Detection Of The Components In Milk Causing Food Intolerance And Its Clinical Application

Objective:Food intolerance is one of the major public issues today,and the identification and purification of food intolerance are critical for prevetion and treatment of anaphylactic diseases.Milk is investigated in this study.To analyze the components in milk causing food intolerance and separate preliminarily, then the separated proteins were used as antigens to make the diagnostic reagents in detecting food intolerance in milk by Double antigen sandwich ELISA and Biotin-Avsdin System ELISA.Methods:Serum samples of the patients, having food intolerance to milk were used to be probes. The proteins in the zones separated by SDS-PAGE and western blotting,which can react with the probes were the antigens. Selective precipitation and gel chromatography were used to purify the intolerance components, immunoreactivities of which were definited by western blotting and ELISA. The main experiment process was coating the antigen and enzyme labelled antigen respectively, the reaction was optimized using cross matching, similarily, indirect ELISA was coating the microwells with biotinylated BAS and avidin, then the biotinylated antigen and HRP labeled anti-human IgG were prepared. Concentrations of the special IgG could be obtained from the standard curve of the double antibody ELISA.Results :The proteins in milk, reacting with serum of food intolerance were immunoglobulin(160k Da), bovine serum albumin(67k Da) and casein(30k Da), while,?-lactoglobulin and?-lactalbumin also had reactions with serum of food intolerance.Casein, ?-lactoglobulin, ?-lactalbumin and the large molecular weight P1 fractions like bovine serum albumin and immunoglobulin could be achieved by isoelectric point precipitation, gel filtration chromatography and ion exchange.Double antigen sandwich ELISA was used to detect antibody in milk, compared with indirect ELISA method of Biomerica US, the sensitivity was 93.3%, specificity was100%, precision was 96.7%, the positive predictive value and negative predictive value were 100% and 93.7% respectively. Also, when used to detect milk special food intolerance, the sensitivity of this model was 92.1%, specificity was 90.0%, precision was 91.0%.Conclusions:1. The food intolerance was different from food allergy, for example, the proteins in food intolerance like immunoglobulin, bovine serum albumin and casein were all big molecular substances, while, the components in different individuals of food intolerance were basically the same.2. The function of food special IgG detection reagent depends on the used known antigen. There are complex proteins in food, so it was necessary to distinguish the proteins and purify preliminarily.3. Double antigen sandwich ELISA was used to detect the food special IgG, it was more specific in detection compared with indirect ELISA. The indirect coating model of food special IgG ELISA method was used to detect food special IgG as well,because it need not coat antigens directly, so we can choose individual detection based on actual need of the patients.