Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

In vitro evaluation of total venom-antivenin immune complex formation and binding parameters relevant to antivenin protection against venom toxicity and lethality based on size-exclusion high-performance liquid chromatography.

Total venom-antivenin immune complex formation and binding parameters relevant to antivenin protection against venom toxicity and lethality can be evaluated using size-exclusion high-performance liquid chromatography (SE-HPLC). Simple integration of regions within SE-HPLC elution profiles was used to compare binding characteristics of Crotalidae Polyvalent Immune Fab (Ovine) antivenin (FabAV) and Crotalus atrox (western diamondback rattlesnake; C. atrox), C. varidis varidis (prairie rattlesnake; C. v. v.), Agkistrodon contortrix contortrix (southern copperhead; A. c. c.), and A. piscivorus leukostoma (western cottonmouth; A. p. l.) venom. Areas associated with bound venom and antivenin ({Area(bnd)}) were evaluated using a logistic dose-response equation to estimate EC(50) and {Area(bnd)}(max). The relative magnitudes of EC(50), which inversely reflect venom-antivenin binding affinity, were C. atrox > C. v. v. > A. c. c. > A. p. l. Less than 50% of FabAV appeared to be reactive with each of the venoms based on {Area(bnd)}(max). Data was also consistent with FabAV binding to multiple sites on polyvalent antigens within the venoms. Evaluation of immune complex formation using SE-HPLC was compared to neutralization of phospholipase A(2) (PLA(2)) activity of C. atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA(2) activity occurred, in general, prior to maximum immune complex formation. Venom-antivenin binding at EC(50) determined via SE-HPLC appeared to be greater than binding associated with neutralization of venom lethality in mice based on LD(50) and ED(50) reported by others. SE-HPLC analysis of venom-antivenin binding could provide a priori information, relevant to reducing the use of animals in evaluating antivenin protection against venom-induced toxicity and lethality.

CroFabtrade mark total anti-venom activity measured by SE-HPLC, and anti-PLA(2) activity assayed in vitro at physiological pH.

One problem in the development and refinement of anti-venoms is ascertaining both overall anti-venom reactivity and which key toxins are neutralized. Here we show by SE-HPLC that the in vitro reaction of CroFab anti-venin with Crotalus atrox venom asymptotically nears completion (>95%) by 11 min at 4 degrees C by following the change in area under chromatographic peaks. The peaks for reactants decrease and the formation of high molecular weight complexes increases with time. To assay the large number of samples a new microplate format phospholipase A(2) (PLA(2)) assay at an initial pH of 7.5 was developed using phosphotidyl choline as the substrate. The change in absorbance is due to the pH change caused by release of fatty acids, and is linear with dilution of enzyme. This choice of substrate limits detection to PLA(2) and nonspecific esterase (if any) activities. The neutralization mixtures show a dose dependent (CroFab anti-venin) inactivation of C. atrox PLA(2) activity approaching a maximum of 85% neutralization. This approach of revealing antibody binding to venom components coupled with enzyme activity measurements is effective and may lead to greater in vitro assessment of antivenin activity in product development, and less routine use of mouse lethality assays.

Application of manual assessment of oxygen radical absorbent capacity (ORAC) for use in high throughput assay of "total" antioxidant activity of drugs and natural products.

INTRODUCTION: Antioxidants are of particular interest in a spectrum of diseases, and thus are an active area of drug discovery and design. It is important to make considered choices as to which assay chemistry will best serve for particular investigations. We examined the manual oxygen radical absorbent capacity (ORAC) assay for "total" antioxidant activity, including a direct comparison to an alternative technique, the AOP-490 assay, using a panel of extracts from 12 phylogenetically unrelated algae. METHODS: The AOP-490 assay was done per manufacturer's protocol. The ORAC assay was done by hand, in 96-well plates, not by machine as had been previously published. Our ORAC calculations were done using an in-experiment antioxidant standard curve. Results were reported as equivalents of the antioxidant Trolox, which was used as a standard. RESULTS: With the AOP-490 kit (from Oxis Research) widespread activity was found, but not in all samples. When the ORAC method was used to assay aliquots of the same extracts there was significant activity detected in all samples, and the rank order of activity by the two methods was not identical. The data showed the wide occurrence of antioxidants in algae. The standard curve with the manual ORAC assay was linear in the range tested (0-100 mM Trolox) and had excellent reproducibility. DISCUSSION: The importance of the beneficial effects of antioxidants is currently an area of active interest for drug development, and thus it is of great value to have an assay that is robust and approximates "total" antioxidant activity in a high throughput format. The ORAC (oxygen radical absorbent capacity) method was adapted to microplates and an eight-channel pipette and was more effective in detecting "total" antioxidant activity than the AOP-490 assay. These results might vary with other types of samples, and would depend on the active agents measured, but do suggest the practical value of the ORAC assay for any laboratory not ready for robotics but using manual 96-well format assays, and the utility of the ORAC assay for evaluating algal, and probably other samples as well.