RESUMO
Objective To investigate the allergization of milk high molecular weight proteins. Methods Thirty cas?es of patients with serum allergic to milk were selected. Their skin prick tests were positive. Results of serum specific IgE (sIgE) test were positive and≥1+. One case of healthy control with negative sIgE test and without history of allertgy, was in?cluded in this study. The serum samples were collected and frozen at-20℃. Sephadex G200 gel chromatography was used to obtain milk high molecular weight proteins. Western blot and ELISA methods were used to detect milk high molecular weight proteins and the activity of serum sIgE. Results Results of SDS-PAGE showed that high molecular weights proteins displayed by Sephadex G200 gel chromatography, mainly including three bands, the molecular weight of 67 ku, 80 ku and 160 ku. Western blot analysis showed that three kinds of high molecular weights proteins can react with milk allergy serum, and the most obvious appeared near the molecular weight 67 ku band. ELISA analysis showed that the positive response rate of high molecular weight proteins with milk allergic patient’s serum was slightly higher than that ofβ-lactoglobulin (46.7%and 43.3%, respectively). Conclusion The milk high molecular weight protein components can induce specific IgE antibod?ies, which have important sensitization in the process of milk allergy.
RESUMO
Objective To explore a novel quantitative detection method for the concentration of specific IgG (sIgG) in food intolerance, taking egg sIgG detection for the example. Methods A total of 173 patients underwent food allergen sIgG detection were included in this study, and 78 healthy subjects were used as negative controls. The microtiter plates were coated with biotinylated bovine serum albumin (BSA) and linked with streptavidin. Then, the biotinylated egg antigen and specimen were successively added into the wells of plate.After washing, enzyme labeled anti-human IgG was added to establish an antigen indirectly coated liquid-phase reaction patterns of ELISA method. The concentration of the biotinylated allergens and enzyme labeled antibody were optimized and the reaction conditions were determined. This method was used to detect the sIgG in serum samples. Results The biotinylated egg white was selected as antigen, the optimal dilution rate was 1∶2 000,and the most suitable enzyme labeled antibody dilution ratio was 1∶12 000 in this method. The within-run and the between-run coefficients of variation were 4.83%-8.55%and 4.88%-7.93%respectively. The specificity is preferable, the species-crossed reaction rates with crab, cow milk and goat milk were＜10%. There was a good correlation between the assay developed in this study and the food intolerance detection kit provided by United States BIOMERICA Inc ( =0.977X+8.45, r=0.961, P＜0.05). Conclusion The detection method can be used to detect serum sIgG for egg intolerance patients with easy operation, highly accuracy and specificity. Furthermore, it showed the capacity of excellent repeatability and flexibility potential, which provided a good foundation for developing a kind of“personalized”random combination of food varieties ELISA kit.