Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia

Scorpion stings are a public health event in Colombia lacking official epidemiological data, and are considered a medical emergency. Despite the two local producers of antivenoms, neither of them is currently manufacturing scorpion antivenoms. We present the characterization of a lab-scale process t...

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Autores:: Estrada-Gómez, Sebastian
Núñez, Vitelbina
Vargas Muñoz, Leidy Johana
Madrid-Bracamonte, Carlos A.
Preciado, Lina Maria

Tipo de recurso:: Article of investigation

Fecha de publicación:: 2022

Institución:: Universidad Cooperativa de Colombia

Repositorio:: Repositorio UCC

Idioma:

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repository_id_str
dc.title.spa.fl_str_mv	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia
title	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia
spellingShingle	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia scorpion venoms LD50 antivenoms IgG Colombia
title_short	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia
title_full	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia
title_fullStr	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia
title_full_unstemmed	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia
title_sort	Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia
dc.creator.fl_str_mv	Estrada-Gómez, Sebastian Núñez, Vitelbina Vargas Muñoz, Leidy Johana Madrid-Bracamonte, Carlos A. Preciado, Lina Maria
dc.contributor.author.none.fl_str_mv	Estrada-Gómez, Sebastian Núñez, Vitelbina Vargas Muñoz, Leidy Johana Madrid-Bracamonte, Carlos A. Preciado, Lina Maria
dc.subject.spa.fl_str_mv	scorpion venoms LD50 antivenoms IgG Colombia
topic	scorpion venoms LD50 antivenoms IgG Colombia
description	Scorpion stings are a public health event in Colombia lacking official epidemiological data, and are considered a medical emergency. Despite the two local producers of antivenoms, neither of them is currently manufacturing scorpion antivenoms. We present the characterization of a lab-scale process to produce the first specific scorpion antivenom for Colombia, formulated to cover scorpion stings produced by Tityus pachyurus, Tityus asthenes, Tityus fuhrmanii, Centruroides spp. To do so, rabbits were immunized by subcutaneous injection with each venom using an immunization program of 3 months. After each rabbit reached the required IgG concentration, rabbits were bled, and plasma was separated by decantation under refrigeration. Immunoglobulins were purified from each hyperimmune plasma using a methodology including precipitation with ammonium sulfate, thermocoagulation, and purification through an ultrafiltration process using a ready-to-use and reusable laboratory crossflow tangential cassette with a polyethersulfone membrane. Each hyperimmune plasma was processed by being separated and freeze-dried at the end of the process. Rabbits were able to produce specific IgG antibodies recognizing the respective immunization venom; even an in vitro interspecies cross-recognition was detected. The separation and purification processes allowed us to obtain IgG products without considerable contaminants (except for albumin). The process was characterized, and critical stages were identified
publishDate	2022
dc.date.accessioned.none.fl_str_mv	2022-09-05T15:12:45Z
dc.date.available.none.fl_str_mv	2022-09-05T15:12:45Z
dc.date.issued.none.fl_str_mv	2022-08-25
dc.type.none.fl_str_mv	Artículos Científicos
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dc.identifier.issn.spa.fl_str_mv	1424-8247
dc.identifier.uri.spa.fl_str_mv	https://doi.org/10.3390/ ph15091047
dc.identifier.uri.none.fl_str_mv	https://hdl.handle.net/20.500.12494/46291
