Development and Validation Method for Potency test of cobra antivenin by indirect Enzyme-Linked Immunosorbent Assay (ELISA)
Potency Test of Cobra Antivenin by Indirect ELISA
Keywords:
Potency test, cobra antivenin, indirect ELISAAbstract
The potency test of cobra antivenin using a mouse neutralizing test is a standard method that is recommended in Thai Pharmacopoeia while the 3Rs principle is recently mentioned according to animal ethics. For that reason, indirect ELISA was developed and validated to replace the use of animals. The results showed that the method was specific to cobra antivenin. The percentage of recovery showed the accuracy of the method at 93.54%. The linearity was acceptable (R2 ≥ 0.99) when the relationship between the cobra antivenin serum sample serial dilution from 1/7,500 to 1/120,000 and the OD was plotted. The method was suitable for testing the sample at concentrations ranging between 0.2 and 0.8 mg/ml. The repeatability and intermediate precision showed percentages of coefficient of variation (%CV) were 7.65% and 5.13% respectively. The robustness was acceptable by varying the analysts, assay plates, and reaction times between antigen and antibody which did not show a significant difference. Taken together, the indirect ELISA method proposed to test the potency of cobra antivenin was reliable. It could be applied to study the correlation between the potency value obtained from indirect ELISA and the standard method in the future.
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