Title of Invention

A PROCESS FOR THE PREPARATION OF ANTI-SNAKE VENUM ANTIBODIES

Abstract

ABSTRACT A process for the preparation of immunoglobulins (IgG), an anti-snake venom antibody from chicken egg yolk comprising in the steps of: (i) immunizing 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 phospate 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 a process for the preparation of immunoglobulins (IgG), an anti-snake venom antibody from chicken egg yolk. BACKBROUND OF THE INVENTION ' The production of antibodies and its purification from mammalian blood has been found lo»«» yielding and laborious. The therapeutic use of specific antibodies is invaluable in certain cases, for eg. administration of anti-Ig6 against snake venom is the only specific treatment currently available for snake bite. The anti-IgG is marketed as the whole serum or partially purified poly¬valent immunoglobulin (IgG) from equine. Due to its scarcity and cost factor, its routine use has been difficult. Even though the benefits of anti-snake venom (ASV) therapy may outweight its risks, there are various side effects of antivenom, such as anaphylactic shock, pyrogenic reaction and serum sickness, incidence increasing with.dose. Purification of IgG from mammalian blood is time consuming and expensive. Hens are recognised as convenient and inexpensive source of antibodies. It has been reported that immunoglobulin 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 isolated from egg yolks 2 weeks after the first immunization and with one booster given, antibody con¬taining eggs can be collected for 100 days. Whei-eas to obtain antibodies from mammals, 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 uH«?ful in many applications, including immunotherapy and immunogiagnostics. PRIOR ART Production of hens antibodies against different types of anti¬gens, eg. proteins, synthetic peptides, etc. has been suggested in the art. However, the production of antibodies against venom in hens is not knoum. Th»? lethality of vipt?r venom has been detected as 0.11 f-xg/g body weight in mice and therefore, the immunisation protocol needs standardization. It has been •".uggested that onih 3-6 repeated injections of gamma irradiated venom, antibodieji could be liaised in rabbits. The purification of immunoglobulins from chicken egg yolk involves the rt?moval of lipi^i^from the diluted egg yolk using dextran sulphate whichi is expensive and has been later on modi¬fied tay a water dilution method and freeze thaw method. Freezing o at "20 C and thawing and centrifugation followed by microfiltra- tion clarified the diluted yolk considerably. Further, ammonium sulphate precipitation was done to concentrate IgG. Unless the lipids are removed completely, the immunoglobulin yield is reduced during the further purification steps. From one egg, upto 100 mg of pure IgG may be obtained and after the immunisation, specific IgG containing egg is laid by a single bird for upto 100 days. OBTECT OF THE INVENTION An object of this invention is to propose a process for producing anti-vip^r venom antibodies using he as the host animal. BRIEF DFBCRH'TliaW OT=^ THE INVENTION According to this irrv^ntion is further provided a process for the preparation of immunoglobulins (IgG), an anti-snake venom antibody from chicken egg yolk comprising in 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 o freezing at a temperature in the range of -50 to -7.0 C, followed by thawing the frozen yolk and centrifugation to produce a clean protein supernatant; iv. subjecting the supernatant to the step of direct gelfil- tration 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 o Ringer's lactate solution. The venom is stored at -15 to -20 C and before immunization, a portion of the venom is irradiated 60 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 pectoral is muscle of the hen. The amount of venom injected is between 300 to 600 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 o remove the lipids and frozen at -50 to -70 C. The diluted and frozen egg yolk is then thawed at a temperature in the range of 4 o to 10 C and w>... V, i luigea at 9 force between 20K to 30K for 4(3 to 50 mins. to produce ^ clear supernatant. As there is no volume enlargement later, the IgG fraction is further isolated by direct gelfiltra-tion, collectinc) and pooling the elution peaks and concentrating the same to obtain the immunoQlobulin. The solvent that may be used for gel f i 1 trat ion is 10 to 100 mj-i phosphate buffered saline at pH ranging from 7.2 to 7.8, In case there is a volume enlargement, ammonium sulphate precipi¬tation is done before gelfi1tration to concentrate the protein and reduce the volume. The immunisation protocol used is simple as only one injection 60 with Co-irradiated antigen is enough to express antibodies in egg yolk, nihil e four to five boost(?rs are recommended in other host animals such as rabbits, to develop a good titre of anti-venom antibodies in blood. Only B doul)le dilution of egg yolk was required to separate the lipds. This reduces the difficulties in handling large volumes egc) yolk during isolation of IgG. Using a simple ge1fi1tration step of IgG has been isolated from the lipid removed egg yolk protein mixture and the isolated immu¬noglobulin is shown to be 90% homogeneous. The anti-viper venom antibodies has been collected 12th day of primary immunisation and with a booster given on 28th day, antibodies were present upto 80 to 100 days in egg yolk. Thus, hens can be used as high yielding host for large scale production of anti-viper venom, by safe immunization with gamma-irradiated venom. The invention will now be explained with the help of non— limiting example. However, such a working example is not intended to limit the scope of the