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

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 × 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.

Mechanism of Anti-Salmonella Rabbit Immunoglobulin Adsorption on Polymer Particles

“…A particularly important role is played by the immunoglobulin corona adsorbed on polymer microparticles referred to as immunolatexes [ 7 , 8 ]. Such conjugates find their application in a variety of tests, including those for bacterial infections such as E. coli and Salmonella [ 9 , 10 , 11 ], viral infections such as HIV [ 12 , 13 , 14 ] and more recently against SARS-CoV-2 [ 15 , 16 , 17 , 18 ]. The widespread application of these tests is primarily attributed to their simplicity and remarkably short execution time, typically taking just a few minutes [ 19 ].…”