Comparison of Antigenicities of Whole Milk and Partial Hydrolysate of Cow's Milk Proteins by Western Blot Analysis

Lee, Soo Young; Hong, Chang Ho

Abstract

For the prevention or the management of milk allergy in infancy, partial or extensive hydrolysates of cow's milk have been used in western countries for a couple of decades. Recently a Korean product of partial hydrolysate(HA-21) became available for the prevention of sensitization to cow's milk proteins. In this study, to compare the antigenicities of whole milk(WM) and HA-21, we performed the IgG and IgE western-blot analysis. Sera were obtained from 17 milk sensitive infants and 2 controls, and crude extract of WM and HA-21 and purified beta-lactoglobulin(BLG), bovine serum IgG(B-IgG), alpha-lactalbumin(ALA) were used for blot- inhibition study. After the non-reduced SDS-PAGE, western-blot studies were done using biotinylated anti-human IgG and IgE antibodies, and the reaction were detected by avidine-phosphatase system. By the SDS-PAGE analysis, WM were separated into bovine-IgG(B-IgG), bovine serum albumin(BSA), 56 KD protein, caseins(CAS), BLG and ALA, but there was no visible bands above 14 KD in the case of HA-21. In preliminary blot analysis with 4 milk sensitive sera, we found the broad and strong IgE-binding protein band in the range of 150-200 KD in all cases. This fraction, named as 'P-band' in this study, was completely inhibited by BLG and partially inhibited by B-IgG using blot-inhibition study. Using the western blot analysis of WM, the P-band revealed the most prevalent IgG and IgE binding protein in 16 of 17 tested sera. More than 15 bands were identified by IgG-blot of WM, but the IgE biding proteins were P-band(16 sera), BSA(12 sera), 56 KD protein(8 sera), and CAS(5 sera). In the case of HA-21, BSA(8 sera) was the only protein bound with IgE antibody and the reaction was very weak compared to that of WM. In conclusion, the major IgE binding protein appears to be the p-band which might contain the epitopes of B-IgG and BLG in this study. The antigenicity of HA-21 is remarkably reduced compared to WM, but still minute residual antigenicity and allergenicity are remained in the extract of HA-21.