weight = 34,300 * 40 mg = 1.166 µmol) was dissolved in 0.125 M phosphate buffer solution (900 µl) * pH = 7.2) and the pH was adjusted to 7.2 with 1 N NaOH solution.

33 DK 1 ^ 8644 B

After adding 50 µl (57.44 µmol) of dimethylformamide solution (hereinafter referred to as DMF) of S-acetylmercaptosuccinic anhydride (200 mg / ral), the mixture was stirred at room temperature for 30 minutes. During the reaction, the pH of the mixture was maintained at 7.2. After completion of the reaction, the solution was placed on a Sephadex G-25 column equilibrated with 0.01 M phosphate buffer saline solution (pH = 7.2) containing 0.01 M Na 2 -acetylmercapto-succinic anhydride. A fraction (100 µl) of the solution containing the reaction product was added to an aqueous solution of 0.5 M hydroxylamine (100 µΐ = 50 µmol; pH = 7.3) and incubated at 37 ° C for 20 minutes to remove it. protective acetyl group. The number of sulfhydryl groups introduced into D-GL was determined by the method of Ellman et al [Arch. Biochem. Biophys., Vol. 82, page 70 (1979) 3 as follows. To the reaction solution (50 µl) was added a methanol solution of 0.01 M 5,5'-dithiobis- (2-nitrobenzoic acid) (evaporated; 0.1 ml) and 1 ml tris-buffer solution (pH = 8.0) and reacted for 20 minutes and the absorbance at 412 nm was measured. The number of S-acetylmercap groups introduced into a D-GL molecule was approx.

15. The S-acetylmercaptosuccinyl-D-GL thus obtained is hereinafter referred to as Ac-S 2 -D-GL. The yield was approx. 77%.

Since D-GL shows no absorbance at 280 nm, the yield and fraction containing the derived D-GL could hardly be determined by absorbance at 280 nm. Therefore, the yield was determined using a reaction product obtained by reaction of N-30 succinimidyl-3- (4-hydroxyphenyl) propionate and D-GL and placed on a Sephadex G-25 column. Since this product absorbs at 280 nm, the determination was conveniently performed by measuring the absorbance at 280 nm.

(6-B) Preparation of m-maleinimidobenzoyl-pentagastrin (hereinafter referred to as MB-pentagastrin):

Pentagastrin (11 mg; 16.1 µmol) was dissolved in 0.1 M phosphate buffer (9.5 ml; pH = 8.0) and then added in one portion to m-maleinimidobenzoyl-N-hydroxysuccinimide.

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34 ester (25.3 mg, 80.5 ymol; hereafter referred to as MBS) dissolved in DMF (1 ml). The mixture was stirred and the reaction was monitored by thin layer chromatography using a solvent system of cyclohexane / ethyl acetate = 5 1: 1 by volume 25 minutes after the beginning of the reaction, dichloromethane (3 ml) was added to the reaction mixture to remove unreacted MBS. The mixture was well stirred and centrifuged.

The top layer of the buffer solution was placed 10 in a second tube. The dichloromethane layer (lower layer) was added a small amount of phosphate buffer, mixed well and centrifuged. The top layer was combined with the said solution. Thin layer chromatography confirmed that the unreacted MBS was removed almost completely from the fraction of the buffer solution containing MB pentagastrin.

Separately, the aforementioned buffer solution (20 μΐ) containing MB-pentagastrin was added to an aqueous solution of 2-mercaptoethanol (20 μΐ; 70 nmol; de-oxygenated with nitrogen stream). The reaction was carried out at room temperature for 20 minutes and the molar amount of 2-mercaptoethanol consumed was determined by Ellman's method and corresponds to the amount of maleimidobenzoyl group introduced into pentagastrin. The number of maleimidobenzoyl groups introduced into a molecule of pentagastrin was 0.9. This compound is then referred to as MB_ Q-pen-u / y tagastrin).

(6-C) Preparation of pentagastrin and D-GL conjugate.

A solution of Ac-S 2 -D-GL prepared in 6-A and

30 a solution of MBn Q-pentagastrin prepared in 6-B

u / y were formed to carry out the reaction in the following manner.

After complete filtration in a stream of nitrogen, the mixture was added a 5 M aqueous solution of hydroxylamine 500 µl; pH = 7.3) and stirred at room temperature for 1 hour.

The reaction solution was then partially extracted to examine the presence of unreacted SH group. There was no such group. That is, there was no SH group that had not been bound to MB pentagastrin. It means

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35 all the SH groups in D-GL had reacted with MB pentagastrin.

Two hours after the addition of the aqueous solution of hydroxylamine, the reaction mixture was added with 2-mer-5-capoethanol to give a final concentration of 1 mM, followed by stirring for 20 minutes. Then, the reaction mixture was dialyzed against 0.01 M phosphate buffer solution (pH = 7.2) for approx. 24 hours using a cellulose membrane and the solution thus obtained was used with or without dilution.

The molar ratio of pentagastrin to D-GL in the thus obtained pentagastrin D-GL conjugate was 15: 1.

This product is hereinafter referred to as pentagastrin β-D-GL.

(7) Preparation of CCK-8-P-keyhole limbed hemocyanin 15 (hereinafter referred to as CCK-8-P-KLH): (7-A) Preparation of S-acetyl-mercaptosuccinyl-KLH (hereinafter referred to as Ac-S-KLH) :

Prepared in a similar manner to that used for the preparation of Ασ-S-D-GL as described in 20 6-Ά.

Prior to the synthesis, KLH (70 mg) was dissolved in a 1% solution of (1.4 ml) and dialyzed against a 0.125 M phosphate buffer solution (pH = 7.2), and the absorbance at 280 nm was measured, of KLH was determined.

S-Acetylmercaptosuccinic anhydride (6.5 µmol) was added to the 0.7 ml KLH solution containing 26.0 mg (corresponding to 260 nmol assuming the molecular weight to be about 100,000; pH = 7.2) and the synthesis was carried out in a manner similar to that described in 6-A. The molar ratio of S-acetyl mercapto group to KLH in the product was 6.8 (hereinafter referred to as AC-S, O-KLH).

O if o (7-B) Preparation of m-maleinimidobenzoyl-CCK-8-P (hereinafter referred to as MB-CCK-8-P):

Prepared in a similar manner to that used for the preparation of MB pentagastrin. CCK-8-P (0.42 mg; 385 nmol) synthesized by the fragment condensation method was dissolved in 0.2 M phosphate buffer (0.5 ml) and reacted

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36 tes with MBS (600 µg / 1.9 ymol) using 50 μΐ DMF solution of 172 mg MBS dissolved in 100 μΐ DMF. The reaction product contained CCK-8-P and MB in a ratio of 1: 1/0 (hereinafter referred to as MB, n-CCK-8 ”P).

5 (7-C) CCK-8-P-KLH:

An AC-Sg g-KLH solution (2.3 ml, 75.9 nmol) prepared under 7-A and an MB, n-CCK-8-P solution prepare J- / u

slightly under 7-B were mixed together and 0/5 M NH90H was added

3 (0.3 cm; 150 µmol). Thereafter, a treatment similar to that described under 6-C was performed. The ratio of CCK-8-P to KLH in the resulting product was approx. 5: 1.

8. CCK-8-P calf serum albumin (hereinafter referred to as CCK-8-P-BSA): 15 (8-A) Preparation of S-acetylmercaptosuccinyl-BSA:

Synthesized in a manner similar to that described under 6-A using BSA (25 mg; 351 nmol) and S-acetylmercaptosuccinic anhydride (1.5 mg; 8.8 ymol). The ratio of BSA to S-acetylmercaptogroup in the product was 7.6: 1.

(8-B) Preparation of MB-CCK-8-P: CCK-8-P (0.5 mg, 457 nmol) was dissolved in 0.1 M phosphate buffer solution (0.5 ml; pH = 8.0) and reacted with MBS (700 µg, 2.29 µmol) using 50 µl of a 25 DMF solution of 1.4 mg MSB dissolved in 100 µl DMF in a manner similar to that described under 6- B. The ratio of MB to CCK-8-P was 1.0: 1. The product was then referred to as MB ^ g-CCK-8-P.

(8-C) Preparation of CCK-8-P-BSA: The solution obtained under 8-A (1.9 ml; 84 nmol) and the solution obtained under 8-B were mixed and added to 0 0.5 MN 2 OH (0.35 cm 2; 175 µmol)

The mixture was treated in a manner similar to that described under 6-C to obtain a conjugate in which the ratio of CCK-8-P to BSA was approx. 5: 1 (hereinafter referred to as CCK-8-P-BSA).

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37 9. Preparation of Pentagastrin-BSA: (9-A) Preparation of MB Pentagastrin:

Pentagastrin (0.5 mg, 750 nmol) was dissolved in 0.1 M phosphate buffer solution (0.5 ml; pH = 8.0) and reacted with MBS (1.05 mg; 3.3 µmol) using 50 μΐ DMF solution prepared by dissolving MBS (2.1 mg) in DMF (100 μΐ) · The reaction was carried out in a manner similar to that described under 6-B. The ratio of MB to pentagastrin was 1.0: 1. The product was named 10 MB MB-pentagastrin.

(9-B) Preparation of pentagastrin-BSA:

A solution (2.54 ml; 112 nmol) prepared under 8-A was mixed with the solution prepared under 9-A. The mixture was added with 0.5 M N 2 OH (0.4 cm 2; 200 μιηοΐ) and treated in a manner similar to that described under β-C. The ratio of pentagastrin to BSA in the product was approx. 5: 1. The product obtained was referred to as the money gas stage BSA.

10. A procedure similar to that described under 6 above was repeated except that D-GL was used with a molecular weight of 115,000 instead of a molecular weight of 34,300 (the molar reaction ratio being same as that described under 6).

Pentagastrine was obtained. - D-GL, which had a density of antigenic determinant combined with D-GL on the molecular surface.

Example 6 A. Immunization schedule and serum collection: (C57BL / 6J x DBA / 2) Female mice; each group consisting of 6 mice was immunized.

All mice in each group were immunized with CCK-8-P-KLH (every 10 µg) in Freund's complete adjuvant (every 0.2 ml), and 3 weeks thereafter they were further immunized with CCK-8-P-KLH (every 10 µg). ) in Freund's incomplete adjuvant (0.2 ml). Two weeks after the secondary immunization, they were booster-immunized with CCK-8-P-KLH

DK 158644 B

38 (each 10 µg) mixed with 2 mg of aluminum hydroxide gel. 2 and 4 weeks thereafter, they were immunized an additional 2 times by injection of 0.2 ml of saline solution of CCK-8-P-KLH (every 10 µg). All treatments were performed at ip.-in-5 section.

Three days before the primary immunization, all the mice in the second group (ip.) Received 0.5 ml of saline solution of pentagastrin β-D-GL (300 µg each). The mice in the first group acted as control and received 0.5 ml of saline 10 without pentagastrin β-D-GL.

Three days before the secondary immunization and three days before the tertiary immunization, all the mice in the third group received 0.5 ml of saline solution of pentagastrin β-D-GL (300 µg each) (ip.).

15 Blood was taken from the retroorbital plexus of each animal to produce the serum.

B. Antibody Titer Assay:

Performed by radioimmunoassay. 100 µl 0.01 M phosphate buffer solution (pH = 7.2 containing 0.15 M 20 NaCl, hereinafter referred to as PBS) containing 10 µg / ml of an antigen (such as CCK-8-P-BSA or pentagastrin BSA) was measured in each bore on a polyvinyl-manufactured round-bottomed microplate for solid-phase radioimmunoassay, and the antigen was bound to the surface of the polyvinylplate by incubation at room temperature for 2 hours. The antigen solution in the bore was discarded and the bore was washed 4 times with waterworks water. After the excess water was discarded, 200 µl of 1% BSA saline solution was added to each bore and allowed to stand overnight at 4 ° C, so that the polyvinyl plate protein combining capabilities were completely depleted. Then the solution was removed from the wells and the plate was purified 4 times with waterworks water. The plate was used for radioimmunoassay after yielding the excess water.

