Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/385—Haptens or antigens, bound to carriers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

• This invention relates to immunogenic compositions; more particularly (but not exclusively) this invention relates to compositions which, on administration to human or non-human animals, provide an immunogenic therapy which suppresses fertility.
• the structure and synthesis of the decapeptide gonadotrophin- releasing hormone (Gn-RH) is known (Matsuo, H. et al. Biochem. Biophys. Res. Commun. 1971, 43 p. 1334). It is also known that the administration of Gn-RH subcutaneously or intradermally in laboratory species as a hapten linked to a carrier protein, for example bovine serum albumen (BSA) (although other carrier proteins have been used), elicits the production of an antibody titre in both males and females of that species.
• BSA bovine serum albumen
• compositions would in at least both domestic and animal husbandry environments provide an alternative to conventional surgical castration and speying ("immunogonadectomy"); moreover, they have the additional advantages that their effects are reversible or can be tailored: for example, reproduction and certain aspects of sexual behaviour (for example, aggression between males) could be suppressed while sufficient steroid hormone is secreted to promote growth without the need to resort (as is presently the case with gonadectomised animals) to anabolic agents, many of which are believed to be carcinogenic.
• immunogenic compositions will be made clear hereinafter.
• FCA Freund's Complete Adjuvant
• FCA Freund's Complete Adjuvant
• This comprises a mineral oil, an emulsifier and killed bacteria (usually mycobacteria), the complete adjuvant imparting a non-specific immunostimulant effect.
• FCA Freund's Complete Adjuvant
• a major problem impeding further progress in this field is the current lack of a suitable adjuvant; that is, one which will not cause tissue damage at the site of injection and which will not impart to the resulting immunogenic composition a low or excessively variable immune response.
• This invention seeks to provide an immunogenic composition which comprises an improved adjuvant.
• an immunogenic composition which comprises:
• an immunogen comprising, as active principle, an antigen; and (ii) an adjuvant which comprises a monomeric adduct formable by adding:
• the immunogen may comprise viruses or cells, for example live or killed bacteria, or other whole or partial biostructures which may be mixed with an adjuvant as aforesaid to form an immunogenic composition of the invention for use, on immunisation of male or female, human or non-human animals, to destroy self-generated such biostructures.
• biostructures include whole cells or cell extracts; for example, whole cell adipocytes to form immunogenic compositions of this invention for use, on immunisation of livestock, to destroy self-generated adipocytes thereby producing leaner animals; whole sperm to form compositions of this invention for use, on immunisation of male human and non-human animals, to destroy self-generated sperm thereby to produce sterility.
• partial biostructures for example cell extracts
• zonae pellucidae the thick, transparent membranes surrounding the fully formed ova In Graafian follicles
• the immunogen may also comprise a macromolecule; for example, a protein or glycoprotein which may be mixed with an adjuvant as aforesaid to form an immunogenic composition of the invention.
• the macromolecule may be a venom; for example, a snake venom to form a composition of the invention for use, on immunisation of humans or livestock, to prevent or mitigate the effects of venomous snake bite. It may be desirable, in the above-mentioned cases, additionally to utilise a carrier especially where the molecular weight of the immunogen is not sufficiently high enough to raise antibody titre.
• the immunogen comprises a hapten at least a part of which, and preferably all of which, is conjugated with a carrier.
• hapten is meant herein any material of poor antigenicity (and generally of low molecular weight) which may be rendered immunogenic by conjugation to a carrier (higher molecular weight) material.
• the hapten may comprise a peptide and/or act as a hormone.
• it may comprise the cyclic tetradecapeptide somatostatin or one of Its analogues (somatostatin 28, Tyr somatostatin, (Tyr') somatostatin or (Tyr”) somatostatin) which, on conjugation to a carrier and admixture with an adjuvant as aforesaid to form a composition of the invention, may be employed, on Immunisation of livestock, as an anabolic agent.
• thyrotrophin releasing hormone may comprise the tripeptide thyrotrophin releasing hormone or one of its analogues (thyrotrophin releasing hormone free acid or histidyl-proline-diketopiperazine). It may comprise a natural or synthetic steroid hormone; for example, progesterone.
• the hapten may comprise the decapeptide gonadotrophin releasing hormone (Gn-RH, LH-RH, gonadovelin or FSH-RH) which, on conjugation to a carrier and admixture with an adjuvant as aforesaid to form a composition of the invention, may be employed, on immunisation of female human or male or female non-human animals, to suppress fertility.
