The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against virginia opossum (Didelphis virginiana) serum

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occuring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon(TM)-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD 2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD 2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD2 monoclonal antibody suggests specificity. DV-2LD2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the fivestep western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD2 did not bind to the nonhemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra (Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five- step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.