GB2075987A

GB2075987A - Antibodies, Clones for the Production of such Antibodies, a Process for the Production of such Antibodies and Clones and a Method of Immunoassay using such Antibodies

Info

Publication number: GB2075987A
Application number: GB8111381A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1980-04-11
Filing date: 1981-04-10
Publication date: 1981-11-25
Also published as: FR2480782A1; GB2075987B; DK158644C; FR2480782B1; DE3114362A1; DK164381A; DE3114362C2; DK158644B

Abstract

An antibody having a high specificity to a first antigen comprising a desired antigenic determinant and having a low cross-reactivity with at least one other antigen, said other antigen comprising at least one antigenic determinant which is structurally related to the desired antigenic determinant of said first antigen, is produced by administering to a mammal a copolymer of D- glutamic acid and D-lysine coupled with said other antigen whereby substantially effective immunological tolerance is induced and subsequently immunizing the mammal with the said first antigen. The process of the invention results the preparation of clones capable of producing the desired antibodies. These clones may themselves be used to produce the desired antibody after removal from the source host. The antibodies thus-obtained have a very low cross-reactivity and high specificity and are particularly useful as specific antibodies for immunoassay of various substances present for example in humans.

Description

SPECIFICATION Antibodies, Clones for the Production of Such Antibodies, A Process for the Production of Such Antibodies and Clones and a Method of immunoassay Using Such Antibodies This invention relates to an antibody having high specificity and low cross-reactivity, a clone capable of producing such an antibody, an antiserum comprising such an antibody, and a process for their production. The invention also relates to an assaying method using the antibody or antiserum comprising the antibody. In addition, the invention relates to a method for preparing a specific antibody by using the said clone.

Clinical laboratories are able to measure a number of substances present in living animals, such as for example hormones with high specificity and sensitivity by immunoassay methods. These methods utilize the competitive antigen-antibody reaction and employ a given amount of an antibody and various different amounts of antigen. For example, the radioimmunoassay method usually consists of the following 4 steps:- 1) Preparation of a labelled antigen and a specific antibody.

2) Reaction of the labelled antigen with the specific antibody in the presence of a non-labelled antigen.

3) Separation of the labelled antigen from the immune complex of the labelled antigen and specific antibody.

4) Determination of the radioactivity and subsequent determination using a calibration curve.

It is also possible to use a labelled antibody and an unlabelled antigen.

Of these steps, step 1 is most important, and the antibody used as the reagent for assay has to be specific to the substance to be assayed. However, this requirement sometimes gives rise to difficulty because, even in cases where specificity is believed to be high, the assay specificity tends to be affected by cross-reaction with various substances having a structure analogous to that of the substance to be assayed.

In order to overcome such difficulty, certain improvements have been employed which, in general, fall into the following categories: 1 ) Prior to the determination, various crossreactive substances are eliminated from the assay samples containing the substance to be assayed by the use of physico-chemical techniques.

2) The cross-reactive antibody contained in the antiserum is removed before use by an immunoadsorbent. Alternatively, it is possible to use a highly purified antibody having a very high specificity for the substance to be assayed.

In order to obtain an antibody having the highest possible specificity, it has been proposed to bind an antigen, prior to injection into an animal host, onto a carrier protein at an appropriate site on its chemical structure in such a manner that the relevant determinant site(s) on the antigen to be recognized by the antibody is exposed on the surface of the carrier molecule. The exposure of a specific determinant site thereby stimulates the formation of an antibody having low cross-reactivity. Known methods of this type are, however, still unsatisfactory because of the need for complicated procedures and a synthesizing step. Moreover, in some cases, it is still difficult to avoid the production of crossreacting antibodies by such methods, and thus such problems may render the immunoassay of the desired substance very difficult.

The present invention is based on the discovery that it is possible to reduce the amount of crossreactive antibody to a minimum and also to obtain an antibody having excellent specificity for a substance, for example to be assayed, by the use of a copolymer of D-glutamic acid and D-lysine (hereinafter referred to as D-GL) which induces effective immunological unresponsiveness (tolerance) to B cells which serve as the precursor for the production of antibodies. When an antigen coupled with D-GL is administered to an animal, the D-amino acids are in general not readily metabolized in the living body, and furthermore D-GL induces substantially no immunological responses in T cells in the living body.Thus it is believed that when an antigen conjugated with D-GL is administered to an animal the antigen-D-GL conjugates specifically bind to surface immunoglobulin receptors on B lymophocytes, and render those cells irreversibly tolerant.

Thus according to one feature of the present invention there is provided a process for producing an antibody having a high specificity to a first antigen comprising a desired antigenic determinant and having a low cross-reactivity with at least one other antigen, said other antigen comprising at least one antigenic determinant which is structurally related to the desired antigenic determinant of said first antigen, which process comprises administering to a mammal a copolymer of D-glutamic acid and Dlysine coupled with said other antigen whereby immunological tolerance, for example substantially effective e.g. effective, immunological tolerance is induced and subsequently immunizing the mammal with the said first antigen.

The process of the present invention results in the production in the living mammal of cells of the same genetic constitution (hereinafter referred to as clones) which are capable of producing the said antibody.

Thus according to the present invention it is possible to obtain a clone capable of producing an antibody having a low cross-reactivity and a high specificity for a specific determinant(s) by administering to an aniamal the conjugate of D-GL with a cross-reactive antigen so as to induce substantially significant immunological tolerance specific for the cross-reactive antigen and then immunizing the animal with a desired antigen, i.e., an antigen which contains specific antigenic determinant(s). Such an animal is capable of producing clones and antibody having a low crossreactivity and high specificity.

The antibody of high specificity as hereinbefore defined may be obtained in the form of purified antibody or in the form of an antiserum containing the said antibody. If desired, however, the antibody may be produced from a clone which is immortalized by hybridization with a suitable tumor cell. It will be appreciated that the clones obtained according to the present invention may if desired be utilized, after removal from the living mammal, in order to produce the desired antibody. Thus once clones are obained the desired antibody may be produced as desired in the absence of the initiating living mammals.

The present invention thus provides a process for the production of the said antibody by the use of a clone obtained according to the process hereinbefore defined. It is well known that, if a suitable clone is available, it is easy to combine such a clone with a suitable tumour cell to obtain a hybrid cell (hybridoma). For example, a hybrid cell formed by combination of a certain clone and a myeloma cell is reported in the literature, (see for example Nature, vol. 256,495497 (1975) & s European J. of Immunol., vol. 6, 511-519 (1976) by Köhler et al; Nature, vol. 266, 550-552 (1977) by Milstein et al. Nature, vol. 266,495(1977) by by Walsh).When such hybrid cells are transplanted to or implanted in another animal, the cells propagate continuously in the living body of such an animal (e.g. in the ascites of a mouse) to produce continuously large amounts of the specific antibody. Thus, it is possible to use the hybridoma as a new source of the desired antibody.

According to a further feature of the present invention there is provided a method for the immunoassay of a substance which comprises the use of an antibody produced by a process as hereinbefore defined or an antiserum containing such an antibody, the said antibody being specific to said substance whereby the assay is effected by reaction of the antibody with the said substance.

It will be appreciated. that the substance to be assayed serves as an antigen.

The immunoassay may be effected by methods known per se, for example radioimmunoassay or assay by the use of non-isotropic labels e.g. enzymes, free radicals, cells, viruses, metal ions and fluorescent and chemiluminescent groups. The immunoassay method of the present invention enables an assay to be effected even in the presence of a crossreacting antigen.

It is preferred for the purpose of this invention to use D-GL having a molecular weight of from about 27,000 to 120,000. The molar ratio of D-glutamic acid to D-lysine is preferably within the range 70:30 to 30:70, for example 60:40. Copolymers of this type are commercially available, for example, from Miles-Yeda, U.S.A., although it is also possible to prepare such copolymers in the conventional manner, for example, by copolymerizing the N-carboxylic anhydride of y-alkyl-D-glutamate and the Ncarboxylic anhydride of E-N-carbobenzyloxy-L-lysine in the presence of a suitable amine, followed by removal of the protecting groups.

Various naturally occurring substances may for example be used for the production of the antibody for immunoassay. Examples of preferred materials for this purpose include steroids, its glucuronates and sulfates thereof; catechol amines; peptides, its subunits and related fragments thereof; and various pharmaceutical agents.

Especially preferred examples of steroids include testosterone (hereinafter referred to as T), Sa- dihydrotestosterone (hereinafter referred to as DHT), androsterone, etiocholanolone, progesterone, 1 7a-hydroxyprogesterone, pregnenolone, dehydroepiandrosterone, oestradiol, oestrone, oestriol, aldosterone, deoxycorticosterone, cortisol, cortisone, corticosterone, 11 -deoxycortisol, cholic acid, deoxycholic acid, lithochoiic acid, and conjugated compounds thereof.

Catecholamines are exemplified by dopamine, norepinephrine, epinephrine and the like.

Peptide hormones are exemplified by gastrin, cholecystokinin-pancreozymin, insulin, proinsulin, C-peptide, glucagon, follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (HCG), somatostatin, thyroid-stimulating hormone (TSH) and their subunits and the related peptides thereof.

