PRODUCTION AND EVALUATION OF ANTI-TETANUS SERUM

Abstract

Tetanus is the most fatal disease of equines and human beings. The disease can be prevented either by active immunization (anti-tetanus toxoid) prior to entry of Clostridium tetani or passive immunization (anti-tetanus serum) post infection. Anti-tetanus serum can be raised, purified and evaluated biologically using mice experimental model. Female sheep (n=10) having 36 - 42 months of age were immunized with commercially available tetanus toxoid (Imatet™, Amson vaccines and Pharma®) to raise anti-tetanus serum. Sera were collected fortnightly and sheep re-injected prior to collect the samples (nine times). Indirect ELISA was performed to determine the antibody (IgG) titer of all the serum samples. Out of 90 samples 21 (23.3%) revealed anti-tetanus antibody (IgG) titer of 100.8 IU or higher and 8 samples (8.9%) had titer of 160.9 IU or higher. Out of these 8 samples, 3 had anti-tetanus antibody (IgG) titer of 190.9 IU or higher. The highest titer observed was 195.4 IU. Immunoglobulins (IgGs) were mixed with saturated ammonium sulphate solution and centrifuged at 10000gfor 30 minutes in refrigerated centrifuge machine and dialyzed against 10 changes of PBS at 4 C. The purified anti-tetanus immunoglobulins (IgGs) were treated with papain to segregate Fab fragments (25~27 kDa) and analyzed by sodium dodecyle sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Anti-tetanus immunoglobulins and Fab fragments were evaluated qualitatively as well as quantitatively by toxin neutralization test (TNT) in mice. It is concluded that anti-tetanus Fab fragments produced in sheep can be used across the species in animals and human beings with trivial harmful effects.