• Gn-RH, LH-RH, gonadovelin or FSH-RH decapeptide gonadotrophin releasing hormone
• the carrier may be any macromolecule which enhances the potency of the immunogen: they may, for example, comprise carbohydrate or peptide moieties such as polysaccharides, glycoproteins or proteins. Their important characteristic is that they raise the size of the conjugate sufficiently for the patient's auto-immune system to consider the conjugate not to be self-generated. In practice, this means that the carrier has a molecular weight greater than about 10,000, typically 60,000 or more: even several millions.
• a preferred group of carriers include natural or synthetic proteins; for example bovine serum albumen, human serum albumen, tetanus toxoid, thyroglobulin, keyhole limpet haemocyanin, guinea pig Y-globulin and horseradish peroxidase, with bovine serum albumen (molecular weight about 70,000) being preferred.
• bovine serum albumen molecular weight about 70,000
• the carrier itself provides a useful immunogenic role; for example, tetanus toxoid which will immunize against tetanus. Any of the abovementioned haptens may be conjugated with any of these carriers.
• the conjugate may be prepared in a manner known per se; for example by reacting the hapten or other immunogen and the carrier with a carbodiimide (Theriogenology 18, p. 65-77 (1982)).
• the organic compound (a) may be substituted, for example by one or more halogen, such as chlorine or bromine, atoms.
• the substituent should not be basic and it is generally convenient to utilise unsubstituted organic compounds (a).
• the organic compound (a) may be aliphatic, alicyclic or aromatic and may comprise olefinic unsaturation. It. is generally convenient to utilise aliphatic, preferably saturated aliphatic, organic compounds (a); for example, mono-, di- or poly-carboxy, hydroxy, amido or mercapto group-containing organic compounds, preferably mono- or dicarboxy or hydroxy group-containing organic compounds.
• the organic compound is preferably a mono- (or di-) carboxy or hydroxy C 1 (or C 2 ) to C 10 , preferably C 1 (or C 2 ) to C 6 , aliphatic hydrocarbyl(ene), preferably a C 2 to C 4 alkan (di) ol.
• a mixture of a plurality of monomeric organic compounds (a) may be used.
• Examples of mono-carboxy or hydroxy hydrocarbyls(a) include monocarboxylic acids, phenols and alcohols, desirably those with less than 18, preferably less than 10, carbon atoms; for example, formic, acetic, propionic, butyric and valeric acids, benzoic and phenylacetic acids; phenol and cresols; methanol, ethanol, propanol, butanol and n-octanol. Alcohols are most suitable, especially the C 1 to C 6 , preferably C 2 to C 4 , alcohols.
• monocarboxylic acids phenols and alcohols
• monocarboxylic acids, phenols and alcohols are linear.
• di-carboxy or hydroxy hydrocarbylenes examples include dicarboxylic acids, dihydric phenols, hydroxyacids and glycols, desirably those with less than 18, preferably less than 10, carbon atoms; for example, 1,2-cyclohexanedicarboxylic, 1,3-cyclohexanedicarboxylic and 1,4-cyclohexanedicarboxylic acids, phthalic, isophthalic and terephthalic acids, 4,4'-dihydroxyphenyl-2,2-pro ⁇ ane, resorcinol, quinol and orsinol, lactic, 2-hydroxyisobutyric, 10-hydroxydecanoic, 12-hydroxyoctadecenoic, 12-hydroxy-cis-9-octadecenoic, 2-hydroxycyclohexane carboxylic, 2-hydroxy-2-phenyl (D) propionic, diphenylhydroxyacetic, 2-hydroxybenzoic, 3-hydroxybenzoic and 4-hydroxybenz
• Glycols are most suitable, especially the C 2 to C 6 , preferably C 2 to C 4 glycols.
• Mixtures of dicarboxylic acids, dihydric phenols, hydroxy acids and glycols may be used.
• the dicarboxylic acids, dihydric phenols, hydroxy acids and glycols are linear.
