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Effect of heating on the immunosuppressor and immunogenic activities of Crotalic venom
Abstracts/Toxicon 38 (2000) 487 595
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titration of snake venoms in vitro. Methods and results: Determination of citotoxicity was based on the technique described by Borefreund and Puerner (J. Tissue Culture Methods 9, 7, 1984). A pre-established number of cells was seeded in a 96-well plate and exposed to the snake venom being tested for a period of time. A solution of neutral red was added for incorporation by the cells alive. After washing and cell lysis the amount of incorporated dye was read spectrophotometrically at 540 nm. The best conditions for the assay were established as 3500 Vero cells/well, 2% FCS in 199 medium and 72 hr of venom exposition. The toxicity of a number of snake venoms have been determined and expressed as the dose capable of killing 50% and 90% of the cells under the test conditions. Conclusion: Cell culture methods can be very useful for the determination of in vitro toxicity of snake venoms and other toxic substances. Acknowledgements: Financial support from CNPq and F A P E M I G .
Effect of heating on the immunosuppressor and immunogenic activities of Crotalic venom. A. Rangel-Santos, I. Mota (Laboratorio de Imunopatologia, Inst. Butantan, S~o Paulo, Brasil). Objective: The effectiveness of horse anti-venom sera depends mostly on the venom immunogenicity. However, some venoms like that of the snake cascavel (Crotalus durissus terrificus) present an immunosupressor effect that hinders the production of an antibody rich serum (Toxicon 35, 607612, 1997: Med. Intl. 5, 8-23, 1996). The objective of this work is to find out whether heating of the venom would abolish its immunosupressor effect without changing its immunogenicity. Methods and results: Different samples of the venom were heated at 560, 700 and 100°C for 30 rain. To evaluate the supressor effect of the venom on the antibody response to a soluble antigen mice were injected subcutaneously with 5 ~g of the non-heated and heated venoms and 1 h later were immunized with human serum albumin (HSA). The effect of heating on the immunogenicity of the venom was studied by immunizing mice with 5 pg of the heated and non-heated venom and determining the anti-venom antibody titre in each case. The antibody titre was assayed by ELISA. The phospholipase and myotoxic activities were also studied using heated and non-heated venom. Heating of the venom at 100 ° but not at 56°C abolished the immunosuppressive effect of the venom but did not change its immunogenicity. The phospholipase and myotoxic activities of venom were both reduced by heating. The same results were obtained when crotoxin, the neurotoxic component of the venom was heated. Conclusions: Heating of the crotalic venom and crotoxin decreased their immunosuppressor effect without changing their immunogenicity. The phospholipase and myotoxic activities were reduced by heating. Acknowledgements: Supported by CNPq-136719/96-1.
Test&g the eficiency of a recombinant myotoxin as a tool for antibody production. C.D. Giuliani, M.R.C. Iemma, H.S. Selistre-de-Araujo (Departamento de Ci~ncias
569
titration of snake venoms in vitro. Methods and results: Determination of citotoxicity was based on the technique described by Borefreund and Puerner (J. Tissue Culture Methods 9, 7, 1984). A pre-established number of cells was seeded in a 96-well plate and exposed to the snake venom being tested for a period of time. A solution of neutral red was added for incorporation by the cells alive. After washing and cell lysis the amount of incorporated dye was read spectrophotometrically at 540 nm. The best conditions for the assay were established as 3500 Vero cells/well, 2% FCS in 199 medium and 72 hr of venom exposition. The toxicity of a number of snake venoms have been determined and expressed as the dose capable of killing 50% and 90% of the cells under the test conditions. Conclusion: Cell culture methods can be very useful for the determination of in vitro toxicity of snake venoms and other toxic substances. Acknowledgements: Financial support from CNPq and F A P E M I G .
Effect of heating on the immunosuppressor and immunogenic activities of Crotalic venom. A. Rangel-Santos, I. Mota (Laboratorio de Imunopatologia, Inst. Butantan, S~o Paulo, Brasil). Objective: The effectiveness of horse anti-venom sera depends mostly on the venom immunogenicity. However, some venoms like that of the snake cascavel (Crotalus durissus terrificus) present an immunosupressor effect that hinders the production of an antibody rich serum (Toxicon 35, 607612, 1997: Med. Intl. 5, 8-23, 1996). The objective of this work is to find out whether heating of the venom would abolish its immunosupressor effect without changing its immunogenicity. Methods and results: Different samples of the venom were heated at 560, 700 and 100°C for 30 rain. To evaluate the supressor effect of the venom on the antibody response to a soluble antigen mice were injected subcutaneously with 5 ~g of the non-heated and heated venoms and 1 h later were immunized with human serum albumin (HSA). The effect of heating on the immunogenicity of the venom was studied by immunizing mice with 5 pg of the heated and non-heated venom and determining the anti-venom antibody titre in each case. The antibody titre was assayed by ELISA. The phospholipase and myotoxic activities were also studied using heated and non-heated venom. Heating of the venom at 100 ° but not at 56°C abolished the immunosuppressive effect of the venom but did not change its immunogenicity. The phospholipase and myotoxic activities of venom were both reduced by heating. The same results were obtained when crotoxin, the neurotoxic component of the venom was heated. Conclusions: Heating of the crotalic venom and crotoxin decreased their immunosuppressor effect without changing their immunogenicity. The phospholipase and myotoxic activities were reduced by heating. Acknowledgements: Supported by CNPq-136719/96-1.
Test&g the eficiency of a recombinant myotoxin as a tool for antibody production. C.D. Giuliani, M.R.C. Iemma, H.S. Selistre-de-Araujo (Departamento de Ci~ncias