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Volume 271, Number 35, Issue of August 30, 1996 pp. 21251-21256
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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-Galactosidase Enzymatic Activity as a Molecular Probe to Detect Specific Antibodies

(Received for publication, April 9, 1996)

From the Institut de Biologia Fonamental and Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain

The main antigenic region of foot-and-mouth disease virus serotype C₁, also called site A, has been inserted in zones of the -galactosidase important for the stabilization of the active site, causing important changes in the K_m 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 -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 -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 -galactosidase activity.

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