• organic compounds (a) substituted by more than two carboxy or hydroxy groups include polycarboxylic acids, polyhydric phenols, hydroxy acids and polyhydric alcohols, desirably those with less than 18, preferably less than 10, carbon atoms; for example, aliphatic polyols such as glycerol, erythritol, pentaerythrltol, sorbitol, dulcltol, inositol, 2-ethyl-2-hydroxy-methylpropane-1,3-diol and 1,2,6-hexanetriol; aromatic polyols such as 1,2,3-trihydroxybenzene, 1,2,4-trihydroxybenzene, 1,3,5-trihydroxybenzene; araliphatic polyols; hydroxy aliphatic, alicyclic and aromatic carboxylic acids, including Krebs cycle acids, such as citric acid, malic acid, tartaric acid, 2-hydroxy-3-methyl (D) succinic acid, ascorbic acid,
• R 1 , R 2 and R 4 which may be the same or different, each represent a hydrogen atom or a substituted or unsubstituted hydrocarbyl or hydrocarbyloxy group;
• R 3 represents a substituted or unsubstituted methylene, ethylene or 1, 3-propylene group ;
• R 5 represents a monovalent group reactive with the organic compound (a) or a hydrogen atom or a C 1 to C 4 alkyl group;
• Q represents an oxygen or a sulphur atom
• X represents:
• Y represents an oxygen atom or an -NR 6 - group wherein R 6 represents any of the values which R 1 may assume; a is 0 or 1; b is 0 or 1: c is 1 or 2; d is 0 or 1; with the proviso that at least one of b or d is 1.
• X may, as shown represent any hydrolysable carboxylic acid ester, carbonate ester or oxalate ester function, or an amide analogue. Preferably, however, X represents -COO- or -CH 2 OCO-. Particularly preferred cyclic unsaturated ethers have the formula:
• R 1 , R 2 and R 4 which may be the same or different, and R 5 are as herein defined.
• R 5 may suitably represent any group provided that it is reactive with the organic compound (a) .
• Preferred examples are vinyl (thio)ether and epoxy groups.
• R 5 represents a group derived from a cyclic unsaturated vinyl (thio)ether, especially of the formula:
• R 1 ' , R 2 ', R 3 ' and R 4 ' which may be the same or different, represent any of the values which R 1 , R 2 , R 3 and R 4 may assume;
• M represents the group -ZX'-;
• Q' represents an oxygen or sulphur atom;
• X' represents any of the values which X may assume
• Z represents a single bond or a carbon-linked mono-, oligo- or homo- or co- poly(substituted or unsubstituted hydrocarbylene or hydrocarbyleneoxy) residue, such as a substituted or unsubstituted arylene, alkylene or alkylene oxide residue.
• Especially preferred cyclic unsaturated ethers are the reaction products formed by subjecting one or a mixture of dihydropyran aldehydes to disproportionation by the Tischenko reaction; they have the formula:
• R 1 ' , R 2 ' and R 4 ' which may be the same or different, represent any of the values which R 1 , R 2 and R 4 respectively may assume.
• a preferred such compound is acrolein tetramer (in which the R i all represent hydrogen atoms).
• Cyclic unsaturated ethers wherein X comprises a -COO- or -CH 2 OCO- group may conveniently be prepared from the tetramer of the corresponding unsaturated aldehyde produced by the Tischenko reaction; namely:
• cyclic ethers wherein X comprises a -COO- group may be prepared by reaction of the tetramer with an alcohol R 5 OH using a transesterification catalyst and reaction conditions:
• Cyclic ethers wherein X comprises a -CH 2 OCO- group may be prepared by reaction of the tetramer with a lower alkyl carboxylic acid ester R 5 COO R 7 in which R 7 represents a lower alkyl group using a transesterification catalyst and reaction conditions:
• the respective by-products may also be transesterified with R 5 COO R 7 or R 5 OH to give, correspondingly:
• the corresponding amides may be prepared analogously.
• Cyclic ethers where X comprises a -COO- group may also be obtained by mild oxidation of the dimer of the corresponding unsaturated aldehyde, followed by esterification of the salt, for example the silver salt.
• Meta-carbonates and oxalates may be obtained, respectively, by esterification:
• Chloroformic esters such as ethyl chloroformate, may also be used.
• acrolein tetramer is readily prepared from acrolein which is a commercially available material; can readily be purified; and has been found to be satisfactory in the practice of this invention.
• Particularly preferred derivatives of the invention are those wherein the derivative has the formula:
• R 8 and R' 8 which may be the same or different, each represent a C 1 to C 6 alkyl group, a C 2 to C 6 hydroxyalkyl group or a homo- or co- poly(propylene or butylene oxide).
• an adjuvant which is a monomeric adduct and wherein the molar ratio of (a) to (b) is no greater than n:l wherein n is the number of ring double bonds in (b).
• compositions of this invention may be prepared by the methods described in our UK Patent Application No. 2112381A.
• the adjuvants formed by reacting acrolein tetramer and C 1 to C 12 monohydric alcohols are oils of relatively high boiling point and of low vapour pressure at ambient temperature. They have a relatively low viscosity (typically 100-300 cP at 20o to 25°C) which allows their administration by injection and, on cooling below -20oC, form colourless glasses. Their densities at 20°C are close to that of water with a progressive decrease as the alkyl chain length is increased. They are insoluble in water and body fluids.