Pharmaceutical agents are exemplified by L-propranolol which is a ,B-blocking agent and I- or dcyclazocine which is an analgesic agent For example, when an anti-T antibody or antiserum is prepared by conventional immunization procedures, DHT acts as the cross-reactive antigen because their structures are closely similar to each other. Similarly, when an anti-DHT antibody or antiserum is prepared, T acts as the cross-reacting antigen. In this manner, various steroids act as the cross-reactive antigens for other steroids.

The above-mentioned substances are in general the haptens or the like (i.e. substances capable of combining with an antibody but incapable of inducing an immune response or capable of inducing only a weak immune response if they are not coupled with a carrier prior to their administration to a living animal). It is thus necessary to couple them to a suitable carrier in order to induce the formation of an antibody. For example, in order to induce an antibody specific for a certain antigen such as antitestosterone (referred to hereinafter as anti-T antibody), this antigen should be coupled with a suitable carrier and used for immunization. However, the anti-T clones and antibodies thus-obtained usually have a strong cross-reactivity with substances having an analogous structure such as DHT.According to this invention, it is possible to inhibit specifically the formation of such crossreactive anti-DHT clones and antibodies by treating the animal with a substance which is the product of coupling a copolymer of D-GL with a cross-reactive antigen, in this case DHT.

Moreover, it was also found that an antibody capable of reacting with the specified determinant of the desired antigen was obtained when an animal is treated with a conjugate of D-GL and a peptide analogous to the said antigen, part of the structure of the peptide being common to that of the desired antigen. An example to demonstrate such an advantage pertaining to this invention includes the case of antibodies to an octapeptide which is a C-terminal peptide comprising eight amino acids of cholecystokinin-pancreozymin (hereinafter referred to as CCK-PZ) which is a gastro-intestinal hormone. The structure (amino acid sequence) of gastrin (human), CCK-PZ and their fragments are shown in Table 1.

Table 1

Gastrin (human): (Pyro)Glu-Gly-Pro-Trp-Leu-Glu-Glu Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (S03H) CCK-PZ: Lys-Ala-Pro-Ser-G ly-Arg-Val-Ser-Met l le-Lys-Asn-Leu-G In-Ser-Leu-Asp-Pro Ser-His-Arg-lle-Ser-Asp-Arg-Asp Tyr-Met-Gly-Trp-Met-Asp- P he-NH2 I SO3H CCK-8-P: Asp-Tyr-Met-G ly-Trp-Met-Asp-Phe-NH2 I SQH Pentagastrin: Gly-Trp-Met-Asp- Phe-NH2 Cholecystokinin-pancreozymin (CCK-PZ) is a type of secretory hormones found in the gastrointestinal tract, mainly in the small intestine, which stimulates the secretion of the pancreatic enzymes and also causes the contraction of the gallbladder.CCK-PZ is a known polypeptide consisting of 33 amino acids and its biological activity results from the fragment of eight amino acids at the C terminal end of the peptide (CCK octapeptide, hereinafter referred to as CCK-8-P). The sequence of the 5 C-terminal amino acids is the same as the sequence of the 5 C-terminal amino acids of gastrin, a hormone capable of stimulating the secretion of acid in the stomach. It is thus known that the anti CCK-8-P antibody is highly cross-reactive with gastrin.

It has recently been found, however, that CCK-8-P and its reactive receptor are also present in the brain, and the investigation of the function of this hormone as a neurotransmitter as well as the secretions which it promotes and its action on various diseases of gastro-intestinal tract is of interest. It is is thus important to provide an antibody capable of specifically reacting with CCK-8-P.

Antibody forming clones and antibodies capable of specifically reacting with CCK-8-P may be obtained according to the present invention by administering to an animal conjugates of D-GL with pentagastrin (i.e. the five amino acids located at the C-terminal end of gastrin and CCK-8-P) so as to inactivate clones which produce antibodies cross-reacting with pentagastrin and/or gastrin-like compounds and then immunizing the said animal with CCK-8-P.

Thus according to this invention, it is also possible to produce gastrin-specific antibody by inhibiting the formation of clones which cross-react with CCK-PZ by treating an animal with a conjugate of D-GL and pentagastrin, which the C-terminal fragment of the desired antigen, and then immunizing the said animal with gastrin.

As is apparent from the Examples of this invention appearing hereinafter, it may also be possible to obtain a desired specific antibody by administering to an immune animal a conjugate of D-GL with a crossreactive peptide even in the case in which specific antigenic determinants can not easily be removed from the whole peptide by peptide fragmentation.

For instance, Attasi et al demonstrated the composition of the antigenic determinants of lysozyme by using synthesized peptides based on his "surface simulation model" [Attasi M. Z., immunochemistry, vol. 15, 909-936 (1978)]. In this case, the antigenic determinant is formed of amino acids located at irregular intervals (for example, assuming that the determinant is formed with amino acids A, B, C and D and that there is a peptide having an entirely different amino acid sequence which however, accidentally comprises amino acids B-C which serve to form a stereochemically common antigenic determinant), it is readily possible to amplify selectively a specific clone directed to the antigenic determinant A-D so as to form an antibody directed to the specific site by administering to an animal a substance formed by coupling antigenic determinant comprising amino acids B-C with D GL and resulting in the elimination of the clones having a cross-reactivity with the antigenic determinant comprising the amino acids B-C.

Such a combined product of D-GL and cross-reactive antigen may 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 made, which derivatives are exemplified by oxime derivatives, succinyl derivatives, and chlorocarboxylic acid derivatives by Erlanger et ai [e.g. B. F. Erlanger et al, J. Biol.

Chem., 228, 713 (1957)], carboxylmethylthioether derivatives [A Weinstein et al., Steroid, 20, 789 (1972)], carboxylmethyletherderivatives [P. N. Rao et al. J. Steroid Biochem., 9,539(1978)] etc.

Preferred coupling methods are exemplified by the carbodiimide method, the mixed anhydride method, the Schotten-Baumann method and the isoxazolium method.

When an NH2 group is introduced into such a compound, the coupling reaction is effected by the glutaraldehyde method, and when an NH2 or SH group is introduced into such a compound, the reaction can be performed by the use of an m-maleimidobenzoyl-N-hydroxysuccinimide ester succinimidyl-4-(N-maleimidomethylcyclohexane)- 1 -carboxylate, succinimidyl-4-(pmaleimidophenyl)butyrate and the like or N-succinimidyl-3-(2-pyridyldithio)propionate N-succinimidyl (4-azidophenyidithio)propionate and the like.

In addition, various other methods conventionally usd to couple peptides with other substances in the field of peptide chemistry may also be used for coupling D-GL with an antigen.

The antigens which may be used for the purpose of this invention are in general haptens or the like, and thus it is necessary to couple the antigen before use with a suitable carrier which is used in conventional immunization methods. Preferred carriers for this purpose are exemplified by keyhole limpet-haemocyanin (KLH), y-globulin and albumin originating from the serum of different animal species used for immunization such as e.g. humans, goats, bovines and the like.

The antigens, their subunits or related fragments should be coupled with a carrier protein in a similar manner to that applied to couple a cross-reactive antigen, its subunit or related fragment with D-GL. Thus, the coupling may be accomplished directly or indirectly. In the latter case, the same intermediate as that 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 effected in conventional manner. Depending upon the type of animal used, a saline solution containing an antigen (a combined product of a hapten or the like and carrier) is administered to the animal by intraperitoneal (ip.) injection. Alternatively, it is possible to inject the antigen solution into the foot pad or a subcutaneous site on the back of the animal. At the primary and secondary immunization, the antigen solution is preferably administered in association with Freund's complete adjuvant and Freund's incomplete adjuvant, respectively. After this, the antigen solution is preferably solely administed in the case of mice, and Freund's complete adjuvant and incomplete adjuvant may for example be used in turns together with the antigen solution in the case of larger animals such as the rabbit.The dose may vary, depending upon the combined ratio of the hapten or the like and carrier, and the molecular weight of the carrier and the like. However, it is usually preferred to immunize animals with an antigen at a dose of 1-100 -100,ug/smaller animals e.g. the mouse or 0.1-1 mg/larger animals e.g. the rabbit/once, which may be repeated for example 2-5 times at 2 4 week intervals. Usually 2-3 days before the primary immunization, a conjugate of D-GL and a cross-reactive antigen may be given to the animal, although in some cases, the D-GL conjugate may be also given after the primary or secondary immunization, depending upon the type of cross-reactive antigen.The dose of the D-GL conjugate with cross-reactive antigen may vary, depending upon the combined ratio of the cross-reactive antigen (hapten or the like) to D-GL, sites of coupling, types of intermediate compound and the like, but is preferably in the range 100-500 ,ug/smaller animals e.g.

mouse or 2-10 mg/larger animals such as e.g. rabbit. The D-GL conjugate may be dissolved in a saline solution and administered intraperitoneally.

