Beta-galactosidase enzymatic activity as a molecular probe to detect specific antibodies

• Published in: The Journal of biological chemistry 1996-08, Vol.271 (35), p.21251-21256
• Main Authors: Benito, A, Feliu, J X, Villaverde, A
• Format: Article
• Language: English
• Subjects: Antibodies - immunology; Antibodies - metabolism; Antigens, Viral - immunology; Aphthovirus - immunology; beta-Galactosidase - metabolism; Enzyme-Linked Immunosorbent Assay; foot-and-mouth disease virus; Molecular Probes; Recombinant Fusion Proteins - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.35.21251

The main antigenic region of foot-and-mouth disease virus serotype C1, also called site A, has been inserted in zones of the beta-galactosidase important for the stabilization of the active site, causing important changes in the Km and the specific activity of the resulting enzymes. The peptide is displayed at the surface of the recombinant proteins and, in all the cases, presents a good antigenicity. Among the recombinant proteins constructed, in proteins M278VP1 and M275SVP1 the peptide is inserted in a large loop of the beta-galactosidase (amino acids 272-288) involved in the formation of the activating interface. In these constructs, the binding of the specific antibodies directed to the foreign peptide causes an increase of the beta-galactosidase activity up to about 200%. This phenomenon has been proved using monoclonal antibodies and also using polyclonal sera generated against the peptide. Different hypothesis of the mechanism of modulation upon antibody binding are discussed. This insertion site seems to be sensitive enough to enzymatic modulation mediated by antibody binding. We propose further exploring this insertion site as a tool for a rapid detection of specific antibodies in a quick and simple homogeneous assay based on the colorimetric determination of beta-galactosidase activity.