• the adjuvant is a liquid at a temperature from 35o to 40oC and at standard pressure.
• Both solid and liquid adjuvants may be extended with pharmaceutically-acceptable oils, such as olive oil and also with other adjuvants of the invention of lower molecular weight in order to alter their properties.
• the present invention is of broad applicability in the formulation of active substances, particularly, but not exclusively, biologically active substances releasable at a sustained rate.
• classes of biologically active substances which may be incorporated in the sustained release compositions of the present invention include flavourings, pharmaceuticals, bacteriostats, viruscides, pesticides such as insecticides, nematicides, molluscicides and larvicides, herbicides, fungicides, algaecides, topical or dermatological agents, antifoulants for marine growth prevention, proteins, for example enzymes, peptides, microbiological and plant hydroculture salts and nutrients and preservatives, veterinary trace metal formulations, and other growth promoting factors used in animal husbandry: for example, antianaemia preparation and anabolic steroids.
• compositions of the present invention comprising, as biologically active substance, at least one pharmaceutical.
• the compositions of this invention thus find wide application in medical and surgical, including veterinary, contexts and in horticulture and agriculture as well as outside
• abortifacients such as prostaglandins, hypnotics, sedatives, tranquilisers, antipyretics, anti-inflammatory agents, preparation for the treatment of allergies, for example anti-histamines, anti-tussives, anticonvulsants, muscle relaxants, anti-tumour agents, for example those for the treatment of malignant neoplasia, local anaesthetics, anti-Parkinson agents, topical or dermatological agents, diuretics, for example those containing potassium, such as potassium iodide, preparations for the treatment of mental illness, for example preparations containing lithium for use in the treatment of manic depression or containing prostaglandins for the treatment of schizophrenia, anti-spasmodics, anti-ulcer agents, preparations containing various substances for the treatment of infection by pathogens including anti-fungal agents, for example metronidazole, anti-parasitic agents and other anti-microbial agents, anti-fungal agents, for example metronidazole, anti-para
• the sustained release compositions of this invention may be used as a contraceptive composition suitably containing, as active substance, at least one natural or synthetic steroid sex hormone for example an oestrogen or progestogen.
• progestogens include the natural progesterone and its synthetic analogues, including 11-dehydroprogesterone, delalutin, 21-fluoro-17-acetoxy-6- ⁇ -methylprogesterone, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, ethisterone, dimethisterone, A-norprogesterone, 19-norprogesterone, 21-norprogesterone, normethandrone, norethynodrel, norethindrone and its acetate, DL- and D-norgestrel, norgestrienone, ethynodiol diacetate, lynstrenol, ethyny
• oestrogens include the natural ⁇ -oestradiol and its synthetic analogues, principally ethinyloestradiol or mestranol, preferably ⁇ -oestradiol.
• the sustained release compositions of this invention are also useful in the treatment of diabetes and pernicious anaemia where, for example, the controlled release of insulin and cobalamin, respectively, may be utilised.
• sustained release compositions of this invention are particularly suited to treatment, both prophylactic and therapeutic, of tropical diseases; for example malaria, leprosy, schistosomiasis and clonorchiasis.
• drugs which can be used as biologically active substance in sustained release compositions of this invention for the treatment of these and other tropical diseases include quinine, sulphonamides, rifamcin, clofazimine, thiambutosine, chlorphenyl derivatives, chlorguamide, cycloguanil, pyrimethamine, sulphadiazine, trimethoprim, quinoline derivatives such as pamaquine, chloroquine, pentaquine, primaquine and amodiquine, pararosaniline, sulphametizole, quinacrine, dapsone, sodium sulphoxone, sulphetrone, sodium hydnocarpate and sodium chaulmoograte. Drugs of particular effectiveness are
• Anti-biotics such as tetracycline (both as free base and hydrochloride or a mixture thereof), have also been found to be efficacious in the treatment of tropical disease in combinations according to this invention.
• the sustained release compositions of this invention are also very well suited to veterinary applications.
• liquid depot preparations of antibiotics for general antibacterial activity and also in the treatment of anaplasmosis in cattle preparations for provision of a wide spectrum of activity against both ectoparasites, for example termites and endoparasites including arthropods, arrested larvae stages of nematodes, lungworms and general strongyles: these may comprise avermectins
• preparations for provision of activity against tremotode, cestode and roundworm infections these may comprise amoscanate and praziquantel: preparations for provision of activity against theileria in cattle: these may comprise biologically active naphthoquinones such as menoctone; preparations for provision of activity against babesiosis in cattle, horses and dogs: these may comprise berenil, amidocarb and diampron; preparations for provision of activity against liver fluke in sheep and cattle and against Haemonchus species: these may comprise closantel.
