A comparative cross-reactivity and paraspecific neutralization study on Hypnale hypnale, Echis carinatus, and Daboia russelii monovalent and therapeutic polyvalent anti-venoms
Fig 4
Effect of H. hypnale, E. carinatus, and D. russelii venoms on coagulant activities.
(A) Plasma re-calcification time of Hhv; 200 μl of citrated human plasma was treated with 1–20 μg/ml of venom for 1 min at 37 0C, clotting was initiated by adding 20 μl 0.25 M CaCl2. (B) Hhv induced the plasma coagulation in the absence of CaCl2; clotting was initiated by adding 1–20 μg/ml of Hhv to the 200 μl of citrated human plasma. (C) Comparative plasma re-calcification time of Hhv, Ecv, and Drv; 200 μl of citrated human plasma was independently treated with 10 μg/ml each of venoms to initiate the clotting. For Drv, clotting was initiated separately by adding 20 μl 0.25 M CaCl2. (D) Activated partial thromboplastin time (APTT); 100 μl of citrated human plasma was treated independently with 5 μg/ml each of Hhv, Ecv, and Drv for 1 min at 37 0C, then 100 μl of reagent (LIQUICELIN-E phospholipids preparation derived from rabbit brain with ellagic acid) was added and incubated for 3 min at 37 0C. The clotting was initiated by adding 100 μl 0.02 M CaCl2. Prothrombin time (PT); 100 μl of citrated human plasma was treated independently with 5 μg/ml each of Hhv, Ecv, and Drv for 1 min at 37 0C. The clotting was initiated by adding 100 μl of PT reagent (UNIPLASTIN–rabbit brain thromboplastin). In all the cases, the plasma devoid of venom was served as control experiments. (E) Thrombin clotting time (TCT); 100 μl of fibrinogen (3 mg/ml) in 10 mM Tris-HCl buffer pH 7.6 was independently treated with 10 μg/ml each of Hhv, Ecv, and Drv to initiate the clotting. Thrombin, 10 U was used as a positive control, and fibrinogen alone was served as a negative control. In all the above experiments, the time taken for the visible clot formation was recorded in seconds. In all the cases, the data is presented as Mean ± SEM (n = 4) and analyzed using one-way/two-way ANOVA followed by Bonferroni post-tests, ‘**** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, and ns (not significant) >0.05. ‘*’ significant compared to the control group (PBS).