Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica.

A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutralize lethal, hemorrhagic and coagulant activities of the venoms of 16 species of Central and South American snakes of the subfamily Crotalinae. Neutralization of lethality was studied by two different methods routinely used in the quality control of antivenoms at Instituto Butantan (IB) and Instituto Clodomiro Picado (ICP). Both antivenoms neutralized the majority of the venoms studied, but the values of effective doses 50% (ED(50)) differed markedly depending on the method used. In general, higher potencies were obtained with the method of ICP, where a challenge dose corresponding to 4 LD(50)s is used, than with the method of IB, where a challenge dose of 5 LD(50)s is employed. All venoms induced hemorrhagic activity in the mouse skin test, which was effectively neutralized by the two antivenoms. All venoms, except those of Porthidium nasutum and Bothriechis lateralis, induced coagulation of human plasma in vitro and both antivenoms were effective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive cross reactivity between these antivenoms and Central and South American crotaline snake venoms.

Effect of beta-propiolactone treatment on the complement activation mediated by equine antisera.

Reduction of complement activation through an alteration of the Fc fragment of immunoglobulins by beta-propiolactone treatment was carried out in equine antisera raised against rabies virus, Bothrops venoms and diphtherial toxin. Results were evaluated by means of an anaphylactic test performed on guinea-pigs, and compared to the ones obtained with the same sera purified by saline precipitation (ammonium sulfate), followed or not by enzymatic digestion with pepsin. Protein purity levels for antibothropic serum were 184.5 mg/g and 488.5 mg/g in beta-propiolactone treated and pepsin-digested sera, respectively. The recovery of specific activity was 100% and 62.5% when using antibothropic serum treated by beta-propiolactone and pepsin digestion, respectively. The antidiphtherial and anti-rabies sera treated with beta-propiolactone and pepsin presented protein purity levels of 5,698 and 7,179 Lf/g, 16,233 and 6,784 IU/g, respectively. The recovery of specific activity for these antisera were 88.8%, 77.7%, 100% and 36.5%, respectively. beta-propiolactone treatment induced a reduction in complement activation, tested "in vivo", without significant loss of biological activity. This treatment can be used in the preparation of heterologous immunoglobulins for human use.

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

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.

Snake antivenoms from hyperimmunized horses: comparison of the antivenom activity and biological properties of their whole IgG and F(ab')2 fragments.

IgG and F(ab')2 fragments were prepared from horse plasma rich in specific antibodies against Brazilian Bothrops or Crotalus venoms. Both preparations, free of gross contamination with non-immunoglobulin proteins, were able to combine in vitro with their respective antigens, forming immune complexes at antigen excess, equivalence or antibody excess, and activating the C system, through either the classical or the alternative pathways. The IgG preparation was more effective in neutralizing the lethal factors in Bothrops or Crotalus venoms, compared with the F(ab')2 fragments. In contrast, IgG and F(ab')2 anti-Bothrops venom were almost equipotent in neutralizing the haemorrhagic and defibrinating activities in the venom. The method used to purify IgG, precipitation of most non-immunoglobulin plasma proteins with caprylic acid, produced antivenoms richer in specific antibodies, with higher specific activity, recovery and yield, compared with the method commonly used to prepare antivenoms containing F(ab')2.

Immunization of equines with phospholipase A2 protects against the lethal effects of Crotalus durissus terrificus venom.

Equines (2 horses and 2 donkeys) immunized with whole Crotalus durissus terrificus venom or its phospholipase A2 component either presented an increased survival time determined 3 days after challenge or were totally resistant to a challenging lethal dose of 200 mg crude venom 270 days after the initial immunization or 90 days after the last booster injection. The resistance was demonstrable on the basis of a good correlation with antibody titers determined by the ELISA method but not with the flocculation and neutralization assays. Since phospholipase A2 is essentially nontoxic, it can be used as a substitute for whole venom for the production of commercial antisera and as an immunizing agent in prophylactic programs.

Immunization of equines with phospholipase A2 protects against the lethal effects of Crotalus durissus terrificus venom

Equines (2 horses and 2 donkeyes) immunized with whole Crotalus durissus terrificus venom or its phospholipase A2 component either presented an increased survival time determined 3 days after challenge or were totally resistant to a challenging lethal dose of 200 mg crude venom 270 days after the initial immunization or 90 days after the last booster injection. the resistance was demonstrable on the basis of a good correlation with antibody titers determined by the ELISA method but not with the flocculation and neutralization assays. Since phospholipase A2 is essentially montoxic, it can be used as a substitute for whole venom for the production of commercial antisera ad as an immuniaing agent in prophylalctic progams