Development of an in vitro potency assay for antivenom against Malayan pit viper (Calloselasma rhodostoma).

An in vitro potency assay of antivenom against Malayan pit viper (Calloselasma rhodostoma, CR) has been developed. The assay is based on the neutralizing activity of the antivenom against the coagulant activity of the venom. The minimum coagulant dose (MCD) of CR venom was 22.12 ± 0.25 μg/ml. The coagulation time induced by 2MCD of the venom was used as the control for calculating the neutralizing activity of each batch of antivenom. The in vitro potency of antivenom, expressed as effective dose (ED), was the antivenom/venom ratio at which the coagulation time was increased three fold of that induced by 2MCD of the venom. Eleven batches of the antivenom were assayed for their lethality neutralizing activity (ED₅₀) by the in vivo assay using mice as well as the developed in vitro assay. The correlation coefficient (r) between the in vitro neutralizing activities (ED) and in vivo neutralizing activities (ED₅₀) was 0.957, (p value < 0.001). This simple and rapid in vitro assay of C. rhodostoma antivenom should be a good alternative method for the assessment of antivenom potency during the immunization program and fractionation process. The assay should be adaptable for use with antivenoms against other similar procoagulant venoms.