Production of antiserum- anti tetanus serum ( ats )

1. Production of antiserum- anti tetanus serum (ats) Dr. Sanjeev D. Patankar Principal ShivprasadSadanandJaiswal College Arjuni/Morgao – Dist. Gondia 441701

2. Anti-serum is nothing but the serum containing antibodies against a speciefic antigen . Antisera are used for immediate protection against a pathogen when there is no time to develop active immunity. It confers a passive immunity to patient in emergencies. Prepared antibodies are used for immunisation. General method for production of anti-sera:- Steps involved in the preparation of anti-sera are as follows- • Preparation of antigen • Selection of animal for immunization. • Immunization of animal. • Bleeding of animal. • Preparation of serum • Purification of serum • Packaging. • Potency testing.

3. PRODUCTION OF ANTI-TETANUS SERUM (ATS) Steps involved in production of anti-tetanus serum are- • Preparation of antigen:- a) Isolation of Cl.tetani. The causative agent of tetanus is Clostridium tetani. It is an anaerobic Gram positive ,rod shaped spore former . I t is non –invasive , toxigenic organism. The pathological symptoms are due to neurotoxin. This is an exotoxin . It is highly antigenic. The detoxified toxin or toxoid or anatoxin can be used for immunisation as it contains antigenic group but no toxigenic group. For toxoid preparation organism must be cultivated in a suitable medium to produce toxin extracellularly. Then it can be detoxified. Thus antigen can be prepared in a following manner- a) Isolation of Clostridium tetani- Clostridium tetani has habitat in dried cow dung. This cow dung is inoculated in Robertson Cooked Meat Medium. Incubate it under anaerobic conditions for several days at 37 degree C. Due to this majority of aerobes are inhibited. By Gram staining a typical morphology of Gram positive rod with terminal spore giving a typical drum stick model. The spores can preserved on sand & calcium carbonae.

4. Spores can be used for cultivation & production of toxin. b) Cultivation of Cl.tetani:- Spores are inoculated in 10ml of Robertson Cooked Meat Medium (RCMM). Incubate it under anaerobic condition at 37 degree C. for 48hrs. The meat pieces of medium become black. Perform Gram staining to confirm the growth of Cl. tetani by establishing the typical drum stick morphology. This 10ml culture is further used for inoculating higher stages of RCMM medium . Finally seed culture is inoculated in glass fermenter. Agitation is done without aeration . Atmosphere of carbon dioxide is maintained to give anaerobic condition. Temperature of 37 degree C is maintained. Time is of 48hrs. Under such conditions Cl.tetani grows in vegetative form & produce exotoxin in medium. C ) Seperation of exotoxin from medium:- Medium is filtered through simple filter. This removes meat pieces & solid matter. The filterate is centrifuged at 5000 to 10000 RPM. Suspended matter setteles down .

5. Clear supernatent is filtered through bacterialogical filter to remove bacterial cells & spores. d) Detoxification of toxin:- Toxin is treated with 0.4% formalin. Mixture is incubated at 37 degree C.for four days . 20ml of this toxoid should prove non-toxic to Guineapig by subcutaneous injection. Such toxoid is used for immunization of horses for anti-serrum preparation. 2) Selection & preparation of animal :- Horses are generally chosen for production of anti-toxin, because horses are easily handelled. They have large volume of blood. Thus more quantity of anti-bodies can be prepared. The horses are observed for three months before their use. Generally they are observed for three Glanders , a disease similar to tuberculosis. Horses used for immunization are separated from herd before seven days actual process starts. 3) Immunization of horses:- Horses are injected with intramuscular injection of tetanus toxoid into neck . There is a gradual increase of dose

6. From 5.0ml to 60ml.or 100ml with a time interval of 8—12 weeks. During this period a rising titre of antibodies is determined by withdrawing samples of blood horse. When animal shows regular increase in titre , due to high concentration of antibody it may be transferred to other parts & thus for a short while it shows decrease in titre. But after that it again shows increase in titre. When 2-3 successive samples shows regular rise in titre then the horse is considered ready for bleeding. 4) Bleeding of horse & seperation of plasma:- When an adequate antitoxin titre has been obtained , the horse bleed. Firstly a pad is placed over the neck of horse. The are then clipped off & skin is treated with disinfectant. A cannula is inserted in the vein & connected by sterile tubing to a four lit. bottle containing anti-coagulant. About 4lit. Of blood is collected aseptically & it is set aside in refrigeration to give natural sedimentation of cell . Alternatively it can be centrifuged in refrigerated centrifuge to separate the cells . This requires strict aseptic conditions during transfer into centrifuge. After separation of cells plasma is siphoned into sterile bottle containing coaggulant such as calcium chloride.

7. The addition of coaggulant gives clotting of plasma & serum is seprated. Serum is filtered through Gradacol membrane to remove fibrin from clotted plasma. The sterile fiterate is nothing but crude antitoxin serum. It contains antibodies, serum albumin thus it must be further purified. The horse may be subjected to further two bleedings within a week of first bleeding. It is then rested for fifteen days & again re-immunized ,three such more bleedings can be taken & then the horse is permanently rested. 4) Refinement & purification of serum:- Crude anti-toxin sera are refined by removal of proteinaceous substances which give rise to undesirable reaction. The two principle methods for purification are used- i) Fractional precipitation. ii) Selective peptic digestion i) Fractional precipitation:-An amount of ammonium sulphate which is sufficient to produce one- third saturated solution is added. The precipitation

8. contains euglobulin & albumin. It is thus rejected. After that ammonium sulphate in concentration to produce half saturated solution. This gives precipitation of pseudoglobulin fraction. This precipitated fraction is dialysed against distiled water to rmove ammonium sulphate. The precipitate is passed in to 0.3% tricresol solution which act as a preservative. The solution is then passed through bacteriological filter.to sterilize it & then it is standardized & filled in ampoules. The sterilized solution may also be freeze dried for longer duration. ii) Selective peptic digestion:- Serum is subjected to peptic digestion at p H 4.2 to 4.6 to enzyme such as papain . The process depends on difference in behavior of enzyme on different serum proteins. The albumin is completely digested. Euglobulin is partly digested & pattly precipitated. The process is continued untill 70 to 80% of serum protein is digested . The antibodies which are associated with pseudoglobulin fraction are then seperated by

9. Dialysis. The disadvantage is that globulin is slightly modified by papain. The globulins can be freeze dried. Potency :- Any antitoxic preparation should not contain less than 1000 Units globulin /gm of protein. Storage :- In sealed sterilized glass container it should be stored at 0—4 degree C.