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In vitro assay to determine the potency of horse anti-crotalic venom serum
Abstracts / Toxlcon 38 (2000) 487-595
571
~tl of serum and r=0.95 (p < 0.01) for EDs0 expressed in mg of protein. Conclusion: Our results indicate the potential use of FUNED's IRV to estimate the potency of samples during the processing of bothropic antivenoms. Acknowledgements: Financial support from F u n d a ~ o Ezequiel Dias.
lntraspecies variations and antigenic cross-reactivity in the venoms o f Bothrops atrox and Lachesis muta muta. M. Colombini, D.F. Cardoso, I. Fernandes, A.M. Moura-
da-Silva (Laborat6rio de Imunopatologia, Instituto Butantan, S~o Paulo, Brazil). Objective: A differential immunoassay to discriminate the accidents by Bothrops atrox and Lachesis muta muta is very important for the diagnostic of ophidic accidents in North Brazil as well as a for a precise epidemiological survey. The aim of this study is to analyze the intraspecies variation and the antigenic crossreactivity in the B. atrox and L. m. muta venoms. These studies are important to select antigens to be used in the production of monoclonal antibodies specific for venoms of each species. These antibodies will be important tools for the differential immunoassay. Methods and results: Venoms of 34 specimens of B. atrox and 4 of L. m. muta were analyzed by SDS PAGE. The intra or interspecies differences were evaluated by analysis of the antigenic pattern of individual venoms stained by Coomassie blue and/or by Western Blotting using serum of mice immunized with a pool of venoms of each species. The electrophoretic pattern of B. atrox venoms showed major bands around 50, 42 and 22 kDa. Qualitative and quantitative variations were observed mainly around 107 50 kDa, 34-29 kDa and below 17 kDa. L. m. muta venoms showed major bands around 97, 28, 24 and 21 kDa. Only little variations were observed for these samples. The Western blotting analysis showed that antigens above 30 kDa were the most immunogenic for both B. atrox and L. m. muta venoms. Very little intraspecies variation was detected by the homologous serum on samples of B. atrox, mainly related to low tool. wt antigens. When antigenic cross-reactivity between the species was further analyzed, the antiserum against L. m. muta recognized several B. atrox venom antigens. However, it failed in recognizing the major antigens of 50 and 42 kDa. The L. m. muta antigens above 30 kDa were equally recognized by homologous and heterologous sera, but the recognition by heterologous serum was weaker than the one with homologous serum and less remarkable between 80 and 50 kDa. Conclusion: Antigenic cross-reactivity as well as intraspecies variability were detected between B. atrox and L. m. muta venoms. Meanwhile, by Western Blotting, it was possible to detect non cross-reactive antigens. These antigens are being isolated and will be used in the immunization of mice to get monoclonal antibodies which will be used in the differential immunoassay. Acknowledgements: Supported by FAPESP (97/13516-0), CNPq.
In
vitro assay
to determine
the potency
o f horse anti-crotalic venom serum.
L.G.D. Heneine a, Jfinior A.D. Carvalho a, C.F. Barbosa b, Santos M.R.A. Dos a (aCentro de Pesquisa e Desenvolvimento; bDiretoria de Produg~o Farmaceutica e
572
Abstracts/Toxicon 38 (2000) 487 595
Industrial, Funda~fio Ezequiel Dias, Rua Conde Pereira Carneiro Carneiro, 80, Belo Horizonte, MG, Brasil). Objectives: Develop an Enzyme linked immunosorbent assay to determine the potency of therapeutic horse anti-crotalic venom serum. Methods and results: A sandwich ELISA was standardised using venom affinity purified IgG from monovalent anti-crotalic serum as coating reagent and as H R P conjugate. The antigen was Crotalus durissus terr~cus venom, used in different concentrations. Assay conditions yielding an absorbance at 492 nm of 1.0 was used to set the competition ELISA, with the test horse antivenom sera diluted 1:10,000 as the inhibitor. Inhibition of the control ELISA, by the different test sera used, correlated well with their antivenom potency as determined in vivo. Correlation values of r ~ 1.0 (p > 0.05) were observed. Test serum included samples from different immunisation cycles and also different batches of the ampouled final product. Conclusion: The results indicate that the method proposed here is accurate. It could be used in different stages of the antivenom production process, either as a screening test for antivenom titre before the horses are bled or as antivenom potency test for the final product. Acknowledgements: We thank the Veterinary Claudio Fonseca, the Immunobiologicals Division and the Biological Control Service, F U N E D . This work was supported by F A P E M I G .
