An ELISA for optimization of horse antivenom digestion by pepsin

Abstract

A study was carried out to optimize the pepsin digestion reaction of horse antivenom. Rabbit antibodies, specific to horse antivenom IgG/IgG(T), horse F(ab')2 and horse Fc, were prepared and purified by using appropriate affinity absorbents. These rabbit antibodies were used to develop ELISAs which quantitated horse antivenom IgG/IgG(T) and antivenom F(ab')2 over a linear concentration range of 50-500 nM. Horse antivenom serum was digested with 0.15% (w/v) pepsin at 3 different pHs (3.25, 3.50, and 3.75) at 25°C for 12 hours. Aliquots of reaction mixtures were taken at intervals to assess the extent of digestion by measuring the concentrations of the immunologically active antivenom IgG/IgG(T) and its F(ab')2 product. It was found that the digestion rate was faster at lower pH. Under these conditions, the starting IgG/IgG(T) was all digested within 2 hours with the concomitant appearance of F(ab')2. Using 0.15% pepsin at pH 3.5, the highest level of immunologically active F(ab')2 was observed with a recovery of antibody activity of about 60%.