Antigenic properties of ovalbumin following heat denaturation at different temperatures: Comparison with enzymatic Denaturation

Abstract

Eighteen monoclonal antibodies (MAbs) raised from mice which were immunized with either native ovalbumin (NOA) or ovalbumin which had been heat denatured at 100∘C, (HDOA) were used to study antigenic modifications induced by either heat or subtilisin treatment. Using enzyme immunoassays (EIA) we have defined three major groups of antigenic sites: Group I thermolabile native epitopes; Group II relatively thermostable native epitopes; Group III epitopes specific to heat‐denatured ovalbumin. Heatdenatured ovalbumin can be separated into monomers and polymers which constitute 1% and 99% of the molecules respectively. Whereas MAbs belonging to Groups I and II bound to the monomeric form (mHDOA), MAbs belonging to Group III only bound to the polymeric form (pHDOA). Plakalbumin (PK) behaved similarly to pHDOA since it was recognized by many MAbs from Group III and a few from Group II. Nevertheless, PK remained as a monomeric molecule, whereas heat‐denatured ovalbumin existed mainly as polymeric aggregates. The epitopes present on ovalbumin after different heating procedures (time/temperature) were found to be stable after cooling, thus allowing specific recognition by different MAbs. The critical modification of the protein structure was found to take place at 75°C. Two main conclusions can be drawn from these results: (i) heat and enzymatic denaturation of ovalbumin led to similar antigenic modifications—these may be explained by the exposure of hydrophobic residues in both HDOA and PK as evidenced from 8‐anilino‐1‐sulphonic acid fluorescence spectra; (ii) the panel of ovalbumin‐specific MAbs was able to differentiate between the various heat treatments which had been applied to ovalbumin within the range 65–85°C, even after subsequent cooling. The aggregation of ovalbumin molecules during the heating process constitutes the main type of antigenic modification.