Development of an indirect ELISA for screening horses based on the potency of antivenom produced against Iranian Cobra snake

In this study, an indirect ELISA test was developed for the determination of the level of antibodies against Cobra venom in equine sera after each injection step. Toxic fractions of Naja naja oxiana snake venom was used as antigen. The test allowed to reveal an increase of antibodies in response to the venom injection and it can be applied to the evaluation of the efficacy of immunization. To the development of the indirect ELISA, dilutions of 1/50, 1/500, and 1/1000 of positive and negative control sera against concentrations of 0.5, 1, and 2 µg/well of the venom were used. From this 2 µg/well of venom and 1:1000 serum dilution was found to be sufficient enough for the estimate of antisnake antibodies. After ELISA development, serums obtained from blood samples of horses immunized with venom were tested to evaluate the trend of increase in antivenom titer at each stage of venom injection. The average optical density of samples after the third injection was 1.213; this is 5.8 times that of the horse samples behind the first step of immunization. All horses had an adequate antivenom titer after the fifth injection. The results suggest that ELISA can be used to determination of antivenom titer in horse sera especially during the initial stages of immunization.