Immunological studies of rabbit antibodies against hemorrhagic fractions of Crotalus viridis viridis venom: role of crossreacting antibodies in neutralization.

1. Crude Prairie rattlesnake (Crotalus viridis viridis) venom was fractionated by HPLC DEAE anion exchange chromatography. Both acidic and basic protein fractions were assayed for hemorrhagic activity in vivo. 2. The acidic fractions that showed the highest hemorrhagic activities were pooled and divided into two samples. One sample was treated with EDTA and heat to inactivate and denature the hemorrhagic toxins; the other sample was left in native form. Both denaturated and native samples were used as immunogens for production of polyvalent antibodies in rabbits. 3. Neutralizing ability for hemorrhage of the antiserum raised against the native sample was about 50% greater than that of antiserum raised against the denatured sample. There was no correlation between ELISA reactivity and neutralizing ability of the antisera. 4. Antiserum raised against native hemorrhagic fractions showed extensive ELISA crossreactivities with the nonhemorrhagic basic proteins. The crossreacting antibodies accounted for 61% of the total ELISA reactivity and 54% of the total neutralizing ability of the antiserum. Treatment of the hemorrhagic fractions with EDTA induced a fundamental conformational change of the molecules, which was reflected in significantly increased ELISA reactivities. Antivenom raised in rabbits had high neutralizing potency compared to Wyeth antivenom (0.38 mg IgG was able to completely neutralize the hemorrhagic lesions induced by 10 micrograms crude C. v. viridis venom). However, when it was quantitatively compared with commercial Wyeth antivenom, no significant advantages were found.