Since it is advantageous to couple the cross-reactive antigen with D-GL so as 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 substantially effective immunological tolerance specific to a certain cross-reactive antigen by administering to an animal a conjugate of D-GL and the cross-reactive antigen. After this, the animal is immunized with the desired antigen, which contains the specific desired antigenic determinants. In this manner, it is possible to obtain a clone capable of producing an antibody which has low cross-reactivity and excellent specificity for the relevant specific antigenic determinants, and an antiserum comprising the said antibody.

It will be appreciated that the above-mentioned findings have been tested not only in smaller animals such as mice but also in larger animals such as rabbits. It is important that the same results have been found in both smaller and larger animals in spite of the differences which might have been expected amongst the animals species. In this connection a substantially larger amount of an antibody is obtained by using larger animals such as the rabbit than by using comparatively small animals such as mice.

The present invention is especially advantageous since previousiy it was difficult to prepare an antibody capable of discriminating analogous structures such as T and DHT.

The process of this invention makes it possible to obtain a large number of clones capable of producing an antibody which is capable of specifically distinguishing an antigen from other crossreacting antigens.

As a result of increased productivity of the specific antibody-producing clone, it may be practically possible to separate and isolate a specified clone. It was previously known in the literature that the probability of separating out and isolating a clone capable of recognizing a specified antigen was approximately 1/106 to 1/10'. This means that such separation and isolation was practically impossible.

By the.process of this invention, it is thus possible to obtain specific antibody-producing clones capable of discriminating specifically between a desired antigenic determinant and structurally related antigenic determinants even by immunization with an antigen containing cross-reactive determinants.

The process of this invention, renders it possible to obtain such specific antibodies at higher probability, and accordingly it is also possible to increase the probability of producing such specific antibodyproducing clones.

The process of this invention may be applied to any and all antibody-producing methods by modifying the method of coupling D-GL with a cross-reacting antigen, the types of the coupling product and the like.

According to still further feature of this invention, this invention provides a simple method for determining a certain substance, of which amount contained in the same is unknown, by the use of an antibody or antiserum obtained by the process as hereinafter described.

The reagents and assay method used in the undergoing Examples are described below:- (1) Synthesis of DHT-3-(o-carboxymethyl) Oxime [Hereinafter Referred to as DHT-3-CMOl: (A) Reaction Formula:

(B) Materials used: 3H-DHT (Radiochemical Centre, Amersham) 6 ,uci (0.015 cog as DHT) DHT (Sigma Chemical Co., U.S.A.) 0.3 g (1 mmole) O-carboxymethyl-hydroxylamine hemihydrochloride (Tokyo Kasei Kogyo K.K. Japan) 0.3 g (2.7 mmole) 2N-NaOH 1.25 ml Ethanol 15 ml (C) Procedure: The materials were put in a round flask equipped with a refluxing apparatus and refluxed by heating for 3 hours. Distilled water (45 ml) was added to the reaction mixture and the solution was adjusted to pH 8 with dilute NaOH solution.The solution was transferred to a separating funnel; ethyl acetate (about 30 ml) added and shaken. Then, the ethyl acetate layer was removed to isolate the unreacted DHT. The aqueous layer was cooled by the addition of ice and acidified to give a pH of about 4 with dilute hydrochloric acid. The DHT-3-(o-carboxymethyl)-oxime thus-produced was extracted by addition of cold ethyl acetate. Anhydrous sodium sulfate was used to dry the ethyl acetate layer which was removed by evaporation. The resultant residue was recrystallized from hot methanol to give the desired product.

(2) Synthesis of testosterone-3-(o-carboxymethyl)-oxime (Hereinafter Referred to as T-3-CMO) The same procedure as that of (1) was repeated to obtain the desired product with the exception that 3H-T and T were used instead of 3H-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] According to the mixed acid anhydride method of Erlanger et al. [J. Biol. Chem., 228, 713 (1957)].

Reaction Formula

Materials Used DHT-3-CMO containing DHT-3-CMO labelled with 3H tracer 20 mg Dioxane (dried) 6 ml Tri-n-butylamine 20 yl Isobutyl chloroformate 10,us Distilled water 6 ml D-GL (Mw=49,000) 30 mg 1 N-NaOH 150,us Procedure: DHT-3-CMO (20 mg) was dissolved in dried dioxane (1 ml). The mixture was stirred with the addition of tri-n-butylamine (20,u) and was then stirred at 11 OC for 5 minutes. After this, isobutyl chloroformate (10 ,ul) was added to the solution and allowed to react at 11 OC for one hour with agitation. Separately, D-GL (30 mg) was dissolved in distilled water (6 ml) and the mixture treated with 1 N NaOH (150 yI). Dioxane (5 ml) was added gradually to the mixture.This D-GL solution was added to the said reaction mixture in one portion with stirring. 10 minutes after this, 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 about 1 OOC for about 2 hours. After this, the reaction solution was dialyzed against water at 40C overnight. On the next morning, the pH of the solution was adjusted to 4.5 with dilute hydrochloric acid to form the precipitates. After the precipitation was completed, the solution was allowed to stand at 40C for 7 hours. The reaction mixture was centrifuged at 40C for 20 minutes (3,000 r.p.m.) and the supernatant was removed. The precipitates were treated with distilled water (10 ml) and its pH adjusted to about 7.0 by the use of 1 N-NaOH so that the precipitates were dissolved.After this, the solution was dialyzed against distilled water at 40C overnight followed by lyophilization.

The number of moles of DHT which combine with 1 mole of D-GL was determined by measuring the radio-activity of 1 mg of 3H-DHT-3-CMO, used as a tracer, and the radioactivity per 1 mg of the reaction product. The molar ratio of DHT to D-GL in the reaction product was 30:1.

When the amounts of the water and dioxane used were equal to the corresponding amounts calculated on the basis of the mixed anhydride method by Erlager et al [J. Biol. Chem., 228, 71 3 (1957)] with respect to Bovine Serum Albumin (hereinafter referred to as BSA), the mixture became gelatinized. Thus, large amounts of water and dioxane were added to the mixture in order to avoid gelatinization of the D-GL therein and moreover to effect the reaction adequately. Thus, conjugation of the D-GL with oxime was effected by the mixed anhydride method so that a sufficient amount of the oxime was combined with the D-GL.

(B) Preparation of testosterone-3-(o-carboxymethyl)-oxime-D-GL [Hereinafter Referred to as T-3 D-GL] The same procedure as above was effected except for the use of T-3-oxime containing 3H labelled T-3-oxime as a tracer to obtain T-3-D-GL (the 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 DHT-3-CMO containing 3H-labelled oxime as tracer 12 mg Dried dioxane 3.0 ml Tri-n-butylamine 10,us Isobutylchloroformate 5 jut Distilled water 4 ml KLH (Mw=100,000) 150 mg 1N NaOH 75jul Procedure Same procedure as described above gave DHT-3-KLH (the molar ratio of DHT:KLH=1 0:1).

(B) Preparation of Testosterone-3-Io-carboxymethyl)-oxime-K [Hereinafter Referred to as T-3 KLH] Materials Used T-3-CMO containing 3H-labelled oxime as tracer 40 mg Dried dioxane 7 ml Tri-n-butylamine 40,t41 Isobutylchloroformate 20jut Distilled water 10 ml KLH 300 mg 1N NaOH 300 my Procedure The same procedure as above gave T-3-KLH (the molar ratio of T:KLH=8:1).

(5) Radioimmunoassay Procedure Reagents 1) 2H-T Standard Solution 1 ,2-3H-T (58 Ci/m mol) was diluted with ethanol to given an ethanol solution contain 20,000 dpm/10,ul (45 pg as T).

2) 3H-DHT Standard Solution 5a-Dihydro-1 ,2,4,5,6,7-3H-T (114 Ci/m mol) was diluted with ethanol to give an ethanol solution containing 40,000 dpm/1 0 yl (45 pg as DHT).

3) 0.05 M tris-buffer solution (pH 8.0) containing 0.05% BSA and 0.1% of Bovine Serum y Globulin (referred to as BGG hereinafter).

4) Saturated ammonium sulfate solution.

5) Unlabelled T standard solution.

6) Unlabelled DHT standard solution.

Procedure 1 Determination of the titre of the anti-testosterone antibody.

A 3H-T standard solution (10 jut) was put in a glass tube and dried by evaporation of the solvent.

To this was added an antiserum (0.2 ml; diluted stepwise with a tris-buffer solution) and well mixed.

The mixture was allowed to stand at room temperature for 2 hours and was then treated with a saturated ammonium sulfate solution (0.2 ml). The solution was well stirred and centrifuged (3,000 r.p.m.) for 20 minutes to give a supernatant, of which 0.2 ml was put into a counting vial. The radioactivity of the sample was determined after addition of a scintillator [2 ml; prepared by dissolving 2 g of 2,5-diphenyloxazole (PPO) toluene (one litre)l.

Procedure 2 Determination of the cross-reactivity of the anti-testosterone antibody with DHT.

Ten yl of a 3H-T standard solution was put into glass tubes and the tubes were divided into two groups. Unlabelled standard solutions of T and DHT were respectively added to the tubes containing 3H-T standard solutions of two groups. However, the amount of the unlabelled steroids added was stepwise increased on each occasion. All of the mixed solutions were dried by evaporation and to each tube, 0.2 ml of antiserum was added and well mixed. Antiserum was used at the dilution which bound 60% of the 3H-T (45 pg).

