Quantitation by enzyme-immunoassay of antibodies against bothrops myotoxins in four commercially-available antivenoms (original) (raw)
Related papers
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 1998
- Venom pools from Bothrops neuwiedi (Bn) and from two subspecies, namely Bothrops neuwiedi pauloensis (Bnp) and Bothrops neuwiedi urutu (Bnu), collected in the States of São Paulo (SP) and Minas Gerais (MG), Brazil, were electrophoretically examined. Basic toxins with different isoelectric points were identified in the venom collected in São Paulo (BnSP). These toxins were absent in the corresponding pools from Minas Gerais (BnMG, BnpMG and BnuMG). (2) BnSP, but not BnMG, BnpMG or BnuMG, showed two myotoxins (pI$ 8.6 and 8.8, respectively) which were isolated by ion-exchange chromatography on CM-Sepharose. (3) From BnMG, three myotoxic isoforms (pI$8.2 and M r = 13600) were isolated by chromatography on CM-Sepharose followed by reversed-phase high-performance liquid chromatography. (4) The chemical and biological characterization of these toxins showed a high similarity with the Lys-49 myotoxins from other bothropic venoms. (5) Doses up to 5 LD 50 (i.p.) of p-bromophenacyl bromide alkylated BnSP-7 caused a total loss of lethality in 18 -22-g mice, thus indicating that the LD 50 was increased by greater than 5-fold. At this dose myotoxicity was also not detectable, but the edematogenic activity on the rat paw apparently did not change.
Antiserum to myotoxin from prairie rattlesnake (Crotalus viridis viridis) venom
Toxicon, 1979
to myotoxin from prairie rattlosnake (Crotahts veldts vuldis) venom. Toxlcon 17, 373-380. 1979 .-A myotoxic component was isolated from rattlesnake (Crotales vuldis vuldis) venom and an antibody to it was produced in rabbits. Gel-filtration and cation exchange chromatography were used to fractionate the crude venom and two components were shown to be locally myotoxic using an to viva assay. One of these components, fraction 3 of the Sephadez cation exchange column, was shown to be homogeneous by electrophoresis. This purified myotoxin was igjected into rabbits, and when the resulting antiserum was reacted with the myotoxin in agar-gel doubladiffusion plates, one precipitin line was formed. Anti-myotoxin serum reacted with only 2 of 14 crude vcnoms forming one precipitin line to both C. v. vlridis and C. derissus terraces. Tho line which formed against these two crude venoms was identical to the one that formed against myotoxin indicating the presence in C. d. terraces venom of a component immunologitxlly very similar to myotoxin from C. v. vlrtafis venom. It is postulated that this component might be crotamine, a known myotoxic component in C. d. terrifrces venom. Antibodies to myotoxin could not be detected in Wyeth's polyvalent Crotalidae antivenin although at least one antibody to crude C. v. vuidls venom was present. It is possible that antiserum to pure myotoxin could prevent the local myonecrosis indtlsd by the pure myotoxin, crude C. v. virldis venom and perhaps by other crotalid venoms.
The American Journal of Tropical Medicine and Hygiene, 1999
A comparative study was performed on the ability of IgG and F(abЈ) 2 antivenoms to neutralize lethal and myotoxic activities of Micrurus nigrocinctus venom. Both antivenoms were adjusted to a similar neutralizing potency in experiments where venom and antivenoms were preincubated prior to injection. No significant differences were observed between IgG and F(abЈ) 2 antivenoms concerning neutralization of lethal effect in rescue experiments, i.e., when antivenom was administered intravenously after envenomation. However, F(abЈ) 2 antivenom was more effective in prolonging the time of death when subneutralizing doses were administered immediately after venom injection. Both products partially reversed the binding of M. nigrocinctus ␣-neurotoxins to acetylcholine receptor in vitro. The IgG and F(abЈ) 2 antivenoms effectively neutralized venom-induced myotoxicity when administered intravenously immediately after envenomation, although neutralization was poor if antivenom injections were delayed. Intramuscular injection of venom promoted diffusion of antivenom antibodies throughout muscle tissue, and F(abЈ) 2 diffused to a higher extent than IgG molecules. Thus, despite the observation that F(abЈ) 2 antivenom was more effective than IgG antivenom in prolonging the time of death when subneutralizing doses were administered immediately after envenomation, no major differences were observed in antivenom neutralization of lethal and myotoxic effects or in their capacity to reverse neurotoxin binding to the acetylcholine receptor.
Toxicon, 1988
alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411-413, 1988 . -The ability of several batches of polyvalent antivenom to neutralize indirect hemolytic activity of Bothrops riper venom was studied using a sensitive plate test . All samples of antivenom tested effectively neutralized this activity . A highly significant correlation was observed between neutralization of indirect hemolytis and neutralization of lethal activity . This simple and sensitive in vitro test could be used to monitor antibody levels in horses immunized to produce polyvalent antivenom .
Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer
Toxicon, 1986
Papmon Jomnale Ltd. J . M . GurtHaaez, B . Lo>uorrre and L . CP~en~s . Isolation and partial charaaeriration of a myotoxin from the venom of the snake Bothropts nummjja. Taadrnn ?A, [885][886][887][888][889][890][891][892][893][894] 1986 . -A myotoxin from the venom of the make Botbropts memmjja was purified to homoaendty by ionexchange chromatogiaphy on CM-Sephadea . The toxin is a basic dinier with a subunit molecular weight of 16,000, as estimated by SDS-polyacrylamide gel dectrophoreaia . The toxin lacks phaspholipaae A, activity when tested on egg yolk Ixithin and :kdetal muscle homogenates . It induces srdetal muscle damage both In vlvo and In vitro. When igjected i .m. it promotes a drastic increase in serum creative kinase levels; the isozyme CK-MM is responsible for this increment . A rapid release of a~eatIne kinase was observed when mouse gastrocnemius muscle wan incubated with the toxin, suggesting that it induces the formation of relatively large 'lesions' in the plasma membrane of muade odls . Moreover, analysis of the done -response data indicated that the myotoxin afftscxs muscle saroolemma by a 'one hit' mec ar,;arn , Skeletal muscle cells are affected by the toxin when calcium is eliminated from the medium . The myotoxin hes an i .v. Ln of 3 .9 mg/kg body wdght in mice. and induces edema when igjated in the foot pad . On the other hand, it is not diraxly hemolytic, andoo:gulant, hemorr myotoxin show: partial immunol ~c nor cytotoxic for lymphocytes" The ogic identity with a myotoxic phospholipase A, isolated from Botlu+optr~venom . The polyvalent antivenom produced in Coca Rica forms a precipitation arc against B. nummjfa myotoxin on immunoelecrtrophoreais .
Journal of …, 1993
The reduction of the tetrazolium compound Mq'T (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) was used as the basis for the development of a simple method for the quantitative estimation of metabolically active skeletal muscle tissue remaining after in vivo venom-induced myonecrosis. Using the venom of the snake Micrurus nigrocinctus as a potent myotoxic agent, this Mq-T-based technique was evaluated in comparison with available methods based on the measurement of creatine kinase (CK) activity, and a quantitative histological technique considered as a reference. Homogenates of the gastrocnemius muscle prepared in the presence of 1% Triton X-100 reduced MTT and this activity correlated closely with the number of viable cells in the tissue, as determined by histological evaluation. After venom injection, residual MTT-reducing activity of muscle homogenates showed higher correlation to the myonecrosis index obtained by histological analysis, than residual muscle CK activity. Using the new MTT-based assay, the ability of an anti-M, nigrocinctus equine antivenom to neutralize venom myotoxins was studied. The myotoxic activity of the venom was completely neutralized using 4 ml antivenom/mg venom, with a 50% effective dose (EDs0) value of about 2.5 ml/mg. The MTI'-based method described should be useful in the estimation and standardization of anti-myotoxic potency of antivenoms, and in the screening of other neutralizing agents, as a convenient and reliable alternative to the time-consuming quantitative histological methods.
Toxicon, 2001
Five-month-old white leghorn chickens were immunized with 50 mg of Common Cobra (Naja naja) and 30 mg of Krait venoms (Bungarus caeruleus) to generate antivenom antibodies against the venom antigen. Chickens received booster doses of increasing concentrations of venom at 14 days time intervals to raise the antivenom level in egg yolk. The antivenom from immunized chicken egg yolk was extracted by polyethylene glycol (PEG) and ammonium sulphate precipitation method which was further purified by DEAE cellulose ion exchange column chromatography. A high molecular weight protein of 180 kDa was detected by electrophoretic analysis which shows the purity of antivenom generated in chicken. Antibodies generated were specific and sensitive to the venom antigen. Various pharmacological activities of Cobra and Krait venoms were carried out by both in-vivo and in-vitro methods. The neutralization of lethality, hemorrhagic, edema, PLA 2 and procoagulant activity was evaluated in assays involving pre-incubation of venom and antivenom prior to testing. The antivenom was effective in neutralizing the toxic and enzymatic activities of venom. The LD 50 of venom for 18 g of mice was found to be 10 mg for Cobra and 3 mg for Krait venoms. The median effective dose (ED 50 ) of anti-Cobra venom was 4.48 mg/ 5LD 50 and 1.0 ml neutralized 0.127 mg of Cobra venom and the median effective dose (ED 50 ) of anti-Krait venom was 3.18 mg/5LD 50 and 1.0 ml neutralized 0.051 mg of Krait venom. The results indicate that antivenom generated in chicken could be used for therapeutic purposes in case of snakebite envenomation.
Snake antivenoms: adverse reactions and production technology
Journal of Venomous Animals and Toxins including Tropical Diseases, 2009
Antivenoms have been widely used for more than a century for treating snakebites and other accidents with poisonous animals. Despite their efficacy, the use of heterologous antivenoms involves the possibility of adverse reactions due to activation of the immune system. In this paper, alternatives for antivenom production already in use were evaluated in light of their ability to minimize the occurrence of adverse reactions. These effects were classified according to their molecular mechanism as: anaphylactic reactions mediated by IgE, anaphylactoid reactions caused by complement system activation, and pyrogenic reactions produced mainly by the presence of endotoxins in the final product. In the future, antivenoms may be replaced by humanized antibodies, specific neutralizing compounds or vaccination. Meanwhile, improvements in antivenom quality will be focused on the obtainment of a more purified and specific product in compliance with good manufacturing practices and at an affordable cost.