Drug – bio-affecting and body treating compositions – Lymphokine
Patent
1984-07-24
1987-04-28
Hazel, Blondel
Drug, bio-affecting and body treating compositions
Lymphokine
435 68, 4351722, A61K 39018
Patent
active
046613480
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a purified babesiosis antigen and method for its preparation.
Australian Patent Specification No. 63616/80 describes a method for preparation of a babesiosis vaccine which includes the steps of: disintegrated erythrocyte suspension; and soluble fraction of the disintegrated erythrocyte suspension
The product of step (iii) may then be combined with an adjuvant for administration purposes.
Babesiosis is a disease that most domestic animals may experience, and it is caused by various species of the tick-born protozoan parasite Babesia. In the cattle industry in particular there are many parts of the world wherein the disease poses serious economic problems.
The vaccine prepared as described above in Australian Patent Application No. 63616/80 is useful in that it may provide protection against babesiosis as described in Patent Application No. 63616/80. However it was based on a relatively impure preparation containing many different antigens and hence it is desirable to purify this preparation to isolate the antigen or antigens which may have when used as a vaccine an effective protective or immunizing ability against babesiosis upon administration to animals and in particular cattle.
Accordingly it is the object of the present invention to provide a babesiosis antigen which when used as a vaccine is suitable for administration to animals which is a more purified antigenic preparation than that described in the prior art.
It is a further object of the invention to provide a method of preparation of the abovementioned antigen.
The method of the invention includes the steps of: derived from a lysate of Babesia infected erythrocytes or soluble fraction thereof; antibody producing cells from an animal having been administered thereto said babesiosis antigenic fraction; antigenic fraction through said absorbent whereby an antigen specific to said antibody may be bound thereto; and
The invention also includes within its scope a babesiosis antigen derived from passing said babesiosis antigenic fraction through an absorbent having antibody specific to said babesiosis antigen bound thereto whereby said antigen is bound to the antibody and subsequently separating said antigen therefrom.
The invention also includes within its scope a vaccine comprising said babesiosis antigen in combination with a suitable adjuvant such as oil based adjuvants including Freunds Complete Adjuvant (FCA), Freunds Incomplete Adjuvant, Adjuvant 65 and Wellcome Mineral Oil Adjuvant. Saponin aqueous solution may also be used as an adjuvant.
In step (1) a suspension of babesia infected erythrocytes may be disrupted or lysed according to the following procedures of weak ionic strength; causing breakdown of the cell membrane; and antibodies.
Preferably in step (i) the cell lysate may be prepared as described by any of the methods referred to in Mahoney (1967) Exp. Parasitol 20, 232-41. Suitably, however, babesia parasite infected cells may be subjected to differential lysis in hypotonic saline. Alternatively infected cells may be lysed in 5 volumes of distilled water by osmotic pressure using a freeze and thaw technique. Alternatively the infected cells may be lysed by sonic disintegration as described in Patent Application No. 63616/80 or by the action of lytic agents such as saponin.
After lysis the cells may be centrifuged suitably at 105,000 g for 30 minutes at 5.degree. C. and the supernatant collected.
In relation to step (i) it will also be appreciated that the soluble fraction (ie SPA) from disrupted babesia infected cells may also be prepared as described in Patent Application No. 63616/80.
In relation to step (ii) suitable animals such as mice or rats (preferably mice) may be immunized by vaccine comprising SPA in combination with a suitable adjuvant. After immunization the mice may have administered thereto antigen in the form of SPA and then killed.
Suitably antibody producing cells may then be extracted from the killed mice and fused with mouse myeloma cells.
The technique as described in Galfre et al Nature 2
REFERENCES:
patent: 4457915 (1984-07-01), Goodger et al.
patent: 4596707 (1986-06-01), Ristic et al.
Goff et al: "The Bovine Immune Response to Tick-Derived . . . ", Veterinary Parasitology, 11 (1982), pp. 109-120.
Todorovic et al: "Comparison of the Dried Blood on Filter Paper and Serum . . . ", Diagnosis of Bovine Babesiosis, Tropenmed. Parasit., 29 (1978), pp. 88-94.
De Vos: "Immunogenicity and Pathogenicity of Three South African Strains . . . ", Onderstepoort J. Vet. Res., 45 (2), pp. 119-124 (1978).
Kuttler et al: "Immunologenicity . . . in Freund's Complete Adjuvant", Am. J. Vet. Res., vol. 41, No. 4, pp. 536-537.
Haggard et al: "Immunologic Effects of Experimental Iodine . . . ", Am. J. Vet. Res., vol. 41, No. 4, pp. 537-538.
Perez et al: "Cell-Mediated Immune Response in Hamsters Infected with Babesia Microti", Veterinary Parasitology, 3 (1977), pp. 161-167.
Molinar et al: "Antigenic and Immunogenic Studies on Cell Culture . . . ", Veterinary Parasitology, 10 (1982), pp. 29-40.
Latif et al: "Effect of Age on the Immune Response of Cattle . . . ", Veterinary Parasitology, 5 (1979), pp. 307-314.
Benach et al: . . . pp. 643-649.
Todorovic et al: "Immunization Against Anaplasmosis and Babesiosis . . . ", Tropenmed Parasit., 30 (1979), pp. 43-52.
Commonwealth Scientific & Industrial Research Organization
Hazel Blondel
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