Title of Invention

ANTI-SNAKE VENOM IMMUNOGLORULINS OBTAINED FROM CHICKEN EGG-YOLK

Abstract

ABSTRACT TITLE: ANTI-SNAKE VENOM IMMUNOGLOBULINS OBTAINED FROM CHICKEN EGG-YOLK Anti-snake venom immunoglobulins obtained from chicken egg-yolk, prepared by the process comprising the steps of; i. immunising an egg laying hen with anti-snake venom prepared in a manner such as herein described; ii. collecting the eggs after the twelfth day of injection; iii. subjecting the yolk of the egg to a step of dilution and freezing at a temperature in the range of -50 to -70°C, followed by thawing the frozen yolk and centrifugation to produce a clean protein supernatant; iv. subjecting the supernatant to the step of direct gelfiltration using 10-100 mM phosphate buffered saline, to obtain a fraction containing immunoglobulin; v. and concentrating the fractions to isolate the immunoglobulin.

Full Text

FIELD OF THE INVENTION This invention relates to anti-snake venom immunoglobul ins obtained from chicken egg-yolk. BACKGROUND OF THE INVENTION The production of antibodies and its purification from mammalian blood has been found low yielding and 1aborious. The therapeutic use of specifie antibodies is invaluable in certain cases, for eg. administration of anti—IgG against snake venom is the only spec i fie treatment currently available for snake bite. The anti-IgG is marketed as the whole serum or partially purified poly-valent immunoglobu]in (IgG) from equine. Due to its seareity and cost factor, its routine use has been difficult. Even though the benefi ts of anti-snake venom (ASV) therapy may outweight i ts risks, there are various side effects of anti venom, such as anaphyl actio shock, pyrogenic react ion and serum sickness, incidence increasing with dose. Purification of IgG from mammalian blood is time consuming and expensive. Hens are recogni sed as convenient and inexpensive source of antihodi es, It has been reported that i mmuriog lobulin concentration that can be yielded from one egg laid by an immunised hen is as high as that can be obtained from 300 ml of rabbit blood. Moreover, specific antibodies can be isol ated from egg yolks- 2 weeks- after the first iramunizat ion and with one booster given, antibody con¬taining eggs can be collected for 100 days. Whereas to obtain antibodies from mammalst frequent boosters need to be given which involve the requirement of more antigen. Chickens are obtainable in inbred strains thus minimizing the genetic variation in anti¬body response, a problem commonly seen in mammalian antibody production. According to an increasing number of publications, the antibodies produced in hens are useful in many applications, OBJECT OF THE INVENTION: An object of this invention is to propose a process for producing anti-viper venom antibodies using her as the host animal. BRIEF DESCRIPTION OF THE INVENTION: According to this invention is provided anti-snake venom immunoglobulins obtained from chicken egg-yolk, prepared by the process comprising the steps of i) immunising an egg laying hen with ant i-snake venom prepared in a manner such as herein described; ii) collecting the eggs after the twelfth day of injection; iii) subjecting the yolk of the egg to a step of dilution and freezing at a temperature in the range of -50 to -70°C, followed by thawing the frozen yolk and centrifugation to produce a clean protein supernatant; iv) subjecting the supernatant to the step of direct gelfiltration using 10-100 mM phosphate buffered saline, to obtain a fraction containing immunoglobulin; v) and concentrating the fractions to isolate the immunoglobulin. In accordance with this invention, the antigen is first collected by allowing the venom from a Russell's viper bite to run into a Ringer' s lactate solution. The venom is stored at -15 to -20 DC and before immunization, a portion of the venom is irradiated and before imminization, a portion of the venom is irradiated with a Co source, with a source strength of 80 KCi for a period of 1 to 6 hours. The irradiated venom is thereafter mixed with Freud's complete adjuvant and injected into the pectoralis muscle of the hen. The amount of venom injected is between 300 to 800 ug. The eggs from the 12th day of injection are collected and the egg yolk is separated from the white. The contents of the yolk are collected and diluted with about two volumes of water to remove the lipids and frozen at -50 to -70DC. The diluted and frozen egg yolk is then thawed at a temperature in the range of 4 to 10°C and centrifuged at t, force between 20K to 30K -for 40 to 50 mins. to produce a clear supernatant. As there is no volume enl argement 1imiting ex amp le. However, such a working example is not intended to 1imi t the scope of the invention. EXAMPLE 1: ISOLATION OF ANTI-VIPER VENOM IMMUNOGLOBULINS FROM CHICKEN EBB YOLK IMMUNIZATION approximately 1.2 ±. 0.2 gm out or which 78 ± 10 mq was precipi¬tated in 40'/. ammonium sulphate fraction. Upto 90'/, of this was IgG. The IgG obtained was s heterogenous protein mixture with M W 174 (kDa)and 155 l:Da. After reduction with mecraptoethanol , the heavy chain fragment showed 55 kDs and light chain 22 fcDa. There mas no marked necrosis at the injection site, and had normal weight gain. Thus, the antibodi PS pur) -f ied by this inven- WE CLAIM: 1. Anti-snake venom immunoglobulins obtained from chicken egg-yolk, prepared by the process comprising the steps of: i. immunising an egg laying hen with anti-snake venom prepared in a manner such as herein described; ii. collecting the eggs after the twelfth day of injection; iii. subjecting the yolk of the egg to a step of dilution and freezing at a temperature in the range of -50 to -70°C, followed by thawing the frozen yolk and centrifugation to produce a clean protein supernatant; iv. subjecting the supernatant to the step of direct gelfiltration using 10-100 mM phosphate buffered saline, to obtain a fraction containing immunoglobulin; v. and concentrating the fractions to isolate the immunoglobulin. 2. Anti-snake venom as claimed in claim 1 wherein the antibodies are anti-viper venom antibodies. 3. Anti-snake venom as claimed in claim 1 wherein hen is used as the host animal. 4. Anti-snake venom as claimed in claim 1 wherein a hen is first' immunised for the development of the antibodies. 5. Anti-snake venom as claimed in claim 1 obtained from the egg-yolk of an egg laying hen, such as a Rhodand Island hen. 6. Anti-snake venom as claimed in claim 1 wherein the eggs are obtained after the twelfth day of immunisation. 7. Anti-snake venom as claimed in claim 1 wherein 600 ug is injected per kilo of body weight of the hen. 8. Anti-snake venom as claimed in claim 1 wherein the hen is immunised with gamma-irradiated venom. 9. Anti-snake venom immunoglobulins obtained from chicken egg-yolk substantially as herein described.

Documents:

0185-mas-2002 abstract duplicate.pdf

0185-mas-2002 abstract.pdf

0185-mas-2002 claims duplicate.pdf

0185-mas-2002 claims.pdf

0185-mas-2002 correspondence-others.pdf

0185-mas-2002 correspondence-po.pdf

0185-mas-2002 description (complete) duplicate.pdf

0185-mas-2002 description (complete).pdf

0185-mas-2002 form-1.pdf

0185-mas-2002 form-19.pdf

0185-mas-2002 form-26.pdf

0185-mas-2002 form-3.pdf

0185-mas-2002 form-5.pdf

0185-mas-2002 petition.pdf