تخليص بتالاكتو گلوبولين از شير گاو و مطالعه آنتي ژنيسيته آن

Simple Methods for Purification of Beta-Lactoglobulin from Cowʹs Milk while Preserving its Antigenicity

Beta-lactoglobulin (BLG) is a major whey protein in the milk of ruminants, e.g. cows, and such non-ruminants as horses. This protein is resistant under various conditions of pH and temperature. There is some evidence of absorption of some intact BLG in humans. Infants being fed with cowʹs milk-based formulas are especially prone to develop adverse reactions against proteins, including BLG, absorbed intact through their intestinal wall that is more permeable than that of adults. Regarding its importance, purification of BLG for immunological and serological studies and for setting up such diagnostic tests as ELISA and RAST is undoubtedly necessary. The aim of this study was to introduce a simple, reproducible, fast and less expensive method for BLG purification while saving its antigenicity, as well. Whey (lactoserum) was prepared by isolating casein from defatted milk using HC1. Globulins were then precipitated in whey by half-saturated ammonium sulfate. The whey and precipitated globulins were used for Sephadex G-50 gel filtration and diethyl aminoethyl cellulose anion exchange chromatography. Using enzyme-linked immunosorbent assay (ELISA), Western blotting and ELISA inhibition assay, antigenicity of the purified protein was then evaluated. In gel filtration, BLG was eluted with the second peak. In anion exchange chromatography of ammonium sulfate precipitated proteins and of whey proteins, it was eluted with the second peak. Our results showed high purity and well-preserved antigenicity of BLG thus purified.