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Antibodies 2018, 7(1), 9;

Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid Latex Agglutination Test

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP 05503-900, Brazil
Department of Molecular Genetics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada
Laboratório de Biofármacos em Células Animais, Instituto Butantan, São Paulo, SP 05503-900, Brazil
Laboratório de Imunologia, Universidade de Brasília, Brasília 70910-900, Brazil
These authors contributed equally to this paper.
Author to whom correspondence should be addressed.
Received: 6 December 2017 / Revised: 10 January 2018 / Accepted: 26 January 2018 / Published: 1 February 2018
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Stx1 toxin is one of the AB5 toxins of Shiga toxin-producing Escherichia coli (STEC) responsible for foodborne intoxication during outbreaks. The single-chain variable fragment (scFv) is the most common recombinant antibody format; it consists of both variable chains connected by a peptide linker with conserved specificity and affinity for antigen. The drawbacks of scFv production in bacteria are the heterologous expression, conformation and stability of the molecule, which could change the affinity for the antigen. In this work, we obtained a stable and functional scFv-Stx1 in bacteria, starting from IgG produced by hybridoma cells. After structural modifications, i.e., change in protein orientation, vector and linker, its solubility for expression in bacteria was increased as well as the affinity for its antigen, demonstrated by a scFv dissociation constant (KD) of 2.26 × 10−7 M. Also, it was able to recognize purified Stx1 and cross-reacted with Stx2 toxin by ELISA (Enzyme-Linked Immunosorbent Assay), and detected 88% of Stx1-producing strains using a rapid latex agglutination test. Thus, the scFv fragment obtained in the present work is a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis. View Full-Text
Keywords: antibody; scFv; Stx1; STEC antibody; scFv; Stx1; STEC

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Luz, D.; Shiga, E.A.; Chen, G.; Quintilio, W.; Andrade, F.B.; Maranhão, A.Q.; Caetano, B.A.; Mitsunari, T.; Silva, M.A.; Rocha, L.B.; Moro, A.M.; Sidhu, S.S.; Piazza, R.M.F. Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid Latex Agglutination Test. Antibodies 2018, 7, 9.

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