In each bore 0.1 ml of antiserum was diluted with 1% BSA-PBS and left overnight at 4 ° C, washed well 3 times with waterworks, washed 6 times with 2 M NaCL-PBS and washed with water. -

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39 working water.

Following the return of the water, 0.1 ml (50,000 dpm / 20 ng as specific antibody protein) of each bore was measured in a diluted solution of rabbit anti 5 mouse IgGFab antibody which had been specifically purified using an immunoadsorbent labeled with 0g. diluted with 1% BSA-PBS and left overnight at 4 ° C. If there is any antibody capable of binding to an antigen on the polyvinyl surface, the antibody will be coupled to the antigen. The presence or absence of such antibody can be determined by the coupling rate of rabbit anti-mouse igGFab antibody labeled with 125 I. Each isotope solution in the bore was removed using a pipette. After thorough washing with water 15, the flexible plate was placed in a rigid plastic plate and the top of the flexible plate was cut with a hot wire and then the radioactivity was counted in each bore.

C. Results: 20 At each time point for serum collection, the antibody titer of the total serum was determined from the mice in each group. Samples collected 11 weeks after primary immunization contained maximum levels of antibody. Therefore, antibodies in the antisera, collected 11 weeks after the primary immunization, were subjected to further detailed analysis.

For this measurement, the total antiserum from Group 1 collected 7 weeks after primary immunization was used as the standard antiserum. The amount of antibody in the test sample obtained from individual mice during the eleventh week was expressed as the ratio of the amount of antibody contained in the antiserum standard and defined as one unit. The serum standard titer was as follows. By assay determination by the following liquid phase radioimmunoassay method using C.CK-8, this serum (at 600 x dilution) was able to bind 0.0015 pmol cholecystokinin (CCK-33).

In the antibodies obtained from the mice in it

DK 158644B

In the first group, which did not receive pentagastrin15-D-GL [shown as "o" in the figure], the amount of antibody reacted with CCK-8-P and antibody reacted with penetastrin was almost equal. . It was found that antibodies obtained from the mice that had received pentagastrin β-D-GL [i.e. the second and third groups of mice indicated with "Δ" and "®" in the figure, respectively) had a very low titer of antibody reactive with pentagastrin. In particular, antibodies obtained from the mice in the third group, 10 and which had received pentagastrin β-D-GL after the primary immunization, viz. 3 days before the secondary and tertiary immunizations, an extremely low titer of antibody reactive with pentagastrin.

From these results, it can be seen that by use of the invention an antibody having a specificity to a specific amino acid sequence in CCK-8 has been obtained.

- SO, H

i.e. i 3

Asp-Tyr-Met

Example 7 The cross-reactivities of the antibodies obtained above were determined with a liquid-phase radioimmunoassay system which allowed the coexistence of CCK-8-P and pentagra 125 steps. CCK-8-P was labeled with I using

Bolton-Hunter's reagent [H. Sankaran et al., J. Biol.

25 Chem., Vol. 254, 9349-9351 (1979)] and is hereinafter referred to as 125 I-BH-CCK-8-P. For small tubes containing different amounts of unlabeled CCK-8-P, 50 μΐ of 0.02 M phosphate buffer solution (pH = 8.0? Containing 1% gelatin) was added to establish a standard CCK-8-P curve. Next, a small test tube containing 50 µl of buffer solution containing various amounts of pentagastrin was prepared to prepare a pentagastrin standard curve.

