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Experimental antivenom against serine proteases from the Bothrops jararaca venom obtained in mice, and its comparison with the antibothropic serum from the Butantan Institute.

Authors
  • Kuniyoshi, Alexandre Kazuo1
  • Kodama, Roberto Tadashi1
  • Cajado-Carvalho, Daniela1
  • Iwai, Leo Kei2
  • Kitano, Eduardo2
  • da Silva, Cristiane Castilho Fernandes1
  • Duzzi, Bruno1
  • Dias da Silva, Wilmar1
  • Portaro, Fernanda Calheta3
  • 1 Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil. , (Brazil)
  • 2 Special Laboratory of Applied Toxinology/ Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Butantan Institute, São Paulo, SP, Brazil. , (Brazil)
  • 3 Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil. Electronic address: [email protected] , (Brazil)
Type
Published Article
Journal
Toxicon : official journal of the International Society on Toxinology
Publication Date
Sep 05, 2019
Volume
169
Pages
59–67
Identifiers
DOI: 10.1016/j.toxicon.2019.09.001
PMID: 31494207
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (<1%) at the dose of 500:1 (weight of antivenom:weight of venom). These results show that a different immunization method using isolated serine proteases improves the toxins neutralizing efficacy and could lead to a better end product to be used as a supplemental medicine to the currently used immunotherapy. Copyright © 2019 Elsevier Ltd. All rights reserved.

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