Body distribution of Loxosceles intermedia spider venom in mice and effects of specific antivenom. M.P. Revelo a, R.C. Aratijo b, M.C. Rodrigues b, R.M. Santos b, F.C. Maciel b, A.P. Ferreira 6, C.R. Diniz b, E.A. Bambirra a, O.C. Mangili °, O16rtegui C. Chilvez b (aDepartamento de Anatomia Patol6gica, U F M G , Brazil; bFundac~o Ezequiel Dias, Laborat6rio de Pesquisa, Belo Horizonte, MG, Brazil; CUFPR, Brazil). Objectives: Report the distribution of Loxosceles intermedia venom in serum and in various tissues of CF1 mice and the efficacy of antivenom in neutralizing the venom. Methods and results: The animals were injected s.c. with 2.5 ~g of L. intermedia venom, divided into groups of four animals and killed at different times from 10 rain to 72 h. Blood samples and samples of different tissues were collected. The ELISA technique was used to detect L. intermedia venom and antivenom in the samples. Maximum venom levels ocurred at 15 rain in the serum and in the site of injection and at 30 rain in the kidney. In other tissues the venom levels were very low and no venom was detected in the brain. After 2 h the venom levels decreased rapidly in serum and in all other tissues until they were no longer detectable, after 4 h in the serum. In another experiment, 20 ~tl of Loxosceles antivenom were injected i.v. together with the venom, and no venom was detected in blood and tissues. In a third experiment, Loxosceles antivenom was injected i.v. 1 h after venom administration, and partial reduction of venom level was detected in both serum and kidney. Conclusions: These studies may provide data for more objective treatment that might result in a more economic, efficient and controlled use of antivenom in spider bite accidents involving humans. Acknowledgements: Financial support from CNPq, F A P E M I G , U F M G , UFPR.
571
~tl of serum and r=0.95 (p < 0.01) for EDs0 expressed in mg of protein. Conclusion: Our results indicate the potential use of FUNED's IRV to estimate the potency of samples during the processing of bothropic antivenoms. Acknowledgements: Financial support from F u n d a ~ o Ezequiel Dias.
lntraspecies variations and antigenic cross-reactivity in the venoms o f Bothrops atrox and Lachesis muta muta. M. Colombini, D.F. Cardoso, I. Fernandes, A.M. Moura-
da-Silva (Laborat6rio de Imunopatologia, Instituto Butantan, S~o Paulo, Brazil). Objective: A differential immunoassay to discriminate the accidents by Bothrops atrox and Lachesis muta muta is very important for the diagnostic of ophidic accidents in North Brazil as well as a for a precise epidemiological survey. The aim of this study is to analyze the intraspecies variation and the antigenic crossreactivity in the B. atrox and L. m. muta venoms. These studies are important to select antigens to be used in the production of monoclonal antibodies specific for venoms of each species. These antibodies will be important tools for the differential immunoassay. Methods and results: Venoms of 34 specimens of B. atrox and 4 of L. m. muta were analyzed by SDS PAGE. The intra or interspecies differences were evaluated by analysis of the antigenic pattern of individual venoms stained by Coomassie blue and/or by Western Blotting using serum of mice immunized with a pool of venoms of each species. The electrophoretic pattern of B. atrox venoms showed major bands around 50, 42 and 22 kDa. Qualitative and quantitative variations were observed mainly around 107 50 kDa, 34-29 kDa and below 17 kDa. L. m. muta venoms showed major bands around 97, 28, 24 and 21 kDa. Only little variations were observed for these samples. The Western blotting analysis showed that antigens above 30 kDa were the most immunogenic for both B. atrox and L. m. muta venoms. Very little intraspecies variation was detected by the homologous serum on samples of B. atrox, mainly related to low tool. wt antigens. When antigenic cross-reactivity between the species was further analyzed, the antiserum against L. m. muta recognized several B. atrox venom antigens. However, it failed in recognizing the major antigens of 50 and 42 kDa. The L. m. muta antigens above 30 kDa were equally recognized by homologous and heterologous sera, but the recognition by heterologous serum was weaker than the one with homologous serum and less remarkable between 80 and 50 kDa. Conclusion: Antigenic cross-reactivity as well as intraspecies variability were detected between B. atrox and L. m. muta venoms. Meanwhile, by Western Blotting, it was possible to detect non cross-reactive antigens. These antigens are being isolated and will be used in the immunization of mice to get monoclonal antibodies which will be used in the differential immunoassay. Acknowledgements: Supported by FAPESP (97/13516-0), CNPq.