In each case, antiserum (50 µl) diluted with mouse serum was added to the test tubes, to which the same phosphate buffer solution (50 µ 50) containing 125 I-BH-CCK-8 (about 5000 cpm) was added and the solution mixed well. using a vortex mixer

DK 158644 B

41 followed by incubation at 4 ° C for 24 hours. Then, 50 µl of rabbit antimus IgGFab antibody diluted to 1: 2 with the same phosphate buffer was added to the test tubes, mixed and incubated at 4 ° C for 24 hours. Then, the tubes were centrifuged (3000 rpm) for 25 minutes. The above liquid was sucked up and the radioactivity of the precipitated substance was counted in a counter.

Instead of the standard solutions of CCK-8-P and 10 pentagastrin, the same phosphate buffer was used to determine the radioactivity of total I-BH-CCK-8-P (count B, expressed as 100) and the binding ratios (B / Bn% ) υ 125 υ of the antibody with I-BH-CCK-8-P in the presence of CCK-8-P or pentagastrin at various concentrations was calculated.

The molar amounts of various peptides required for 50% inhibition of binding of the antibody by 125 * 3I-BH-CCK-8-P are as follows.

In the first group, CCK-8 was 1.1 pmol and penta-20 gastrin was 20.1 pmol. Thus, the cross-reactivity calculated by Abraham's method was 5.5% (1.1 / 20.1 x 100).

In the second group, whose mice had received pentaga step D-GL prior to primary immunization, the cross-reactivity was 2.7% (4 pmol / 150 pmol x 100).

In the third group, whose mice had received pentagastrin - ^^ - D-GL after the primary immunization, viz. Three days before the secondary and tertiary immunizations, no cross-reactivity with gastrin was found. That is, 1.7 pmol of CCK-8-β was required for 50% inhibition when said antibody was used, but no inhibition was found at 10,000 pmol of pentagastrin. This means that pentagastrin is in fact unable to react with the antibody produced by the mice of the third group.

From these results, it can be seen that CCK-8-P can be assayed specifically without significant effect of co-existing pentagastrin using the antibodies of the mice of the third group that had received pentagastrin15-D-GL.

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42

Example 8

A procedure similar to that described in Example 6 was repeated except replacing the mice with rabbits.

Using Freund's complete or incomplete adjuvant containing 100 µg CCK-8-P-KLH and 10 ml saline containing 10 mg pentagastrin ^^ D-GL, the results obtained were substantially similar to those obtained using 10 mice.

Example 9

The same procedure of Example 6 was repeated except that pentagastrin β -D-GL was replaced by pen-tagastrin β-D-GL with a molecular weight of 115,000. The results obtained were essentially the same as the results obtained in Example 6.

4. Brief description of the drawing: In the figure, the abscissa indicates the amount of antibody reacted with pentagastrin and the ordinate indicates the amount of 20 antibody reacted with CCK-8-P. The entry "o" shows antibody from the mice (the first group) that did not receive penta-gastrin β-D-GL. The indication "A" shows the amount of antibody obtained from the mice (the second group) that received pentaga-step15 ~ D-GL prior to the primary immunization. The indication 25 "·" shows antibody obtained from the mice (the third group) that received pentagastrin β-D-GL after the primary immunization.

Each value is conveniently expressed as the ratio of each antibody amount to that present in the pooled serum collected from mice in the first group 7 weeks after the primary immunization.

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43

Example 1O

Preparation of strains producing specific anti-CCK-8-β antibodies.

A) Procedure 8

Spleen cells (each 2x10) of mice from the first (control) group and the third group (treated with pentagastrin-D-GL) immunized by the procedure described in Example 6 (A) were used each time as starting materials. In each case, the cells were digested with P3-X63-Ag8-U1 (P3U1) tumor cells 10 (2 x 107) in polyethylene glycol 4000.

The cell membranes were removed and diluted with a PEG solution (1 ml) to which was added dropwise a mixture of 9 g of PEG 4000 and 20 ml of HANKS to give a cell suspension, which was then allowed to stand at room temperature for 8 minutes. Then MEM (15 ml) was slowly added to the cell suspension within 5 minutes and then additional MEM to a total of 50 ml.

