2-SITE ENZYME IMMUNOMETRIC ASSAYS FOR DETERMINATION OF NATIVE AND DENATURED BETA-LACTOGLOBULIN

Two enzyme immunometric assays suitable for measuring native and denat ured P-lactoglobulin (BLg) have been developed. The assays were perfor med in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduc ed and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the oth er milk proteins and tryptic fragments of BLg. The validity of the ass ays in different media such as cow's milk and cow's milk products, sal ine buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-d enatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentr ation of native BLg was associated with an increase in denatured BLg s pecifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were ab le to establish a modified sandwich immunoassay permitting very sensit ive detection of cow's milk in goat's milk. (C) 1998 Elsevier Science B.V. All rights reserved.