Effect of Heat Treatment on the Antigen-Binding Activity of Anti-Peroxidase Immunoglobulins in Bovine Colostrum

Abstract

After intramammary immunization with horseradish peroxidase, bovine colostrum containing anti-peroxidase immunoglobulins (Ig) was obtained. Thermoresistance of the antigen-binding region of these Ig was studied using a direct competitive ELISA. This technique is based on the competition between the anti-peroxidase IgG coated to the plate and the anti-peroxidase IgG contained in the colostrum to bind peroxidase, the antigen, and the enzyme responsible for development of the color of the assay. Thus, the degree of denaturation of the antigen-binding region in the IgG molecule can be determined because this region is directly involved in the assay. The kinetic and thermodynamic parameters for heat-induced denaturation of IgG in colostrum were determined over a temperature range of 69 to 81°C. The denaturation of IgG was best described assuming an apparent reaction order of 1.5. D values, the time required to reduce the antigen-binding activity of IgG by 90%, were 8504, 1387, 285, and 152s at 69, 72, 77, and 81°C, respectively. Similarly, Z value, the degrees necessary to reduce the D value in one logarithmic cycle, was estimated to be 6.6°C, and the activation energy value was 386.83 kJ/mol. These results should be taken into account in the design of heat treatments of milk in order to preserve the biological function of Ig.