In
vitro assay
to determine
the potency
o f horse anti-crotalic venom serum.
L.G.D. Heneine a, Jfinior A.D. Carvalho a, C.F. Barbosa b, Santos M.R.A. Dos a (aCentro de Pesquisa e Desenvolvimento; bDiretoria de Produg~o Farmaceutica e
572
Abstracts/Toxicon 38 (2000) 487 595
Industrial, Funda~fio Ezequiel Dias, Rua Conde Pereira Carneiro Carneiro, 80, Belo Horizonte, MG, Brasil). Objectives: Develop an Enzyme linked immunosorbent assay to determine the potency of therapeutic horse anti-crotalic venom serum. Methods and results: A sandwich ELISA was standardised using venom affinity purified IgG from monovalent anti-crotalic serum as coating reagent and as H R P conjugate. The antigen was Crotalus durissus terr~cus venom, used in different concentrations. Assay conditions yielding an absorbance at 492 nm of 1.0 was used to set the competition ELISA, with the test horse antivenom sera diluted 1:10,000 as the inhibitor. Inhibition of the control ELISA, by the different test sera used, correlated well with their antivenom potency as determined in vivo. Correlation values of r ~ 1.0 (p > 0.05) were observed. Test serum included samples from different immunisation cycles and also different batches of the ampouled final product. Conclusion: The results indicate that the method proposed here is accurate. It could be used in different stages of the antivenom production process, either as a screening test for antivenom titre before the horses are bled or as antivenom potency test for the final product. Acknowledgements: We thank the Veterinary Claudio Fonseca, the Immunobiologicals Division and the Biological Control Service, F U N E D . This work was supported by F A P E M I G .
Body distribution of Loxosceles intermedia spider venom in mice and effects of specific antivenom. M.P. Revelo a, R.C. Aratijo b, M.C. Rodrigues b, R.M. Santos b, F.C. Maciel b, A.P. Ferreira 6, C.R. Diniz b, E.A. Bambirra a, O.C. Mangili °, O16rtegui C. Chilvez b (aDepartamento de Anatomia Patol6gica, U F M G , Brazil; bFundac~o Ezequiel Dias, Laborat6rio de Pesquisa, Belo Horizonte, MG, Brazil; CUFPR, Brazil). Objectives: Report the distribution of Loxosceles intermedia venom in serum and in various tissues of CF1 mice and the efficacy of antivenom in neutralizing the venom. Methods and results: The animals were injected s.c. with 2.5 ~g of L. intermedia venom, divided into groups of four animals and killed at different times from 10 rain to 72 h. Blood samples and samples of different tissues were collected. The ELISA technique was used to detect L. intermedia venom and antivenom in the samples. Maximum venom levels ocurred at 15 rain in the serum and in the site of injection and at 30 rain in the kidney. In other tissues the venom levels were very low and no venom was detected in the brain. After 2 h the venom levels decreased rapidly in serum and in all other tissues until they were no longer detectable, after 4 h in the serum. In another experiment, 20 ~tl of Loxosceles antivenom were injected i.v. together with the venom, and no venom was detected in blood and tissues. In a third experiment, Loxosceles antivenom was injected i.v. 1 h after venom administration, and partial reduction of venom level was detected in both serum and kidney. Conclusions: These studies may provide data for more objective treatment that might result in a more economic, efficient and controlled use of antivenom in spider bite accidents involving humans. Acknowledgements: Financial support from CNPq, F A P E M I G , U F M G , UFPR.