Procedure 3 Determination of the titre of the anti-DHT antibody.

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

Procedure 4 Determination of the cross-reactivity of anti-DHT antibody with T.

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

Example 1 Preparation of anti-testosterone specific antibody having low cross-reactivity (mouse): Three groups of mice (each group) consisting of 4-5 female mice; C57BL/6 strain; 8-10 weeks old) were used in this Example. Saline solution alone was adminstered to each mouse of the first group (control group) and no DHT-3-D-GL was given. Three days before primary immunization with T3-KLH, DHT-3-D-GL (500 jug) was administered to each mouse of the second group by i.p. injection.

T-3-KLH (100,ug/mouse) in saline together with Freund's complete adjuvant was administered (i.p.) to all the mice and 3 weeks after this, T-3-KLH in saline together with Freund's incomplete adjuvant was administered in the same amount. The same amount of T-3-KLH in saline was also administered to each mouse after 5, 7, 9 and 17 weeks.

Three days before the secondary immunization and 3 days before the tertiary immunization (5 weeks after the primary immunization) with T-3-KLH, DHT-3-GL (500 jug) was administered on each occasion, to the mice of the third group.

Serum was collected from retro-orbital plexus of the mice successively at two week intervals to determine the titre and cross-reactivity of the antibody. The cross-reactivity was determined by Abraham's method. The results are shown in Table 1 below.

Table 1 (A) Titre of anti-testosterone antibody*, and (B) its cross-reactivity with DHT4* Weeks Group 1 Group 2 Group 3 pre-treated with DHT-3 (not pre- D-GL f500 jug, (notpre- treated) ip.) treated)*** 9 A 3331 1004*** Formation of B 30.3+4.9 7.1+1.9*** antibody: + 11 A 4320 1711 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 38.8+10.1 7.4+4.1 Notes: *-Titre is expressed by the reciprocal number of the dilution of the serum capable of binding 50% of 3H-testosterone (45 pg).

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

***-Mean value of antibody titre is expressed by the geometric mean, and crossreactivity is expressed by the arithmetic mean + standard deviation.

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

As is apparent from this table, the antibody titer of the second group was slightly lower than that of the first (control) group, and the kinetics of antibody formation of the second group was similar to that of the control group. On the other hand, in the third group no significant antibody formation was observed throughout the entire period. The cross-reactivity with DHT of the second group which was pretreated with DHT-3-D-GL was substantially lower than that of the control group.

The relationship between the antibody titre and cross-reactivity of the antibody obtained from each of the test mice was plotted. However, although the data are not shown here, no significant relationship between the antibody titre and cross-reactivity was found. In general, it was found that the treatment with DHT-3-D-GL results in a decrease in cross-reactivity with DHT and this was not correlated with a decrease in the antibody titre. In other words, a decrease in the cross-reactivity did not result in a significant decrease in titre.

From these facts it is postulated that, when clones having a cross-reactivity with DHT are removed by pretreatment with DHT-3-D-GL, subsequent immunization with T-3-KLH may selectively enhance the formation of antibody-forming clones having a higher specificity with respect to T. The formation of antibodies having a low cross-reactivity according to the method of this invention is an important advantage as described hereinbefore.

Example 2 Preparation of a specific anti-DHT antibody having low cross-reactivity (mouse): Five groups of mice (each group consisting of 5-7 female mice; C57BL/6 strain; 8 weeks old) were used in this Example. Mice of the first group (control group) were not treated with T-3-D-GL and the primary immunization was effected using DHT-3-KLH. Three and 5 weeks respectively after this, the secondary and tertiary immunizations were effected, followed by additional immunization treatments which were effected at 2 weeks intervals. Mice of other groups were also immunized in this manner simultaneously with the mice of the control group.

Three days before the primary immunization with DHT-3-KLH, T-3-D-GL (500,ug/mouse) was administered to the mice of the second group by ip. injection. As another control group, mice of the third group were treated with DHT-3-D-GL (500 jug/mice) by i.p. injection, 3 days before the primary immunization with DHT-3-KLH.

Three days before the secondary immunization which was effected 3 weeks after the primary immunization using DHT-3-KLH, T-3-D-GL and DHT-3-D-GL (each 500 jug/mouse) were respectively administered (ip.) to the mice of the 4th and 5th groups. From 7 weeks after the primary immunization, serum was collected from each of the mice of 5 groups at 2 week intervals to determine the antibody titre and cross-reactivity. The results obtained by using the serum collected 13 weeks after the primary immunization as shown in Table 2.

Table 2 Cross Group Antibody titer* reactivity** 1 3457 90.3+12.9 2 1286 38.2+14.6 3 1079 86.3+23.7 4 0 5 0 *1 and *2: Cf. footnote of Table I Table 2 indicates that anti-DHT antibody having a 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 was observed in the 4th and 5th groups which had been treated with T-3-D-GL or DHT-3-D GL after the primary immunization.

In the 3rd group, the mice were pre-treated with DHT-3D-GL and were then immunized with DHT-3-KLH, the thus-formed anti-DHT antibody showed a high cross-reactivity with T as in the case of the control mice and almost the same magnitude of antibody titre was observed in the 2nd and 3rd groups. From these results, it can be concluded that an anti-DHT antibody having a low cross-reactivity with T was obtained by treatment with T-3-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 about 3 kg) were used.

T-3-D-GL (10 mg/rabbit) was administered (ip.) to the rabbits of the first group three days before primary immunization with 100 jug of DHT-3-KLH emulsified in complete Freund's adjuvant, and 3 weeks after this, the rabbits were given subcutaneously a secondary immunization with 200 jug of DHT-3-KLH in incomplete Freund's adjuvant.

They were then consecutively boosted subcutaneously four times with 200 jug of DHT-3-KLH in complete Freund's adjuvant and incomplete Freund's adjuvant alternately at monthly intervals.

The control rabbits (the 2nd group) were also immunized with DHT-3-KLH in a similar manner to that applied to the first group. However, T-3-D-GL was not given to them. The rabbits of the 3rd group were treated with (ip.) T-3-D-GL (10 mg/rabbit) 3 days before the secondary and tertiary immunizations with DHT-3-KLH, which were effected 3 and 7 weeks respectively after the primary immunization.

The rabbits of the 4th group were treated (ip.) with the DHT-3-D-GL (10 mg/rabbit) 3 days before the primary immunization with T-3-KLH. The method of immunization with T-3-KLH was effected in a similar manner to that used for the first group.

In the 5th group (control group), the rabbits were immunized with T-3-KLH in a similar manner to that used for the 4th group. However, no DHT-3-D-GL was administered to them.

In the 6th group, the rabbits were treated (ip.) with DHT-3-D-GL (10 mg/rabbit) 3 days before the secondary and tertiary immunizations with T-3-KLH (effected 3 and 7 weeks after the primary immunization, respectively).

Ten days after the above-mentioned booster injection procedures, serum was collected from each rabbit and determined to give the following results.

In the 3rd and 6th groups, no significant formation of the antibody was observed throughout the entire period. Anti-DHT and anti-T antibodies obtained respectively from the 2nd and 5th groups (control groups) showed substantially the same reactivity on both the hapten used for immunization and the cross-reactive hapten. That is, the anti-DHT antibodies of the rabbits of the 2nd group showed a 100% cross-reactivity with T and the anti-T antibodies of the rabbits of the 5th group showed a 95.2% cross-reactivity with DHT. On the other hand, the rabbits of the first and 4th groups gave significantly low cross-reactivities, and the anti-DHT antibodies from the rabbits of the first group showed a 11.9% cross-reactivity with T and the anti-T antibodies from the rabbits of the 4th 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 obtained from the rabbit: 1) Anti-T and anti-DHT antibodies having low cross-reactivity were obtained from the rabbits of groups 1 and 4 of the above-mentioned Examples. The following tests were effected to investigate the practical feasibility of using these antibodies for determining the T and DHT present in human serum.

For this purpose, given amounts of T and DHT were respectively added to samples of human serum, and the relationship between the added amount and the amount of T or DHT which was determined by radioimmunoassay using the said antiserum was investigated. In order to determine the level of T present in human serum, T (1, 2 and 4 ng) or DHT (0.2; 0.2; and 0.3 ng) was added to a pooled serum (1 ml; collected from female and having a low T value), which was then treated with 3H-T (2000 dpm) or 3H-DHT (2000 dpm) in order to determine the recovery in the extraction step. On each occasion, the mixture was well mixed and a mixture of hexane/ether (3:2,3 ml) added, followed by shaking for one minute with a vortex mixer. After this, the test tube was allowed to stand in a dry ice/acetone bath for 10 seconds and the organic solvent layer transferred to another test tube.The organic solvent was evaporated off and the residue was dissolved with ethanol (2 ml) with shaking. A part of the ethanol solution was put into a small test tube. The amount of the solution transferred to the test tube was adjusted so as to enable the determination of T from the inhibition curve caiibrated by using the above-mentioned antiserum. The solution was dried by evaporation and subjected to radioimmunoassay using the low cross-reacting anti-T antibody which was obtained by DHT-3-D-GL treatment. On the other hand, an ethanol solution (0.5 ml) was put into a vial and dried by evaporation.

