A sensitive and specific immunoassay for the measurement of the antibodies present in horse antivenoms endowed with the capacity to block the phospholipase A2-dependent hemolysis induced by snake venoms

ABSTRACT

Phospholipase A2(PLA2), a component of most snake venom toxins, cleaves 3-sn-phosphoglycerides releasing lysophosphatidyl-choline. The indirect quantitative assay method for PLA2 was standardized for specific antivenom titration in a fast and sensitive assay by the similarity with the hemolysis induced by PLA2 and by complement system in sheep erythrocytes. The curves obtained by plotting the degree of hemolysis against the doses of snake venom are concave to the abscissa to the abscissa axis following an equation similar to that previously described for the hemolysis induced by the C system. We observed that venoms of some Bothrops, Crotalus and Micrurus species contained around 1 X 10(3) to 10(4) Z/mg of venom, while the venom of Naja contained over one million Z/mg. Antibodies against PLA2 were titrated by incubating amounts of venom predetermined to give 1 to 5 Z with various dilutions of the antivenoms, and the remaining active PLA2 was determined in the hemolytic assay. We observed the following a) the antivenoms contained specific antibodies against the PLA2 present in the corresponding venoms; b) cross-reactivity was not detected among PLA2 epitopes from venoms and nonspecific antivenoms and c) the assay quantitatively performed determined the specific antibodies directed to epitopes on the molecule of PLA2. The method described in this highly specific, sensitive and reproducible, besides being fast and inexpensive.