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

Abstract

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.

Discriminating Foot-and-Mouth Disease Virus-Infected and Vaccinated Animals by Use of β-Galactosidase Allosteric Biosensors

“…Enzymatic assays. ␤-Galactosidase enzymatic activity was analyzed according to Miller's method (35) as described previously (3) using chlorophenol red The enzymatic activity modulation assay for detection of anti-FMDV antibodies was performed as described previously (3). Briefly, different amounts of recombinant protein were incubated at 28°C with 1% (wt/vol) bovine serum albumin, in the presence or absence of different dilutions of anti-FMDV antibodies or animal sera (Table 1).…”

Designing allosteric peptide ligands targeting a globular protein

“…[23][24][25][26][27][28] In addition, it manifests concentration dependent enzymatic activation by landscape phage derived antibody peptides. [29][30][31][32][33][34][35] These findings, GAL's commercial availability, and the technical ease of its ELISA 36,37 encouraged its selection as the target globular protein.…”

“…These peptides displayed positive modulatory influences on GAL's binding-activation kinetics that were not unlike the reported allosteric responses to antibody of both native and engineered GAL (Figure 4). [23][24][25][26][27][28] In the second kind of experiment, mAB and the peptides were held constant, 50 L/well (at 500 g/mL), and GAL was added at four concentrations: 2.3, 4.8, 9.6, and 19.2 g/mL ( Figure 5). The kinetic studies with results portrayed in Table II were of the second kind and conducted at 238C.…”

Engineering Regulable Escherichia coliβ-Galactosidases as Biosensors for Anti-HIV Antibody Detection in Human Sera

“…Proteins were detected in crude cell extracts by Western blot as described (4). Essentially, small amounts of cultures (around 20 l) were submitted to rapid centrifugation and solubilized cell sediments loaded on 7.5% SDS-polyacrylamide gel electrophoresis.…”

“…The three-dimensional structure of ␤-galactosidase (3) permits the permissiveness of solvent-exposed loops to heterologously inserted peptides to be explored. In this context, we previously constructed some recombinant ␤-galactosidases displaying foot-and-mouth disease virus (FMDV) 1 B-cell epitope peptides accommodated in solvent-exposed surfaces (4,5). The peptide insertion results in hybrid ␤-galactosidases with reduced enzymatic activity.…”