A scintillator was added to the residue and counted to determine the recovery in the extracting step.

This recovery ratio was used to correct the value obtained by the radioimmunoassay.

in order to determine the level of DHT present in the serum, the pooled serum obtained from female humans (1 ml) was treated with DHT (0.2 and 0.5 ng) or T (0.2 and 0.5 ng) and 3H-T (2000 dpm) or 3H-DHT (2000 dpm) was then added in order to determine the recovery. Hexane/ether (3:2, 3 ml) was added to the mixture and extracted in a similar manner to that described above. Ethanol (2 ml) was added to the residue of the extract and a part of the ethanol solution was put into a small test tube.

The amount of the solution in the test tube was adjusted to be sufficient to enable the determination of DHT using the inhibition curve of DHT obtained by using the above-mentioned antiserum. The solution was dried by evaporation and subjected to radio immunoassay using the antiserum, of low cross reactivity which was obtained by the treatment with T-D-GL. On the other hand, the ethanol solution (0.5 ml) was put into a vial and ethanol was removed by evaporation. To the residue was added the scintillator and counting was effected to determine the recovery in the extracting step. This recovery ratio was used to correct the value obtained by the radioimmunoassay.

As a result, it was found that when an anti-T antibody having low cross-reactivity was used to measure added T (1, 2 and 4 ng) of normal female human serum originally having a 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 and 0.3 ng) was added to the female human serum instead of T, in order to investigate the influence of DHT upon the amount of T assayed, no significant influence was found and the level of T in the normal female human serum was almost unchanged.

In a separate experiment, DHT (0.2 and 0.5 ng) was added to the normal female human serum and the amount of DHT in the serum was determined by the use of an anti-DHT antibody having low cross-reactivity. It was found that the amount of DHT in the normal female human serum before addition was 0.21 ng, then changed after addition to 0.41 and 0.72 ng, respectively after addition in each occasion. These values were sufficiently in conformity with the added amounts of DHT.

On the other hand, when T (0.2 and 0.5 ng) was added to the normal female human serum instead of DHT to investigate the influence of T upon the level of DHT assayed, no significant change of the DHT level in the normal female human serum was made by addition of T, It is apparent that T and DHT levels in the serum can be exactly determined by using the antibodies having low cross-reactivity described in Example 3.

2) Assay of a sample containing a mixture of specific antigen and cross-reacting antigen: The DHT level in human blood was again determined by using a low cross-reactive antibody originating from rabbits, as described above. On each occasion, DHT and T (5 and 10 ng; 10 and 5 ng; or 10 and 10 ng, respectively) (0.1 m) were simultaneously added to female human pooled serum and the determination was effected in a similar manner to that described in (1) above. As a result, it was found that, as shown in Table 3, even when a large amount of T, i.e. a cross-reactive antigen is present in the sample, the amounts of DHT were 5.67, 10.17 and 9.20 ng on each occasion.Thus it can be said that DHT may be assayed accurately by the use of the low cross-reactive anti-DHT antibody obtained in Example 3, even when a large amount of T which is the cross-reactive antigen is present in the sample.

Table 3 Amount of DHT added (nag) Amount of T added (ngJ DHTFound lngl 5 10 5.67 10 5 10.17 10 10 9.20 Similarly, T and DHT (respectively 5 and 10 ng; 10 and 5 ng or 10 and 10 ng) were added to the serum (0.1 ml) and T levels were determined by 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, from which it is apparent that when above mentioned anti-T antibody of low cross-reactivity is used, T is accurately assayed in the presence of DHT which serves as a cross-reactive antigen.

Table 4 Amount of T Amount of DHT TFound added (nag) added (ng) (nag) 5 10 6.80 10 5 10.3 10 10 10.59 3) Assay of human sample (direct method): The term "direct method" denotes the assay method which does not require prior separation of T and DHT. For comparison, the values obtained by a conventional method are also indicated. In the latter method, T and DHT are separated by paper chromatography prior to determination.

3.1) Assay of T in human serum: (A) Preparation of Sample Male human serum (each 0.1 ml) and female human serum (each 0.5 ml) were put into test tubes, respectively, and 3H-T (each 3000 dpm) added in order to determine the recovery in the extraction step or extraction-chromatography step. Distilled water (each 0.5 ml) was added to the male human serum. A mixture of hexane/ether (3:2, each 3 ml) was added to the serum samples and well mixed by using a vortex mixer for one minute. The test tubes were then put into 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 human serum samples and well mixed, and the solution was transferred into a radioimmunoassay tube. The amount of the solution in the test tube was adjusted to fall into a range of 50 to 400 pg, that is, 100 jul in thecase of normal range and 50 jul in the case of the serum of high T level such as test no. 1, 2 and 3 in Table 5 described hereinafter. In order to determine the recovery in the extraction step, the solution (100 jul) was transferred into a vial and dried by evaporation. After addition of a scintillator, the residue was counted.

In the case of female human serum, ethanol (0.7 ml) was added to each residue, and the ethanol solution (0.5 ml) transferred to a radioimmunoassay tube. In order to determine the recovery in the extraction step, the solution (100 jul) was put into a vial and dried by evaporation. The residue was counted after addition of a scintillator.

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

(C) Paper Chromatography The dried extract prepared in a similar manner to that used in the direct method was applied to a paper with three 50 jul rinses of chloroform. On an identical strip of paper, T (10 jug) was applied as a reference marker and run with each set of determinations. The paper strips were set in a chromatograph tank containing Bush A (a solvent system of cyclohexane/methanol/water=1 0:8:2).

After 2 hours for equilibration, they were developed in the upper layer of Bush A.

After developing for 16 hours, the spot of T used as a reference was detected by ultraviolet absorption at 254 nm and a 3 cm long section of the test paper corresponding to the location of the said spot of reference marker was cut off from the paper and extracted with ethanol (3 ml). Ethanol was removed by evaporation under a nitrogen stream, and the residue was used for the radioimmunoassay in a similar manner to that described in the direct method.

(D) Radioimmunoassay In order to prepare a T standard curve, ethanol solution containing 0. 20, 50, 100, 200 or 400 pg.

of T was put into test tubes in duplicate. To these test tubes which contained test samples, was added an ethanol solution (each 10,us); containing 20,000 dpm of 3H-T/1081 and then the ethanol was removed by evaporation. An anti-T antiserum originating from the rabbit was diluted with a 0.05M trisbuffer solution [pH=8.0; containing 0.05% BSA and 0.19/0 BGG] to the concentration (Bo=60%) which is capable of binding 60% of 20,000 dpm of 3H-T (45 pg. as T), and 0.2 ml of this antiserum solution was put into each test tube, followed by mixing by the use of a vortex mixer.Separately, this tris-buffer solution (each 0.2 ml) was transferred to two test tubes, each contained 20,000 dpm of 3H-T alone, and well mixed to obtain a total dpm (Bo=100%). After standing at room temperature for 2 hours, saturated ammonium sulfate (0.2 ml) was added to the solution and well mixed. Centrifugation was then effected at 3000 rpm for 10 minutes, and 0.2 ml of the supernatant was then transferred into a vial.

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

3.2) Assay of DHT in the female human serum; Sampling of serum, extraction and separation of DHT and T by paper chromatography were conducted in the same manner as for the assay of T using a female human serum sample. The location of a reference DHT marker was detected by spraying with a solution of equal volumes of a 1% absolute ethanol solution of m-dinitrobenzene and 2.5 N sodium hydroxide in absolute ethanol.

Radioimmunoassay of DHT was effected by the use of the standard ethanol solution of DHT and an ethanol solution of 3H-DHT (40,000 dpm/10,ul) in a similar manner to that used for radioimmunoassay of T. In both direct and conventional paper chloromatography methods, the result from the radioimmunoassay was corrected by the recovery ratio.

In the direct method, the recovery ratio of T or DHT was within a range of 95 to 100%, and therefore the recovery correction by the addition of 3H-T or 3H-DHT may not be needed for routine work.

The results are shown in Tables 5 and 6.

(E) Results and analysis: Table 5 Serum used Amount of T found (ng/ml) Test (*male; No. ** female) Direct method Paper chromatography 1 * 10.35 9.60 2 * 13.34 15.36 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 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 13 ** 0.340 0.356 14 ** 0.500 0.490 15 ** 0.250 0.240 16 ** 0.154 0.218 It is apparent from this table that the results from two assay methods are substantially in conformity with each other.

Table 6 (Serum UsedSerumfrom Female Human) pHT found (ng/ml) Test No. Direct method Paper chromatography 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 6 0.250 0.260 7 0.221 0.232 8 0.201 0.198 In this table, the results from two methods are substantially in conformity with each other.

As apparent from these experimental results, it is possible by the direct method of this invention and using a low cross-reactive antibody, to assay simply and accurately the desired substance even in the presence of a cross-reacting antigen. In this case, it is no longer necessary to separate out the cross-reacting antigen, prior to assay, by paper chromatography as used in conventional methods.

