Problems of antivenene standardization revealed by the flocculation reaction.

Mixed with either of the specific venoms, refined antivenene (anti-Bitis arietans-Naja flava) will show optimal flocculation at a number of different venom: serum ratios. This is the natural consequence of individual antigen-antibody reactions in a heterogeneous system.Toxin may be recovered from floccules formed at three optimal N. flava venom: serum ratios by heating the floccules at 70 degrees C for ten minutes at pH 2. These three immunologically distinct heat-stable toxins have been called the alpha, beta, and gamma toxins of N. flava venom.The flocculation reaction obtained with the viperine venom cannot be interpreted in terms of protective potency.Most venom-antivenene systems fail to conform with the "law of combination in multiple proportions", i.e., they give non-linear titration curves. The shape of such curves, convex towards the dose axis, is readily explained by the simultaneous titration of multiple toxin-antitoxin systems.In the particular case of N. flava venom-antivenene, the titration curve obtained with whole venom is determined by the relative anti-alpha, anti-beta, and anti-gamma contents of the serum. Further study may pave the way to the standardization of the anti-alpha, anti-beta, and anti-gamma potencies of sera, but attempts at standardizing antivenene potency against any venom are futile unless the toxic "make-up" of the venom is understood.The cross-protection afforded by sera produced against either N. flava venom, or the venom of Sepedon haemachates is limited but indicates an antigenic similarity which may be clarified by work in progress.