The benefits from treatment with antivenom sera are indubitable. However, the mechanism for toxin neutralization has not been completely elucidated. A mixture of anti-bothropic and anti-crotalic horse antivenom has been reported to be more effective in neutralizing the effects of Bothrops jararacussu snake venom than anti-bothropic antivenom alone. This study determined which regions in the three PLA₂s from B. jararacussu snake venom are bound by antibodies in tetravalent anti-bothropic and monovalent anti-crotalic commercial horse antivenom. Mapping experiments of BthTX-I, BthTX-II and BthA-I using two small libraries of 69 peptides each revealed six major IgG-binding epitopes that were recognized by both anti-bothropic and anti-crotalic horse antivenom. Two epitopes in BthTX-I were only recognized by the anti-bothropic horse antivenom, while anti-crotalic horse antivenom recognized four unique epitopes across the three PLA₂s. Our studies suggest that the harmful activities of the PLA₂s present in the venom of B. jararacussu are neutralized by the combinatorial treatment with both antivenom sera through their complementary binding sites, which provides a wide coverage on the PLA₂s. This is the first peptide microarray of PLA₂s from B. jararacussu snake venom to survey the performance of commercial horse antiophidic antivenom. Regions recognized by the protective antivenom sera are prime candidates for improved venom cocktails or a chimeric protein encoding the multiple epitopes to immunize animals as well as for designing future synthetic vaccines.
Peptide microarrays have become increasingly more affordable in recent years with the SPOT technique being one of the most frequently used methods for synthesis and screening of peptides in arrays. Here, a protocol is presented for the identification of the amino acid sites involved in the conversion of human IgG to IgE response during the passive administration of therapeutic, anti-snake venom sera. Similarly, the minimal region of both the IgG and IgE binding epitopes, important for its interaction with ligand, were identified. As the ratio of concentrations for IgG to IgE in human serum is 1:10,000, also presented is a reproductive protocol of chemiluminescence-scanning for the detection of both immunoglobulins.
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