In the process of this invention a specific antibody as well as a clone capable of producing such an antibody may be obtained by pre-treatment of an animal with a conjugate of a cross-reactive antigen and D-GL so as to induce an immunological unresponsiveness to a cross-reactive antigen. The present invention is not, of course, restricted to the above-mentioned specific embodiments using T-3 D-GL and DHT-3-D-GL, and may be applied in a wide variety of cases, such as exemplified by Example 5 hereinafter, in which substantially the same results were obtained even when T and DHT were coupled to the carriers at a different site. In the following example, T and DHT were again used as model systems and their coupling site was changed from the 3rd to the 15th position, so that the structure of the hapten exposed on the surface of carrier molecules was changed.

Example 5 Properties of an antiserum obtained by using a conjugate of 1 SP-ca rboxyethyl me rcapto- testosterone {hereinafter referred to as 1 SP-CEM-T) and 1 5-carboxyethylmercapto-5a- dihydrotestosterone (hereinafter referred to as 1 CSM-5a-DHT) with KLH and D GL: In this example, 1 5-CEM-Twas synthesized by the method reported by Rao, P. N. et al: [Steroid, 28, p. (1976)] and 15p-CEM-5cr-DHT was synthesized by the method reported by Rao, P. N., et al: [Steroid, 29 p.171 (1977)1.

The conjugation of 15p-CEM-T to D-GL and KLH and the conjugation of 15,B-CEM-5cg-DHT to D GL and KLH were performed in a similar manner to that used for the above-mentioned conjugation of DHT-3-CMO or T-3-CMO to D-GL or KLH. The conjugate thus obtained are referred to as T-1 5-D-GL, T 1 5-KLH, DHT-15-D-GL and DHT-1 5-KLH, respectively.

In this example, 6 groups of mice (each group consisting of 7 mice; C57BLS6 strain; 8-10 weeks old) were used.

The first group is a control group and saline (without DHT-1 5-D-GL) was administered to the mice. DHT-1 5-D-GL (each 500 jug/mouse) was administered to the second group three days before primary immunization. The 3rd group is another control group and T-1 5-D-GL (each 500 yg/mouse) was administered (i.p.) to the mice 3 days before the primary immunization with T-1 5-KLH. T-1 5-KLH (100 ssg/mouse) was used for immunization of the first, second and third groups. After three weeks, the secondary immunization of these mice was effected, and then additional booster immunizations were effected every 2 weeks after the secondary immunization.The 4th, 5th and 6th groups of mice were immunized with DHT-15-KLH. Three days before the primary immunization with DHT-15-KLH, T-15-D- GL (500 jug/mouse) was administered (i.p.) to the mice of the 5th group. Simultaneously with 5th group, the mice of the 4th group served as a control group and saline instead of T-1 5-D-GL was administered to this group. DHT-15-D-GL (500 jug/mouse) was administered (i.p.) to the mice of the 6th group (another control group) 3 days before primary immunization with DHT-15-KLH.

Seven weeks after primary immunization, serum was collected from each mouse at 2 week intervals in order to measure the antibody titre and cross-reactivity. The antiserum obtained 13 weeks after primary immunization had the highest antibody titre, from which the following results were obtained.

Properties of anti-T antisera from the first, second and third groups: When expressed by the reciprocal of the dilution of antiserum capable of binding 50% of 3H-T (45 pg), the anti-T antibody titres of the mice of the first (control) and second groups are within the ranges of 1,600 to 9,000 and 1,000 to 3,500, respectively, and the mice of the third group which had been given T-1 5-D-GL showed no formation of the antibody.

When determined by Abraham's method, the antiserum from the mice of the first group showed cross-reactivities within a range of from 4.1 to 8.2, whereas cross-reactivities of from 0.27 to 0.94 were obtained in the antiserum from the mice of the second group. The results of the 2nd group are superior to the cross-reactivities with DHT obtained by any other anti-T antisera reported by others.

Thus it can be said that the cross-reactivity with DHT of the obtained antiserum was practically negligible according to the present invention. It is known that 1.81% is the lowest cross-reactivity of any anti-T antiserum reported in the literature in so far as DHT is concerned and that Rao et al obtained this low value by the use of anti-T antiserum prepared by immunizing rabbits with 1 SP-CEM-T-BSA [P.

N. Rao et al: Steroid, 28 p. 101 976)j which is also used in the above-mentioned example. The cross- reactivities of the mice of the first group in this example are substantially equivalent to this lowest value.

Properties of anti-DHT antisera from the 4th, 5th and 6th groups: When expressed by the reciprocal of the dilution capable of binding 50% 3H-DHT (45 pg), the anti DHT antibody titres of the 4th group (control group) were within a range of from 1,500 to 5,600 and the corresponding value of the 5th group to which T-1 5-D-GL was administered were from 1,000 to 7,600. The mice of the 6th group to which DHT-1 5-D-GL was administered showed no formation of antibodies.

When the antisera obtained from the mice of the 5th group, which had been treated with T-15-D- GL, were investigated by Abraham's method, the cross-reactivities with T of the thus-obtained antisera were within a range from 6.5 to 19.0% in contrast to the cross-reactive values of from 48.3 to 68.5 in antisera from the mice of the 4th group (control group). These facts indicate that the administration of T-1 5-D-GL significantly reduced the cross-reactivity with T.

No formation of antibodies was found in the mice of the 6th group to which DHT-1 5-D-GLwas administered. The reason therefore is believed to be the specific inactivation of anti-DHT antibodyproducing clones by administration of DHT-D-GL.

The reagents and assay method used in the undergoing Examples 6-9 are explained as follows.

in these Examples, the invention is applied to peptide determinants.

(6) Preparation of Pentagastrin-D-GL Conjugate Reference: Liu Et Al, Biochemistry, viol. 18. 690 (1979) (6-A) Synthesis of S-acetylmercaptosuccinyl-D-GL (Hereinafter Referred to as Ac-S-D-GL) D-GL (Mw=34,300; 40 mg=1.166 jumol) was dissolved in a 0.125m phosphate buffer solution (900 jut; pH=7.2) and the pH was adjusted to 7.2 with 1 N NaOH solution. After the addition of 50 jul (57.44 jumol) of a dimethylformamide solution (hereinafter referred to as DMF) of Sacetylmercaptosuccinic anhydride (200 mg/ml), the mixture was stirred at room temperature for 30 minutes. During the reaction, the pH of the mixture was kept at 7.2.After completion of reaction, the solution was applied to a Sephadex G-25 column, equilibrated with 0.01 M phosphate buffer saline (pH=7.2) containing 0.01 M Na2-EDTA so as to separate the reaction product from unreacted Sacetylmercaptosuccinic anhydride. A fraction (100 jut) of the solution containing the reaction product was added with an aqueous solution of 0.5M hydroxylamine (100,ul=50 jumol; pH=7.3) and incubated at 370C for 20 minutes to remove the protecting acetyl group. The numbers of sulfhydryl groups introduced in D-GL were determined by the method of Ellman et al [Arch. Biochem. Biophys., vol. 82, p.

70 (1959)] in the following manner. The reaction solution (50 jut) was added to a methanol solution of 0.01 M 5,5'-dithiobis-(2-nitrobenzoic acid) (deoxidized; 0.1 ml) and 1 ml of tris-buffer solution (pH=8.0) and reacted for 20 minutes, and the absorbance at 412 nm was measured. The number of Sacetylmercapto groups introduced into D-GL was about 1 5/molecule. The thus-obtained Sacetylmercaptosuccinyl-D-GL was hereinafter referred to as Ac-S,5-D-GL. The yield was approximately 77%. As D-GL has no absorbance at 280 nm, it was not possible to measure the recovery of the fraction containing the D-GL derivative by the absorbance at 280 nm.The recovery was therefore determined by using, as monitoring substance, a reaction product which was obtained by the reaction of N-succinimidyl-3-(4-hydroxyphenyl)propionate and D-GL, and applied to a column of Sephadex G25. As this product does have an absorbance at 280 nm, the determination was conveniently measured by the absorbance at 280 nm.

(6-B) Preparation of m-maleimidobenzoyl-pentagastrin (Hereinafter Referred to as MBpentagastrin) Pentagastrin (11 mg; 16.1 jumol) was dissolved in 0.1 M phosphate buffer (9.5 ml; pH=8.0) and was then added in one one portion to m-maleimidobenzoyl-N-hydroxysuccinimide ester (25.3 mg, 80.5 jumol; hereinafter referred to as MBS) dissolved in DMF (one ml). The mixture was stirred, and the reaction was monitored by thin layer chromatography using a solvent system of cyclohexane/ethyl acetate=1 :1 by voiume. Twenty-five minutes after the beginning of the reaction, dichloromethane (3 ml) was added to the reaction mixture to remove unreacted MBS. The mixture was well agitated and centrifuged.

The upper layer of the buffer solution was put into another test tube. The dichloromethane layer (lower layer) was treated with a small amount of a phosphate buffer, well mixed and centrifuged. The upper layer was combined with the said solution. By thin layer chromatography, it was confirmed that the unreacted MBS was almost completely removed from the fraction of the buffer solution containing MB-pentagastrin.

