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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom

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Author(s):
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Castro, J. M. A. [1] ; Oliveira, T. S. [1] ; Silveira, C. R. F. [1] ; Caporrino, M. C. [1] ; Rodriguez, D. [2] ; Moura-da-Silva, A. M. [1] ; Ramos, O. H. P. [3] ; Rucavado, A. [4] ; Gutierrez, J. M. [4] ; Magalhaes, G. S. [1] ; Faquim-Mauro, E. L. [1] ; Fernandes, I. [1]
Total Authors: 12
Affiliation:
[1] Inst Butantan, Lab Imunopatol, BR-05503900 Sao Paulo - Brazil
[2] Inst Butantan, Lab Biotecnol 4, BR-05503900 Sao Paulo - Brazil
[3] CEA, iBiTecS, Serv Ingn Mol Prot, Gif Sur Yvette - France
[4] Univ Costa Rica, Fac Microbiol, Inst Clodomiro Picado, San Jose - Costa Rica
Total Affiliations: 4
Document type: Journal article
Source: Toxicon; v. 87, p. 81-91, SEP 2014.
Web of Science Citations: 9
Abstract

BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G(4)S)(3) polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of recombinant antibody fragments for improving the neutralization of local tissue damage in snakebite envenoming. (C) 2014 Elsevier Ltd. All rights reserved. (AU)

FAPESP's process: 12/01028-3 - Production and characterization of scFv recombinant antibodies against toxins from Bothrops and Crotalus venoms
Grantee:Irene Fernandes
Support Opportunities: Regular Research Grants
FAPESP's process: 04/08297-3 - AbEvo - (Antibodies Evolution): intelligent system for the development of antibodies based on the structure of the antigen
Grantee:Oscar Henrique Pereira Ramos
Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE