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

Author(s):Show less -

Luz, Daniela ^[1] ; Shiga, Emerson A. ^[1] ; Chen, Gang ^[2] ; Quintilio, Wagner ^[3] ; Andrade, Fernanda B. ^[1] ; Maranhao, Andrea Q. ^[4] ; Caetano, Bruna A. ^[1] ; Mitsunari, Thais ^[1] ; Silva, Miriam A. ^[1] ; Rocha, Leticia B. ^[1] ; Moro, Ana M. ^[3] ; Sidhu, Sachdev S. ^[2] ; Piazza, Roxane M. F. ^[1]

Affiliation

^[1] Inst Butantan, Lab Bacteriol, BR-05503900 Sao Paulo, SP - Brazil

^[2] Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Dept Mol Genet, Toronto, ON M5S 3E1 - Canada

^[3] Inst Butantan, Lab Biofarmacos Celulas Anim, BR-05503900 Sao Paulo, SP - Brazil

^[4] Univ Brasilia, Lab Imunol, BR-70910900 Brasilia, DF - Brazil

• Document type: Journal article
• Source: ANTIBODIES; v. 7, n. 1 MAR 2018.
• Web of Science Citations: 0

Abstract

Stx1 toxin is one of the AB(5) 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 (K-D) of 2.26 x 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. (AU)

• FAPESP's process: 11/12928-2 - New challenge for the diagnosis of diarrheagenic Escherichia coli: recombinant antibody obtention against different virulence factors
• Grantee: Roxane Maria Fontes Piazza
• Support Opportunities: Regular Research Grants
• FAPESP's process: 17/17006-2 - Shiga toxin-producing Escherichia coli (STEC) intoxication and colonization in Zebrafish model: An in vivo alternative for pathogenicity studies and therapeutic antibodies validation
• Grantee: Daniela Luz Hessel da Cunha
• Support Opportunities: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 13/03160-9 - New strategies for recombinant antibodies generation against Stx1 and Stx2 toxins produced by Shiga toxin-producing E. coli
• Grantee: Daniela Luz Hessel da Cunha
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate