Preparation of Monoclonal Antibody against Bovine β-Lactoglobulin and Its Unique Binding Affinity

Abstract

Five hybridoma cell lines, 21B3, 31A4, 61C1, 61B4, and 62A6 were established and characterized to produce IgG₁ antibodies with specificity for bovine β-lactoglobulin (β-LG) which is a potent milk allergen. A representative monoclonal antibody (MAb) 21B3 had a unique affinity; it bound more strongly to RCM (reduced carboxymethylated) β-LG than to native β-LG. The affinity increased drastically when β-LG was heated at 70°C or above for lOmin at pH 7.0, while β-LG heated at pH 3.0 did not alter its affinity even when heated at 80°C for 10min. An enzyme-probe method suggested that unfolding of β-LG by heating at pH 7.0 seemed to start at 70°C, and β-LG heated at 90°C and RCM β-LG were extensively unfolded. The results indicated that the phase of unfolding and increase of affinity by heating were coincidental. Finally the relationship of the unique affinity of MAb and the β-LG conformation was discussed.