Separately, the above-mentioned buffer solution (20 jut) containing MBS-pentagastrin was added to an aqueous solution of 2-mercaptoethenol (20 jul; 70 n mol; deoxidized with nitrogen stream). The reaction was effected at room temperature for 20 minutes, and the molar quantity of 2 mercaptoethanol consumed was determined by Ellman's method, which correspond to the quantity of maleimidobenzoyl group introduced into pentagastrin. The number of maleimidobenzoyl group introduced into one molecule of pentagastrin was 0.9. The compound was hereinafter referred to as M B,,-pentagastrin.

(6-C) Preparation of a Conjugate of Pentagastrin and D-GL A solution of Ac-Srs-D-GL prepared in 6-A and a solution of MB09-pentagastrin prepared in 6-B were mixed together to effect the reaction in the following manner. After complete deoxidation in a nitrogen stream, a 5M aqueous solution of hydroxylamine (500 jul; pH=7.3) was added to the mixture and the mixture stirred at room temperature for one hour. Then part of the reaction solution was removed to investigate the presence of unreacted groups. None of such groups was found. That is, there were no SH groups which had not bound with MB-pentagastrin. Thus, it was confirmed that all of the SH groups held by D-GL had reacted with MB-pentagastrin.

Two hours after addition of the aqueous solution of hydroxylamine, 2-mercaptoethanol was added to the reaction mixture to give a final concentration of 1 mM, followed by stirring for 20 minutes.

After this, the reaction mixture was dialyzed against 0.01 M phosphate buffer solution (pH=7.2) for about 24 hour using a cellulose membrane, and the thus-obtained solution 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 was hereinafter referred to as pentagastrin,6-D-GL.

(7) Preparation of CCK-8-P-keyhole Limpet hemocyanin (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 Ac-S-D-GL as described in 6-A.

Prior to the synthesis, KLH (70 mg) was dissolved in a 1% solution of K2CO3 (1.4 ml) and dialyzed against a 0.125M phosphate buffer solution (pH=7.2) and the absorbance at 280 nm was measured so that the amount of KLH was determined.

S-acetylmercaptosuccinic anhydride (6.5 ssmol) was added to the KLH solution of 0.7 ml containing 26.0 mg (corresponding to 260 nmol assuming that the molecular weight is about 100,000; pH=7.2), and the synthesis was effected in a similar manner to that described in 6-A. The molar ratio of the S-acetylmercapto group to KLH in the product was 6.8 (hereinafter referred to as Ac S08-KLH).

(7-B) Preparation of m-maleimidobenzoyl-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 n mol) synthesized by the fragment condensation method was dissolved in 0.2M phosphate buffer (0.5 ml) and subjected to reaction with MBS (600 jug, 1.9 ymol) by using 50 jut of a DMF solution. (1.2 mg MBS dissolved in 100 jul DMF). The reaction product contained CCK-8-P and MB a ratio of 1:1.0 (hereinafter referred to as MB, o-CCK-8-P).

(7-C) CCK-8-P-KLH An Ac-S,,-KLH solution (2.3 ml; 75.9 n mol) prepared in 7-A and an MB, oCCK-8-P solution prepared in 7-B were mixed together and 0.5M NH20H (0.3 cc; 150 jumol) added. After this, an analogous treatment to that described in 6-C was effected. The ratio of CCK-8-P to KLH in the resultant product was about 5:1.

(8) CCK-8-P-bovine Serum Albumin (Hereinafter Referred to as CCK-8-P-BSA) (8-A) Preparation of S-acetylmercaptosucci nyl-BSA Synthesized in a similar manner to that described in 6-A using BSA (25 mg; 351 n mol) and Sacetylmercaptosuccinic anhydride (1.5 mg; 8.8 jumol). The ratio of BSA to S-acetylmercapto group in the product was 7.6:1.

(S-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 subjected to the reaction with MBS (700 jug, 2.29 pmol) by using 50 jul of DMF solution (1.4 mg MSB dissolved in 100 jul DMF) in a similar manner to that described in 6-B. The ratio of MB to CCK-8-P was 1.0:1. The product was referred to as MBt o-CCK-8-P hereinafter.

(8-C) Preparation of CCK-8-P-BSA The solution obtained in 8-A (1.9 ml; 84 nmol) and the solution obtained from 8-B were mixed together and added with 0.5M NH2OH (0.35 cc; 175 jumol). The mixture was treated in a similar manner to that described in 6C to obtain a conjugate which comprises the ratio of CCK-8-P to BSA was about 5:1 (referred to as CCK-8-P-BSA hereinafter).

(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 mi; pH=8.0) and subjected to reaction with MBS (1.05 mg; 3.3 jumol) by the use of 50 jul of DMF solution (SO jul) prepared by dissolving MBS (2.1 mg) in DMF (1Q0jul). The reaction was effected in a similar manner to that described in 6-B. The ratio of MB to pentagastrin was 1.0:1. The product was referred to as MB.O-pentagastrin hereinafter.

(9-B) Preparation of Pentagastrin-BSA A solution (2.54 ml; 112 nmol) prepared in 8-A was mixed with the solution prepared in 9-A.

0.5M NH2OH (0.4 cc; 200 ssmol) was added to the mixture and treated in a similar manner to that described in 6-C. The ratio of pentagastrin to BSA in the product was about 5:1. The product obtained was referred to as pentagastrin6-BSA.

(10) A similar procedure to that described in the above-mentioned 6 was repeated except for the use of a D-GL having a molecular weight of 11 5,000 instead of one having a molecular weight of 34,300 (The reaction ratio being the same as that described in 6). Pentagastrin33-D-GL was obtained having the same density as the antigen determinant combined with D-GL on the molecular surface.

Example 6 (A) Schedule of Immunization and Collection of Serum (C57BV6JxDBA/2) F1 female mice, each group consisting of 6 mice were immunized.

All mice in each group were immunized with CCK-8-P-KLH (each 10 jug) in Freund's complete adjuvant (each 0.2 ml), and 3 weeks after this, they were further immunized with CCK-8-P-KLH (each 10 jug) in Freund's incomplete adjuvant (0.2 mi). Two weeks after the secondary immunization, additional booster immunizations with CCK-8-P-KLH (each 10 jug) mixed with 2 mg of aluminium hydroxide gel were effected. Two and 4 weeks after this, they were further immunized twice by injection of 0.2 ml of saline solution containing CCK-8-P-KLH (each 10 jug). All treatments were effected by i.p. injection.

Three days before primary immunization, 0.5 ml of saline solution of pentagastrin,s-D-GL (each 300 yg) was administered to all the mice of the second group. The mice of the first group served as the control and 0.5 ml of saline without pentagastrin,,-DGL was administered to each mouse in this group.

Three days before the secondary immunization and 3 days before the tertiary immunization, 0.5 ml of saline solution of pentagastrin,6-D-GL (each 300 yg) was administered (i.p.) to all the mice of the 3rd group.

Blood was taken out from the retro-orbital plexus of each animal to prepare the serum.

(B) Assay of Antibody Titre Effected by radioimmunoassay. Each 100 jut of a 0.01 M phosphate buffer solution (pH=7.2; containing 0:15M NaCI referred to as PBS, hereinafter) containing 10 ug/ml of an antigen (as CCK-8 P-BSA or pentagastrin-BSA) was dispensed into each well of a polyvinyl-made round bottom microplate for solid phase radioimmunoassay and the antigen was bound to the surface of the polyvinyl plate by incubation at room temperature for 2 hours. The antigen solution in the well was discarded and the well was washed 4 times with tap water. After flicking off the excess water, 200 jul of 1% BSA saline was added to each well and allowed to stand at 40C overnight so that the protein-combining ability of the polyvinyl plate was completely quenched.After this, the solution was removed from the wells and the plate rinsed with tap water 4 times. The plate was used for radioimmunoassay after flicking off the excess water.

0.1 ml of the antiserum diluted with 1% BSA-PBS was dispensed into each well which was allowed to stand at 40C overnight, washed well with tap water three times, washed with 2M NaCI-PBS 6 times, and washed with tap water.

After flicking out the water, rabbit anti-mouse IgG Fab antibody which had been specifically purified by using an immunoadsorbent and labeled with 1251, was diluted with 1% BSA-PBS and each 0.1 ml (50,000 dpm/20 ng as specific antibody protein) of the diluted solution was dispensed into each well and allowed to stand at 40C overnight. If there is any antibody capable of binding with an antigen on the surface of polyvinyl, then the antibody will be coupled with the antigen. The presence or absence of such an antibody can be detected by the degree of coupling of the rabbit anti-mouse IgG Fab antibody labelled with 1261. Each isotope solution in the well was removed by using a pipette.After washing throughly with water, the flexible plate was placed in a plastic rigid plate, and the top of the flexible plate was cut off with hot wire, and then the radioactivity in each well was counted.

(C) Results At each time of collection of the serum, the antibody titre of the pooled serum from the mice of each group was assayed. Samples collected 11 weeks after the primary immunization contained maximum levels of antibody. Therefore, antibodies in the antisera collected 11 weeks after primary immunization were subjected to further detailed analysis.