dc.identifier.bibliographicCitation.spa.fl_str_mv	: Estrada-Gomez, S.; Núñez, V.; Vargas-Muñoz, L.J.; Madrid-Bracamonte, C.A.; Preciado, L.M. Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia. Pharmaceuticals 2022, 15, 1047. https://doi.org/10.3390/ ph15091047
identifier_str_mv	1424-8247 : Estrada-Gomez, S.; Núñez, V.; Vargas-Muñoz, L.J.; Madrid-Bracamonte, C.A.; Preciado, L.M. Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia. Pharmaceuticals 2022, 15, 1047. https://doi.org/10.3390/ ph15091047
url	https://doi.org/10.3390/ ph15091047 https://hdl.handle.net/20.500.12494/46291
dc.relation.isversionof.spa.fl_str_mv	https://www.mdpi.com/1424-8247/15/9/1047
dc.relation.ispartofjournal.spa.fl_str_mv	Pharmaceuticals
dc.relation.references.spa.fl_str_mv	WHO. Guidelines for the Production, Control and Regulation of Snake Antivenoms Immunoglobulins; WHO: Geneva, Switzerland, 2018. Borges, A.; Lomonte, B.; Angulo, Y.; Acosta de Patino, H.; Pascale, J.M.; Otero, R.; Miranda, R.J.; De Sousa, L.; Graham, M.R.; Gomez, A.; et al. Venom diversity in the Neotropical scorpion genus Tityus: Implications for antivenom design emerging from molecular and immunochemical analyses across endemic areas of scorpionism. Acta Trop. 2020, 204, 105346. [CrossRef] [PubMed] Ward, M.J.; Ellsworth, S.A.; Nystrom, G.S. A global accounting of medically significant scorpions: Epidemiology, major toxins, and comparative resources in harmless counterparts. Toxicon 2018, 151, 137–155. [CrossRef] [PubMed] Barona, J.; Otero, R.; Nunez, V. Toxicological and immunological aspects of scorpion venom (Tytius pachyurus): Neutralizing capacity of antivenoms produced in Latin America. Biomedica 2004, 24, 42–49. [CrossRef] [PubMed] Estrada-Gomez, S.; Cupitra, N.I.; Arango, W.M.; Munoz, L.J. Intraspecific variation of Centruroides edwardsii venom from two regions of Colombia. Toxins 2014, 6, 2082–2096. [CrossRef] Otero, R.; Navio, E.; Cespedes, F.A.; Nunez, M.J.; Lozano, L.; Moscoso, E.R.; Matallana, C.; Arsuza, N.B.; Garcia, J.; Fernandez, D.; et al. Scorpion envenoming in two regions of Colombia: Clinical, epidemiological and therapeutic aspects. Trans. R. Soc. Trop. Med. Hyg. 2004, 98, 742–750. [CrossRef] Barona, J.; Batista, C.V.; Zamudio, F.Z.; Gomez-Lagunas, F.; Wanke, E.; Otero, R.; Possani, L.D. Proteomic analysis of the venom and characterization of toxins specific for Na+ - and K+ -channels from the Colombian scorpion Tityus pachyurus. Biochim. Biophys. Acta Proteins Proteom. 2006, 1764, 76–84. [CrossRef] Estrada-Gomez, S.; Gomez-Rave, L.; Vargas-Munoz, L.J.; van der Meijden, A. Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family. Toxicon 2017, 130, 104–115. [CrossRef] Estrada-Gómez, S.; Vargas-Muñoz, L.J. MS/MS analysis of four scorpion venoms from Colombia: A descriptive approach. J. Venom. Anim. Toxins incl. Trop. Dis. 2021, 27, e20200173. [CrossRef] Rincón-Cortés, C.A.; Olamendi-Portugal, T.; Carcamo-Noriega, E.N.; Santillán, E.G.; Zuñiga, F.Z.; Reyes-Montaño, E.A.; Vega Castro, N.A.; Possani, L.D. Structural and functional characterization of toxic peptides purified from the venom of the Colombian scorpion Tityus macrochirus. Toxicon 2019, 169, 5–11. [CrossRef] Gómez, J.P.; Quintana, J.C.; Arbeláez, P.; Fernández, J.; Silva, J.F.; Barona, J.; Gutiérrez, J.C.; Díaz, A.; Otero, R. Picaduras por escorpión Tityus asthenes en Mutatá, Colombia: Aspectos epidemiológicos, clínicos y toxinológicos. Biomedica 2010, 30, 126–139. [CrossRef] Bedoya Roqueme, E.; Quirós-Rodríguez, J. On Current Status of the Scorpion Fauna (Chelicerata: Scorpiones) from Cordoba, Colombian Caribbean: A Checklist; Discovery Publication: New Delhi, India, 2020; Volume 21 Gómez, J.P.; Otero, R.; Núñez, V.; Saldarriaga, M.; Díaz, A.; Velásquez, P. Aspectos toxinológicos, clínicos y epidemiológicos del envenenamiento producido por el escorpión Tityus fuhrmanni Kraepelin. MedUNAB 2002, 5, 159–165. [CrossRef] Mendoza-Tobar, L.L.; Meza-Cabrera, I.A.; Sepúlveda-Arias, J.C.; Guerrero-Vargas, J.A. Comparison