• the immunogenic composition of this invention is formulated as an injectable dosage form.
• an immunogenic composition of this invention may be used to boost antibody titre in human or non-human animals which have already been sensitised by immunogenic compositions comprising a conventional adjuvant or a differing adjuvant of this invention.
• the fertility-suppressing compositions of this invention are particularly advantageous in relation to domestic pets and animal husbandry where the fertility suppression may be effected at any desired time (for example late on in the animal's life or immediately prior to an anticipated season) and is reversible.
• the following Examples illustrate the invention. EXAMPLE 1
• a conjugate of gonadotrophin-releasing hormone (Gn-RH ex Hoechst) and bovine serum albumen BSA, Cohn Fraction V, molecular weight ⁇ 70,000 (ex Sigma Chemicals Ltd.) was first- prepared in the following manner. Gn-RH, BSA and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (CDI, ex Sigma Chemicals Ltd) were mixed in the weight ratio 1:1:3 and left overnight. The conjugate so formed was then divided into two aliquots. The first was emulsified with an adjuvant which was the methanol adduct with acrolein tetramer which was prepared as described in our UK Patent Application No.
• each animal was administered a single subcutaneous injection in the neck such that each animal received 200 ⁇ g of Gn-RH, and unknown proportion of which was conjugated to an equal weight of BSA.
• the total injection volume was 0.6 ml.
• Testes lengths were also measured using calipers at weeks intervals from injection to 10 weeks next thereafter.
• the antibody titre (determined as described in Theriogenology 18, p. 65-77 (1982)) from the blood samples, together with mean values at weeks 0, 4, 8 and 23, are shown in Figures 1 and 2; the testes lengths, together with mean values at weeks 0, 4, 8 and 23, are shown in Figures 3 and 4.
• Antibody titres rose in all immunized rams following the administration of the injection (the rise being more pronounced where the comparative composition was injected) which correlated with a decrease in testes lengths.
• Oil 1 was the methanol adduct with acrolein tetramer
• Figure 5 is a graph (lower scale) of variation of antibody titre (% binding) with time obtained after injection with a comparative composition formulated with FIA; the upper scale shows the onset of oestrus (closed circles) for each ewe;
• Figure 6 depicts the same information for a composition of this invention comprising oil 1;
• Figure 7 depicts the same information for a composition of this invention comprising oil 2;
• Figure 8 depicts the same information for a composition of this invention comprising oil 3.
• ewes No. 592 which received a comparative composition formulated with FIA
• antibody titres rose sharply in all immunized ewes following the administration of the injection.
• the occurrence of oestrous cycles which can be regarded as an indication of reproductive function in ewes, also ceased in all but one ewe (No. 467 which received a composition of the invention formulated with oil 3) following the rise in antibody titres.
• Oestrous cycles recommenced in one ewe (No. 1484 which received a composition of the invention formulated with oil 1) which correlated with a decline in antibody titre.
• Oestrous cycles continued in control ewes throughout the experimental period.
• biodegradable oils used in the compositions of the present invention provide suitable vehicles for the stimulation of antibody synthesis in previously immunized animals which are sensitive to stimulation by the original antigen.
• EXAMPLE 3 This Example, the experimental design of which was essentially that used in the previous Examples, was effected to investigate the ability of three compositions of this invention comprising biodegradable oils with differing rates of breakdown to stimulate de novo antibody synthesis in animals which had not been previously exposed to the antigen.
• Antibody titres were detected in all rabbits which had received compositions of the invention following second injections although, at this stage, the titres were lower than those produced by the comparative composition comprising FCA.
• mice Male TFW outbred mice of a 20 gm body weight received 0.1 ml/mouse subcutaneously of an inoculum of 400 ⁇ g E.. carinatus venom dispersed in an oil which was an ethanol adduct with acrolein tetramer.
• a second group of three mice received an inoculum of 400 ⁇ g lyophilised venom in an oil which was a dodecanol adduct with acrolein tetramer.
• mice were bled and serum tested for the presence of antibodies to E. carinatus venom.
• the ELISA test was employed in microtitre plate using wells coated with E.
• carinatus venom (100 ⁇ g/ml); a commerical anti mouse conjugate was used.
• the level of antibody in a sample of blood was determined from the amount of alkaline phosphatase binding to the venom coated well surface. 30 and 60 minute incubation for alkaline phosphatase were employed. Enzyme activities were determined from the optical density of the well content (read at 405 nm, using P.B.S as blank).

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