Natural immunity is commonly affiliated with inherent antibodies in animals that have not been deliberately immunized. Most natural antibodies are of the IgM class. However, the natural immunity of snakes and certain warm- blooded animals to snake venoms involves non-immunoglobulin proteins. These proteins have a molecular weight range of 50,000 to 100,000 daltons and isoelectric points of 4-4.5. The neutralization capacities of the natural products are strong when compared to specific immunoglobulins and neutralize many hemorrhagic factors in different venoms. So far, the mechanism has been only partially elucidated, and further studies are required. The goals of the proposed research are to (a) compare the antigenic relationship of different hemorrhagins, (b) determine the range of the specificity and cross-reactivity of the antihemorrhagins isolated, (c) determine if the antihemorrhagic titer increase when the animal is challenged, (d) determine the exact mechanism (s) in the neutralization process, (e) determine the relationship of hemorrhagins to proteolytic inhibitors, (f) determine if a smaller nonantigenic segment of the antihemorrhagic molecule be used to neutralize hemorrhagic toxins, and (g) determine if the antihemorrhagic factors or a smaller segment can be used clinically. To achieve these goals, the PI will purify and characterize antihemorrhagic and hemorrhagic factors from warm-blooded animals and liquid chromatography (HPLC) will be used. To determine purity, electrophoresis, isoelectric focusing, and HPLC will be used. Electrophoresis, HPLC, and radioactive labeling will be used to study the mechanism of venom neutralization with the purified antihemorrhagic elucidation of the mechanism of neutralization with the purified antihemorrhagic and hemorrhage factors. Special attention will be devoted to elucidation of the mechanism of neutralization. Subsequent research will focus on determining the feasibility of inserting an opossum gene segment into E. Coli for the production of antihemorrhagic factors which could neutralize venom. Students, as in the past, will be involved in all phases of the research, including presentation at professional meetings and publishing in professional journals.
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