Antigenic relationships and relative immunogenicities of isolated metalloproteinases from Echis ocellatus venom

The antigenic relationship between snake venom metalloproteinases (SVMPs) was analysed using rabbit antisera raised against the native forms of two SVMPs purified from Echis ocellatus venom. Using enzyme-linked immunosorbent assay (ELISA), western blotting and two-dimensional SDS-PAGE, our findings show that antibodies raised against EoVMP1, a non-haemorrhagic class P-I 24 kDa SVMP, and EoVMP2, a haemorrhagic class P-III 56 kDa SVMP, demonstrate cross-reactivities which relate to the domain hierarchy observed in class P-I to P-III/IV SVMPs. A third 65 kDa P-III metalloproteinase (designated EoVMP3) was also isolated from E. ocellatus venom using hydrophobic interaction, size exclusion and anion exchange chromatography. In comparative immunoassays, EoVMP2 and EoVMP3 bound strongly to the commercial monovalent ovine Fab fragment antivenom EchiTAb™ (raised against the same venom), but EoVMP1 showed no cross-reactivity. This could indicate that antivenoms may lack antibodies to potentially important venom components.