Epitopic characterization of native bovine β-lactoglobulin - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement Access content directly
Journal Articles Journal of Immunological Methods Year : 2002

Epitopic characterization of native bovine β-lactoglobulin

Gilles Clement
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Didier Boquet
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Yveline Frobert
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Hervé Bernard
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Luc Negroni
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Jean-Marc Chatel
Karine Adel-Patient
Christophe Creminon
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Jean-Michel Wal
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Jacques Grassi
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Two monoclonal antibodies (mAbs) (mAb 97 and mAb 117) selected from a panel of 52 mAbs directed against beta-lactoglobulin (BLG) have previously been used to develop a two-site enzyme immunometric assay (EIA) specific for the native form of the protein [J. Immunol. Methods 220 (1998) 25]. In the present work, the conformational epitopes recognized by these two mAbs and by the 50 others have been studied. Firstly, an epitope map was drawn using a surface plasmon resonance (SPR) biosensor: the epitopes were organized in a circle of 11 overlapping and 1 nonoverlapping antigenic regions. Secondly, 55 site-directed BLGA mutants were prepared and tested by ELISA and competitive immunoassay to localize these 12 antigenic regions on the protein molecule. Among them, 20 mutants showed a 10- to 7500-fold decrease in relative affinity for the mAbs of one or several neighbouring regions: their circular dichroism (CD) spectra were identical to the spectrum of wild-type (WT) BLGA. At least one mutant was found for each of the 11 overlapping antigenic regions which circled the molecule and for the nonoverlapping one which was localized near the entrance of the calyx. The two mAbs initially chosen were each directed towards very conformation-dependent epitopes and were thus suitable for monitoring native BLG in food products and manufacturing processes. Other mAb pairs could be used to follow the fate of specific regions of the molecule during denaturation or proteolytic digestion.



Dates and versions

hal-04083984 , version 1 (27-04-2023)



Gilles Clement, Didier Boquet, Yveline Frobert, Hervé Bernard, Luc Negroni, et al.. Epitopic characterization of native bovine β-lactoglobulin. Journal of Immunological Methods, 2002, 266 (1-2), pp. 67-78. ⟨10.1016/S0022-1759(02)00149-7⟩. ⟨hal-04083984⟩


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