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

Autor: Andrea Queiroz Maranhão, Emerson Andrade Shiga, Gang Chen, Daniela Luz, Ana Maria Moro, Sachdev S. Sidhu, Bruna Alves Caetano, Letícia B. Rocha, Roxane M. F. Piazza, Wagner Quintilio, Thais Mitsunari, Miriam A. Silva, Fernanda B. Andrade
Jazyk: angličtina
Rok vydání: 2018
Předmět:
Zdroj: Antibodies, Vol 7, Iss 1, p 9 (2018)
Antibodies
ISSN: 2073-4468
Popis: 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.
Databáze: OpenAIRE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje