Russell’s viper (Daboia russelii) venom was a gift from Colombo University, Sri Lanka. Saw-scaled viper (Echis carinatus) venom was purchased from Sigma. Carpet viper (Echis ocellatus) venom was donated by Robert Harrison (Liverpool School of Tropical Medicine). Russell’s viper venom factor X activator toxin (RVVFX) was purchased from Haematologic Technologies Inc. Rabbit anti-snake antibodies were purchased from the West Australian Institute of Medical Research. Hen anti-snake IgY antibodies to P. textilis venom were a gift from Frank Madaras (Venom Science Pty Ltd, South Australia). Australian commercial antivenoms were produced by CSL
Ltd, including brown snake (BSAV; 1000 U), tiger snake (TSAV; 3000 U), black selleck compound snake (BlSAV; 18,000 U), taipan (TAV; 12,000 U) and death adder (DAAV; 6000 U). One unit (1 U) of antivenom activity is defined to be the amount required to bind/neutralise 10 μg of venom from the snake species against which the antivenom is raised. Indian polyvalent antivenom was obtained from VINS Bioproducts (Batch No. 1054 Manufactured 09/2008 Expiry 08/2012).
Indian polyvalent antivenom is raised against four snake venoms – D. russelii, Notechis naja, E. carinatus and Bungarus caeruleus. All commercial antivenoms are of equine origin. Rabbit anti-horse IgG conjugated with horseradish peroxidise, goat anti-rabbit IgG conjugated with horseradish peroxidise, bovine serum albumin (BSA) and tetramethylbenzidine (TMB) were all purchased from Sigma. All other chemicals used were of analytical grade. Carbonate buffer JNK screening Dolichyl-phosphate-mannose-protein mannosyltransferase is 50 mM, pH 9.5. Blocking solution is 0.5% BSA in phosphate buffered saline (PBS) at pH 7.4. Washing solution is 0.02% Tween 20 in PBS.
High binding microplates from Greiner (#655061) were used. Plates were read on a BioTek ELx808 plate reader at 450 nm. All procedures were carried out at room temperature. A known concentration of venom in blocking solution was added to serial dilutions of antivenom in PBS (450 μl), such that the final venom concentration in the mixture was 500, 250, 100, 50 or 0 ng/ml. The mixture was allowed to stand for one hour then applied in triplicate to a microplate as below. Control solutions containing antivenom only were included to allow for subtraction of background absorbance. Plates were coated with anti-snake venom IgG (100 μl, 1 μg/ml in carbonate buffer) for 1 h at room temperature then at 4 °C overnight. They were then washed once, and blocking solution (300 μl) was applied for 1 h. Plates were washed again and the incubated mixture of venom and antivenom (100 μl) was added. After a further hour, the plates were washed three times and a solution of labelled anti-horse IgG (100 μl, 1 μg/ml in blocking solution) was applied.