The cells were centrifuged from the suspension and then resuspended in a 10% FCS-RPMI solution and partitioned into a culture tray (1 ml / well) for culture at 37 ° C overnight using a humidified CO2 incubator. which contained 7% CO 2 in air with a relative humidity of 85-95%. The next day, HAT medium (1 ml) was added to each well. Each morning for the next two 25 days, HAT medium (1 ml) was aspirated from the well and replaced with fresh HAT medium (1 ml). The same replacement was done over the next two weeks at three day intervals. Then, the culture mixture was taken and tested to determine the antibody secretion using solid phase radioimmunoassay as described in Example 6 (B). For strain formation, the positive culture media was subjected to limit dilution using 10% FCS-RMPI and the culture mixtures containing strains were tested according to the solid phase radioimmunoassay described in Example 6 (B) to determine the specificity of the strains thus obtained.

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44 produced antibodies.

B) Results

Using spleen cells from mice of the first group, 18 strains were obtained with which anti-CCK-8-β antibodies could be produced. All antibodies thus obtained reacted with CCK-8-P as well as with pentagastrin. Therefore, for the antibodies thus obtained, it was determined that they were monoclonal antibodies specific to the pentagastrin portion of CCK-8-P. On the other hand, spleen cells from the third group of 15 strains capable of producing anti-CCK-8-β antibodies were obtained. Of these 15 strains, 15 12 strains were able to produce anti-CCK-8-P antibodies that did not react with pentagastrin and other related peptides and react specifically to CCK-8-P, while the antibodies produced by the remaining 3 strains were cross-reactive. with pentagastrin. Therefore, by using spleen-20 cells from mice treated in accordance with the invention with penta-gastrin D-GL, it is possible to obtain a greater number of strains producing the desired specific anti-CCK-8-P antibodies than using spleen cells treated in the usual way. The 25 specific anti-CCK-8-β antibodies thus obtained showed no cross-reactivity with pentagastrin.

Claims (9)

A method of producing an antibody or antiserum of high specificity to a first antigen having a desired antigenic determinant by low cross-reactivity with at least one second antigen containing at least one antigenic determinant structurally related to the desired antigen. determinant of the first antigen, by immunizing a mammal or culturing appropriate mammalian cells from that mammal after its immunization, characterized by administering to the mammal a copolymer of D-glutamic acid and D-lysine coupled with the second antigen, and upon induction of a clear immunological tolerance, the mammal immunizes with the first antigen. Process according to claim 1, characterized in that the copolymer has a molecular weight of from approx. 27000 to approx. 120,000th Process according to Claim 1 or 2, characterized in that the molar ratio of D-glutamic acid to D-lysine in the copolymer is from approx. 70:30 to approx. 30:70. Method according to any one of claims 1-3, characterized in that the second antigen is a steroid, catecholamine, peptide, 1-propranolol or 1- or d-cyclazocin or, as appropriate, a subunit or fragment. of these substances. Process according to claim 4, characterized in that the steroid is selected from testosterone, 5a-dihydrotestosterone, androsterone, etiocholanolone, progesterone, 17a-hydroxy-progesterone, pregnenolone, dehydroepiandrosterone, estradiol, estrone, estriol, aldosterone, deoxycorticosterone, cortisol, cortisone, corticosterone, 11-deoxycortisol, cholic acid, deoxycholic acid, litho-cholic acid and conjugated compounds thereof. Process according to claim 4, characterized in that the catecholamine is selected from dopamine, norepinephrine and epinephrine and derivatives thereof. DK 158644 B Process according to claim 4, characterized in that the peptide is selected from gastrin, cholecystokinin-pancreozymine, insulin, proinsulin, glucagon, follicle stimulating hormone (FSH), luteinizing hormone 5 (LH), human chorionic gonadotrophin (HCG), somatostatin, hormone (TSH) or a subunit and related peptides. Use of the method according to any one of claims 1-7 for obtaining mammalian cells 10 capable of producing an antibody according to claim 1. Use of the antibody or antiserum for immunoassay produced according to any one of claims 1-7 or by mammalian cells according to claim 8.