In this measurement, pooled antiserum from group 1 collected 7 weeks after the primary immunization was used as the standard antiserum. The amount of antibody in the test sample obtained from individual mice at 11 weeks was expressed as the ratio to the amount of antibody contained in the standard antiserum which was defined as 1 unit The titre of the standard serum was as follows.

When assayed by the following liquid phase radioimmunoassay method using CCK-8, this serum (with x600 dilution) was capable of binding 0.0015 pmol of cholecystokinin (CCK-33).

With regard to the antibodies obtained from the mice of the first group, to which no pentagastrin,5-D-GL was administered, (shown by the mark "o" in Fig. 1) the amount of antibody reacting with CCK-8-P and the amount of antibody reacting with pentagastrin were almost equal. It was found that antibodies obtained from the mice administered with pentagastrin,s-D-GL (i.e. the second and third groups of mice shown by the marks "ski' and '," in Fig. 1, respectively) showed a very low titre of antibody reactive with pentagastrin. In particular, the antibodies obtained from the mice of the third group, to which pentagastrin,5-D-GL was given after the primary immunization (i.e. 3 days before the secondary and tertiary immunizations) showed an extremely low titre of antibody reactive with pentagastrin.

From these results, it is apparent that by use of the present invention, an antibody having a specificity for the specific amino acid sequence of CCK-8, i.e.

has been obtained.

Example 7 The cross-reactivities of the antibodies obtained above were determined by liquid phase radioimmunoassay system which allows the coexistence of CCK-8-P and pentagastrin. CCK-8-P was labelled with 1251 using Bolton-Hunter reagent [H. Sankaran et al. J. Biol. Chem., Vol. 254, 9349- 9351 [1 979)j and hereinafter referred to as 1251-BH-CCK-8-P. 50 yl of 0.02M phosphate buffer solution (pH=8.0; containing 1% gelatin) containing various amounts of unlabelled CCK-8-P was added to small test tubes to establish a CCK-8-P standard curve. Separately, 50 jul of a buffer solution containing various amounts of pentagastrin was added to a small tube to establish a pentagastrin standard curve.

On each occasion, antiserum (50 m,ul) diluted with normal mouse serum was added to the test tubes, to which was then added the same phosphate buffer solution (50 ,ul) containing '251-BH-CCK-8 (about 5000 cpm) and the solution was well mixed using a vortex mixer, followed by incubating at 40C for 24 hours. After this, 50 jul of rabbit anti-mouse IgG Fab antibody diluted to 1:2 with the same phosphate buffer was added to the test tubes, mixed and incubated at 40C for 24 hours. After this, the test tubes were centrifuged (3000 r.p.m.) for 25 minutes. The supernatant was aspirated, and the radioactivity of the precipitate counted in a counter.

Instead of the standard solutions of CCK-8-P and pentagastrin, the same phosphate buffer was used in order to determine the radioactivity of total 1251-BH-CCK-8-P (count, B,, expressed as 100), and the bound ratios (B/Bo%) of the antibody with 1251-BH-CCK-8-P in the presence of CCK-8-P or pentagastrin at different concentrations were calculated.

The molar amounts of various peptides required for 50g- inhibition of binding of the antibody with '25l-BH-CCK-8-P are as follows.

In the first group, CCK-8 was 1.1 pmol, and pentagastrin was 20.1 pmol. Thus, the crossreactivity calculated by Abraham's method was 5.5% (1.1/20.1 xl 00).

In the second group, in which pentagastrin,s-D-GL was administered before the primary immunization, the cross-reactivity was 2.7% (4 pmol/1 50 pmolx 100).

In the third group, in which pentagastrin,s-D-GL was administered after the primary immunization, i.e. 3 days before the secondary and tertiary immunization, no cross-reactivity with gastrin was found. That is, 1.7 pmol of CCK-8-P was required for 50% inhibition when the said antibody was used, but no inhibition was found by 10,000 pmol of pentagastrin. This means that pentagastrin is actually incapable of reacting with the antibody produced by the mice of the third group.

From these results, it is apparent that CCK-8-P may be specifically assayed without significant effect of coexisting pentagastrin by using the antibodies originating from the mice of the third group, to which pentagastrin,s-D-GL had been administered.

Example 8 A similar procedure to that described in Example 6 was repeated except for the use of rabbits instead of mice.

By the use of Freund's complete or incomplete adjuvant containing 100 jug of CCK-8-P-KLH and 10 ml of saline containing 10 mg of pentagastrin,s-D-GL, the results obtained were substantially similar to the results obtained by the use of mice.

Example 9 The same procedure of Example 6 was repeated except for the substitution of pentagastrin,s-D- GL with pentagastrin33-D-GL of molecular weight of 1 15,000. The results obtained were substantially the same as the results obtained by Example 6.

In Fig. 1, the abscissa shows the amount of antibody reacted with pentagastrin, and the ordinate shows the amount of antibody reacted with CCK-8-P. The mark "o" shows antibody obtained from the mice (the first group) to which no pentagastrin 15-D-GL was given. The mark 'A" shows the amount of antibody obtained from the mice (the 2nd group) to which pentagastrin15-D-GL was administered before the primary immunization. The mark t" shows the antibody obtained from the mice (the 3rd group) to which pentagastrinrs-D-GL was administered after primary immunization.

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

Claims

1. A process for producing an antibody having a high specificity to a first antigen comprising a desired antigenic determinant and having a low cross-reactivity with at least one other antigen, said other antigen comprising at least one antigenic determinant which is structurally related to the desired antigenic determinant of said first antigen, which process comprises administering to a mammal a copolymer of D-glutamic acid and D-lysine coupled with said other antigen whereby substantially effective immunological tolerance is induced and subsequently immunizing the mammal with said first antigen.

2. A process as claimed in claim 1 wherein the antibody is obtained in the form of an antiserum containing the said antibody.

3. A process as claimed in claim 1 wherein the antibody is obtained from a clone capable of producing said antibody.

4. A process as claimed in any one of the preceding claims wherein the mammal is a mouse, rat or guinea pig.

5. A process as claimed in any one of the preceding claims wherein the mammal is selected from rabbits, sheep, goats, horses, pigs and cattle.

6. A process as claimed in any one of the preceding claims wherein the copolymer of D-glutamic acid and D-lysine has a molecular weight of from about 27,000 to 120,000.

7. A process as claimed in any one of the preceding claims wherein the copolymer of D-glutamic acid and D-lysine has a molar ratio of D-glutamic acid to D-lysine of from about 70:30 to 30:70.

8. A process as claimed in any one of the preceding claims wherein the said other antigen is a steroid, catecholamine, peptide, pharmaceutical agent, or where appropriate a subunit and fragment thereof.

9. A process as claimed in claim 8, wherein the steroid is testosterone, Sa-dihydrotestosterone, androsterone, etiocholanolone, progesterone,17cg-hydroxyprogesterone, pregnenolone, dehydroepiandrosterone, oestradiol, oestrone, oestriol, aldosterone, deoxycorticosterone, cortisol, cortisone, corticosterone, 1 1-deoxycortisol, cholic acid, deoxycholic acid, lithocholic acid and conjugated compounds thereof.

10. A process as claimed in claim 8, wherein the catecholamine is dopamine, norepinephrine or epinephrine and their derivatives thereof.

11. A process as claimed in claim 8, wherein the peptide is gastrin, cholecystokininpancreozymin, insulin, proinsulin, C-peptide, glucagon, follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotrophin (HCG), somatostatin, thyroid stimulating hormone (TSH) or a subunit and related peptide thereof.

12. A process as claimed in claim 8 wherein the said pharmaceutical agent is 1 -propanolol or Ior d-cyclazocine.

1 3. A process as claimed in any one of claims 2 to 12 wherein the antibody as defined in claim 1 is isolated from the antiserum by methods known per se.

14. A process as claimed in any one of claims 3 to 12 wherein a clone capable of producing an antibody as defined in claim 1 is isolated from the said mammals by methods known peruse.

15. A process as claimed in any one of the preceding claims for the preparation of an antibody as defined in claim 1 substantially as herein described in any one of the Examples.

1 6. An antibody as defined in claim 1 when prepared by a process as claimed in any one of claims 1 to 13 and 15.

1 7. An antiserum containing an antibody as defined in claim 1 when prepared by a process as claimed in any one of claims 2 to 12.

18. A clone capable of producing an antibody as defined in claim 1, when prepared by a process as claimed in claim 3.

19. A clone capable of producing an antibody as defined in claim 1 when prepared by a process as claimed in claim 14.

20. A process for producing an antibody as defined in claim 1 which comprises the formation of a hybrid cell from a clone as defined in claim 1 8 or claim 1 9 and the implantation of said hybrid cell into a mammal whereby to produce an antibody as defined in claim 1.

21. A method for the immunoassay of a substance which comprises the use of an antibody produced by a process as claimed in any one of claims 1 to 13 and 1 5 or an antiserum containing such an antibody, the said antibody being specific to said substance whereby the assay is effected by reaction of the antibody with the said substance.