invention, EXAMPLE 1: ISOLATION OF ANTI-VIPER VENOM IMMUNOGLOBULINS FROM CHICKEN EGG YOLK IMMUNIZATION A Eix month old egg laying Rhodand Island hen, weighing 1 kg was immunized with 600 ug of viper venom. For collection of antigen, about 0.5 ml venom was allowed to run into a 4.5 ml of Ringer's lactate from a Russell's viper bite. The venom was stoj^ed at -20 and before immunization, 0.5 ml venom was taken into a small vial 60 and exposed to Co irradiation with source strength of 12 krad for 6 h. The irradiated venom was mixed with an equal volume of Freud's complete adjuvant and injected into the pectoral is muscle of the hen. ]■ SOI AT I ON OF IMMUNOGLOBULINS The eggs from the 12th day of injection was collected and the ecjg yolk was separated from the white. The contents of the yolk was collected by puncturing the yolk membrane, and diluted with o two volumes of distilled water and frozen at -50 to -70 C. The o diluted and fro:-::en egg yolk was then thawed slowly at 4 C. The o suspension was then centrifuged for 40 to 45 mins. at 4 C and at a 9 value ranging from 20K to 30K. As there was no volume enl ar'gement , later, the IgG fraction was further isolated by direct geTfi1tration on Biogel P-150, obtained from Biorad, USA. The fractions from the first elution peak consisting of IgG were pooled and concentrated by lyophi1ization. Compared to the freeze and thaw method known in the art, the con¬ditions of the present invention clarified the yolk solution, easily. The total protein concentration in one egg yolk was approximately 1,2 ±. 0.2 gm out of which 78 + 1)3 mg was precipi¬tated in 4«?)'/, ammonium sulphate fraction. Upto 90% of this was Igfa. The IgG obtained was a heterogenous protein mixture with M W 174 kDa and 155 kDa. After reduction with mecraptoethanol, the heavy chain fragment showed 55 kDa and light chain 22 kDa. The purified antibodies wer^e evaluated for their functional effi¬cacy. IN VITRO NEUTRALISATION OF VENOM WITH IMMUNOGLOBULIN FROM EGG The thromb in-1 i ke activity of the venom on |-UAman blood fibrinogen reflects on the coagulation assay of plasma in presence of venom. Thus, it was found that when 10 to 20 ;.ig of venom was added to 1 ml blocjd, the thrombin time was prolonged hy 275/',, u.iit;h respect to the contro] normal blood lOasma. Antibodies isolated from chicken egg was treated with venom for hflilf «n hciur and when the mixture uias added to the whole blood, the effect cjf venom was reversed, and showed normal coisgulation values. The LD50 d in 24 hours, the survivors had severe necrosis at the site of injection. The weight gain of these animals- were also slow with maximum gain of 10% compared to normal controls in 7 days. When 4 mg/kg body weight ujas injected, all the animals died in 8 h. The 2 mg/kg body weight dose gave only 25% mortality in 24 h. When an equivalent concentration There was no marked necrosis at the injection site, and had normal weight gain. Thus, the antibodies purified by this inven¬tion is able to neutralise the tonicity of venom. WE CLAIM: 1, A process for the preparation of immunoglobulins (IgG), an anti-snake venom antibody from chicken egg yolk comprising in the steps of : i. immunising an egg laying hen with anti-snake venxjm 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 o 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 gelfil- tration using 10-100 mli phosphate buffered saline, to obtain a fraction containing immunoglobulin; V. and concentrating the fractions to isolate the immunoglobulin. 2. The process as claimed in claim 1 wherein said egg laying hen is in particular a Rhodand Island hen which is immunized with 300 to 800 ug of the venom. 3. The process as claimed in claim 1 wherein 600 ug of venom is injected to for each kilo of the weight of the hen, 4. The process as claimed in claim 1 wherein the venom for the step of immunisation is prepared comprising the steps of running the venom from a Russell's viper bite into Ringer's lactate solu- o tion to obtain a mixture, storing the mixture at -15 to -20 C, irradiating the same, and mixing with Freud's complete adjuvant to obtain the venom to be used for immunisation. 5. The process as claimed In claim 4 wherein the mixtuTB is 60 irradiated with a Co source of source strength of about 80 KCi. 6. The process as claimed in claim 4 wherein the mi>!ture is irra¬ diated for 1-6 h. 7. The process as claimed in claim 4 wherein the irradiated mixture is mixed with an equal volume of Freud's adjuvant. 8. The process as claimed in claim 1 wherein the contents of the ec(9 yolk is diluted with about two volumes of distil lt?d water. 9. The process as claimed in claim 1 wherein the frozen egg o yolk is thawed at a temperature in the range of 4 to 10 C, 10. The process as claimed in claim 1 wherein the centrifugation is carried out at a g force of between 20K to 30K for a period of 40 to 50 mins.. 11. The process as claimed in claim 1 wherein for the step of gelfi1tration, phosphate buffered saline at a pH in the range of 7.2 to 7.8 is used. 12. The process as claimed in claim 1 wherein the desired fractions from gelfi1tration comprises the first elution peaks. 13. The process as claimed in claim 1 wherein said desired fractions from gelfi1tration are concentrated by lyophiIisation. 14. A process for the preparation of immunoglobulins (IgG), an anti-snake venom antibodyfrom chicken egg yolk substantially as herein described.

Documents:

2697-MAS-1998 CORRESPONDENCE OTHERS 27-07-2012.pdf

2697-mas-1998 abstract.pdf

2697-mas-1998 claims.pdf

2697-mas-1998 correspondence-others.pdf

2697-mas-1998 correspondence-po.pdf

2697-mas-1998 description (complete).pdf

2697-mas-1998 form-1.pdf

2697-mas-1998 form-26.pdf

2697-mas-1998 form-4.pdf

2697-mas-1998 form-5.pdf

2697-mas-1998 petition.pdf