of the Scorpionism Caused by Centruroides margaritatus, Tityus pachyurus and Tityus n. sp. aff. metuendus Scorpion Venoms in Colombia. Toxins 2021, 13, 757. Brenes, E.G.; Gomez, A. Scorpion maintenance in captivity for venom extraction in Costa Rica. Rev. Biol. Trop. 2016, 64, 1019–1027. [CrossRef] Solano-Godoy, J.A.; González-Gómez, J.C.; Torres-Bonilla, K.A.; Floriano, R.S. Comparison of biological activities of Tityus pachyurus venom from two Colombian regions. J. Venom. Anim. Toxins Incl. Trop. Dis. 2021, 27, e20210005. [CrossRef] de Armas, L.F.; Sarmiento, D.L.; Florez, E. Composición del género Centruroides Marx, 1890 (Scorpiones:Buthidae) en Colombia, con al descripción de una nueva especie. Bol. De La Soc. Entomol. Aragonesa 2012, 50, 105–114 Guidlolin, R.G.; Marcelino, R.M.; Gondo, H.H.; Morais, J.F.; Ferreira, R.A.; Silva, C.L.; Kipnis, T.L.; da Silva, J.; Fafetine, J.; Dias da Silva, W. Polyvalent horse F(Ab’)2 snake antivenom: Development of process to produce polyvalent horse F(Ab’)2 antibodies anti-african snake venom. Afr. J. Biotechnol. 2010, 9, 2446–2455. Guidolin, F.R.; Caricati, C.P.; Marcelino, J.R.; da Silva, W.D. Development of Equine IgG Antivenoms against Major Snake Groups in Mozambique. PLoS Negl. Trop. Dis. 2016, 10, e0004325. [CrossRef] Krifi, M.N.; Savin, S.; Debray, M.; Bon, C.; El Ayeb, M.; Choumet, V. Pharmacokinetic studies of scorpion venom before and after antivenom immunotherapy. Toxicon 2005, 45, 187–198. [CrossRef] Istvan, L.J.; Istvan, L.S. GelAnalyzer, 19.1. 2021. Available online: http://www.gelanalyzer.com/index.html (accessed on 23 June 2022). Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. [CrossRef] Fernandez, J.; Gutierrez, J.M.; Angulo, Y.; Sanz, L.; Juarez, P.; Calvete, J.J.; Lomonte, B. Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization. Biochimie 2010, 92, 273–283. [CrossRef] Otero-Patino, R.; Segura, A.; Herrera, M.; Angulo, Y.; Leon, G.; Gutierrez, J.M.; Barona, J.; Estrada, S.; Pereanez, A.; Quintana, J.C.; et al. Comparative study of the efficacy and safety of two polyvalent, caprylic acid fractionated [IgG and F(ab’)2 ] antivenoms, in Bothrops asper bites in Colombia. Toxicon 2012, 59, 344–355. [CrossRef] Ouchterlony, O. Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand. 1949, 26, 507–515. [CrossRef] Doumas, B.T.; Bayse, D.D.; Carter, R.J.; Peters, T., Jr.; Schaffer, R. A candidate Reference Method for determination of total protein in serum. I. Development and validation. Clin. Chem. 1981, 27, 1642–1650. Doumas, B.T.; Watson, W.A.; Biggs, H.G. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta Int. J. Clin. Chem. 1971, 31, 87–96. [CrossRef] Zheng, K.; Wu, L.; He, Z.; Yang, B.; Yang, Y. Measurement of the total protein in serum by biuret method with uncertainty evaluation. Measurement 2017, 112, 16–21. [CrossRef]
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dc.publisher.spa.fl_str_mv	Universidad Cooperativa de Colombia, Facultad de Ciencias de la Salud, Medicina, Medellín y Envigado Angel Josabad Alonso-Castro
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spelling	Estrada-Gómez, SebastianNúñez, VitelbinaVargas Muñoz, Leidy JohanaMadrid-Bracamonte, Carlos A.Preciado, Lina Maria152022-09-05T15:12:45Z2022-09-05T15:12:45Z2022-08-251424-8247https://doi.org/10.3390/ ph15091047https://hdl.handle.net/20.500.12494/46291: Estrada-Gomez, S.; Núñez, V.; Vargas-Muñoz, L.J.; Madrid-Bracamonte, C.A.; Preciado, L.M. Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia. Pharmaceuticals 2022, 15, 1047. https://doi.org/10.3390/ ph15091047Scorpion stings are a public health event in Colombia lacking official epidemiological data, and are considered a medical emergency. Despite the two local producers of antivenoms, neither of them is currently manufacturing scorpion antivenoms. We present the characterization of a lab-scale process to produce the first specific scorpion antivenom for Colombia, formulated to cover scorpion stings produced by Tityus pachyurus, Tityus asthenes, Tityus fuhrmanii, Centruroides spp. To do so, rabbits were immunized by subcutaneous injection with each venom using an immunization program of 3 months. After each rabbit reached the required IgG concentration, rabbits were bled, and plasma was separated by decantation under refrigeration. Immunoglobulins were purified from each hyperimmune plasma using a methodology including precipitation with ammonium sulfate, thermocoagulation, and purification through an ultrafiltration process using a ready-to-use and reusable laboratory crossflow tangential cassette with a polyethersulfone membrane. Each hyperimmune plasma was processed by being separated and freeze-dried at the end of the process. Rabbits were able to produce specific IgG antibodies recognizing the respective immunization venom; even an in vitro interspecies cross-recognition was detected. The separation and purification processes allowed us to obtain IgG products without considerable contaminants (except for albumin). The process was characterized, and critical stages were identifiedsebastian.estrada@udea.edu.covitelbina.nunez@udea.edu.coleidy.vargasmu@campusucc.edu.coaseguramientotls@udea.edu.comaria.preciado@udea.edu.co16Universidad Cooperativa de Colombia, Facultad de Ciencias de la Salud, Medicina, Medellín y EnvigadoAngel Josabad Alonso-CastroMedicinaMedellínhttps://www.mdpi.com/1424-8247/15/9/1047PharmaceuticalsWHO. Guidelines for the Production, Control and Regulation of Snake Antivenoms Immunoglobulins; WHO: Geneva, Switzerland, 2018.Borges, A.; Lomonte, B.; Angulo, Y.; Acosta de Patino, H.; Pascale, J.M.; Otero, R.; Miranda, R.J.; De Sousa, L.; Graham, M.R.; Gomez, A.; et al. Venom diversity in the Neotropical scorpion genus Tityus: Implications for antivenom design emerging from molecular and immunochemical analyses across endemic areas of scorpionism. Acta Trop. 2020, 204, 105346. [CrossRef] [PubMed]Ward, M.J.; Ellsworth, S.A.; Nystrom, G.S. A global accounting of medically significant scorpions: Epidemiology, major toxins, and comparative resources in harmless counterparts. Toxicon 2018, 151, 137–155. [CrossRef] [PubMed]Barona, J.; Otero, R.; Nunez, V. Toxicological and immunological aspects of scorpion venom (Tytius pachyurus): Neutralizing capacity of antivenoms produced in Latin America. Biomedica 2004, 24, 42–49. [CrossRef] [PubMed]Estrada-Gomez, S.; Cupitra, N.I.; Arango, W.M.; Munoz, L.J. Intraspecific variation of Centruroides edwardsii venom from two regions of Colombia. Toxins 2014, 6, 2082–2096. [CrossRef]Otero, R.; Navio, E.; Cespedes, F.A.; Nunez, M.J.; Lozano, L.; Moscoso, E.R.; Matallana, C.; Arsuza, N.B.; Garcia, J.; Fernandez, D.; et al. Scorpion envenoming in two regions of Colombia: Clinical, epidemiological and therapeutic aspects. Trans. R. Soc. Trop. Med. Hyg. 2004, 98, 742–750. [CrossRef]Barona, J.; Batista, C.V.; Zamudio, F.Z.; Gomez-Lagunas, F.; Wanke, E.; Otero, R.; Possani, L.D. Proteomic analysis of the venom and characterization of toxins specific for Na+ - and K+ -channels from the Colombian scorpion Tityus pachyurus. Biochim. Biophys. Acta Proteins Proteom. 2006, 1764, 76–84. [CrossRef]Estrada-Gomez, S.; Gomez-Rave, L.; Vargas-Munoz, L.J.; van der Meijden, A. Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family. Toxicon 2017, 130, 104–115. [CrossRef]Estrada-Gómez, S.; Vargas-Muñoz, L.J. MS/MS analysis of four scorpion venoms from Colombia: A descriptive approach. J. Venom. Anim. Toxins incl. Trop. Dis. 2021, 27, e20200173. [CrossRef]Rincón-Cortés, C.A.; Olamendi-Portugal, T.; Carcamo-Noriega, E.N.; Santillán, E.G.; Zuñiga, F.Z.; Reyes-Montaño, E.A.; Vega Castro, N.A.; Possani, L.D. Structural and functional characterization of toxic peptides purified from the venom of the Colombian scorpion Tityus macrochirus. Toxicon 2019, 169, 5–11. [CrossRef]Gómez, J.P.; Quintana, J.C.; Arbeláez, P.; Fernández, J.; Silva, J.F.; Barona, J.; Gutiérrez, J.C.; Díaz, A.; Otero, R. Picaduras por escorpión Tityus asthenes en Mutatá, Colombia: Aspectos epidemiológicos, clínicos y toxinológicos. Biomedica 2010, 30, 126–139. [CrossRef]Bedoya Roqueme, E.; Quirós-Rodríguez, J. On Current Status of the Scorpion Fauna (Chelicerata: Scorpiones) from Cordoba, Colombian Caribbean: A Checklist; Discovery Publication: New Delhi, India, 2020; Volume 21Gómez, J.P.; Otero, R.; Núñez, V.; Saldarriaga, M.; Díaz, A.; Velásquez, P. Aspectos toxinológicos, clínicos y epidemiológicos del envenenamiento producido por el escorpión Tityus fuhrmanni Kraepelin. MedUNAB 2002, 5, 159–165. [CrossRef]Mendoza-Tobar, L.L.; Meza-Cabrera, I.A.; Sepúlveda-Arias, J.C.; Guerrero-Vargas, J.A. Comparison of the Scorpionism Caused by Centruroides margaritatus, Tityus pachyurus and Tityus n. sp. aff. metuendus Scorpion Venoms in Colombia. Toxins 2021, 13, 757.Brenes, E.G.; Gomez, A. Scorpion maintenance in captivity for venom extraction in Costa Rica. Rev. Biol. Trop. 2016, 64, 1019–1027. [CrossRef]Solano-Godoy, J.A.; González-Gómez, J.C.; Torres-Bonilla, K.A.; Floriano, R.S. Comparison of biological activities of Tityus pachyurus venom from two Colombian regions. J. Venom. Anim. Toxins Incl. Trop. Dis. 2021, 27, e20210005. [CrossRef]de Armas, L.F.; Sarmiento, D.L.; Florez, E. Composición del género Centruroides Marx, 1890 (Scorpiones:Buthidae) en Colombia, con al descripción de una nueva especie. Bol. De La Soc. Entomol. Aragonesa 2012, 50, 105–114Guidlolin, R.G.; Marcelino, R.M.; Gondo, H.H.; Morais, J.F.; Ferreira, R.A.; Silva, C.L.; Kipnis, T.L.; da Silva, J.; Fafetine, J.; Dias da Silva, W. Polyvalent horse F(Ab’)2 snake antivenom: Development of process to produce polyvalent horse F(Ab’)2 antibodies anti-african snake venom. Afr. J. Biotechnol. 2010, 9, 2446–2455.Guidolin, F.R.; Caricati, C.P.; Marcelino, J.R.; da Silva, W.D. Development of Equine IgG Antivenoms against Major Snake Groups in Mozambique. PLoS Negl. Trop. Dis. 2016, 10, e0004325. [CrossRef]Krifi, M.N.; Savin, S.; Debray, M.; Bon, C.; El Ayeb, M.; Choumet, V. Pharmacokinetic studies of scorpion venom before and after antivenom immunotherapy. Toxicon 2005, 45, 187–198. [CrossRef]Istvan, L.J.; Istvan, L.S. GelAnalyzer, 19.1. 2021. Available online: http://www.gelanalyzer.com/index.html (accessed on 23 June 2022).Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. [CrossRef]Fernandez, J.; Gutierrez, J.M.; Angulo, Y.; Sanz, L.; Juarez, P.; Calvete, J.J.; Lomonte, B. Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization. Biochimie 2010, 92, 273–283. [CrossRef]Otero-Patino, R.; Segura, A.; Herrera, M.; Angulo, Y.; Leon, G.; Gutierrez, J.M.; Barona, J.; Estrada, S.; Pereanez, A.; Quintana, J.C.; et al. Comparative study of the efficacy and safety of two polyvalent, caprylic acid fractionated [IgG and F(ab’)2 ] antivenoms, in Bothrops asper bites in Colombia. Toxicon 2012, 59, 344–355. [CrossRef]Ouchterlony, O. Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand. 1949, 26, 507–515. [CrossRef]Doumas, B.T.; Bayse, D.D.; Carter, R.J.; Peters, T., Jr.; Schaffer, R. A candidate Reference Method for determination of total protein in serum. I. Development and validation. Clin. Chem. 1981, 27, 1642–1650.Doumas, B.T.; Watson, W.A.; Biggs, H.G. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta Int. J. Clin. Chem. 1971, 31, 87–96. [CrossRef]Zheng, K.; Wu, L.; He, Z.; Yang, B.; Yang, Y. Measurement of the total protein in serum by biuret method with uncertainty evaluation. Measurement 2017, 112, 16–21. 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Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia

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