Comparative studies by different dyeing methods on antiserum preparation against hemorrhagins from Agkistrodon acutus venom / 中国免疫学杂志

Objective:In order to look for a good method for preparation of hemorrhagin antiserum. Methods: Three kinds of hemorrhagins including AaH Ⅰ, AaH Ⅱ, and AaH Ⅳ were purified from Agkistrodon acutus venom according to predecessors's methods and crude AaH Ⅰ, AaH Ⅱ and AaH Ⅳ were obtained. Preparation electrophoresis was used to purify AaH Ⅰ,AaH Ⅱand AaH Ⅳ further. As for an hemorrhagin, six different dyeing methods were used to dye PAGE gel and the gel contained hemorrhagin was obtained respectively. The ground gel contained hemorrhagin was used to immune mice and its antiserum was obtained. Antiserums quality was tested through ELISA test and neutralization of the hemorrhagic activities of corresponding hemorrhagin. Results:Effective IgG concentration in different antiserum was different and effective IgG made through non toxic type protein fast stain reagent kit was higher than others. Conclusion:Non toxic type protein fast stain reagent kit is the best dyeing method among the six dyeing methods.

Isolation, characterization and antigenic cross-reactivities of the major hemorrhagin from Cryptelytrops purpureomaculatus venom.

The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 μg in mice) but was not lethal to mice at a dose of 1 μg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex

Acutolysin C, a weak hemorrhagic toxin from the venom of Agkistrodon acutus with leucoagglutination activity

The properties and agglutination activity of acutolysin C, a hemorrhagic metalloproteinase obtained from Agkistrodon acutus venom, were studied herein. Acutolysin C is a basic glycoprotein consisting of a single polypeptide chain with a molecular weight of 23.1 kDa and pI 8.7, containing one Zn2+ and one Ca2+ per molecule. It possesses caseinolytic, weak lethal (LD50 = 7.6 mg/kg) and weak hemorrhagic (MHD = 12.0 µg) activities, but does not present fibrinolytic, fibrinogenolytic, arginine esterase and phospholipase A2 actions. In addition, it revealed agglutination activity on some animal lymphocytes, including five species of mammals, six of birds, three of reptiles and one of amphibians, but had no effect on lymphocytes from two species of reptiles, one amphibian and nine species of fish. It had no effects on the erythrocytes and platelets of all 26 animal species tested. Both leucoagglutination and caseinolytic activities were inhibited by EDTA; while cysteine, 2-mercaptoethanol, 1,4-dithiothreitol, glutathione, serum against acutolysin C and serum against homologous snake venom as well as glucose, sucrose, mannose, lactose and galactose had no effects on inhibition. The lowest concentration of acutolysin C that induced mouse lymphocyte agglutination was 2.5 µg/mL. Acutolysin C is an interesting substance since it is the first member of the hemorrhagin family to be shown to have leucoagglutination activity. (AU)

Serum kinetics of Calloselasma rhodostoma (Malayan pit viper) venom components in a rabbit

The serum kinetics of Calloselasma rhodostoma (Malayan pit viper) venom - specifically two of its components, the major hemorrhagin (rhodostoxin) and a thrombin-like enzyme - was examined in a rabbit by double-sandwich enzyme-linked immunosorbent assay (ELISA). The animal received intramuscularly a 1.0-mg/kg dose of C. rhodostoma venom. The venom level in serum peaked 12 hours after the injection, followed by a gradual decline and finally reached low rates 72 hours after administration. The serum kinetic profile of venom components, however, did not correspond to the profile of the whole C. rhodostoma venom. The serum levels of the C. rhodostoma thrombin-like enzyme increased slowly and peaked only 48 hours post-injection. Then both thrombin-like enzyme and rhodostoxin remained at relatively high levels 72 hours after administration. Data suggest that various venom components bind to tissue at the injection site with different affinities and that conjugated venom components were continuously released into circulation at different rates. The prolonged high serum levels of both thrombin-like enzyme and hemorrhagin are consistent with the clinical picture of prolonged clotting deficiency in severe cases of C. rhodostoma envenomation. Our results also suggest that since venom components are being released into and eliminated from the circulation at different rates, the "average composition" of the venom antigen in the circulation changes over time. This implies that data from ELISA quantification of antigen levels from serum venom employing "whole venom" as reagent must be interpreted with care.(AU)