The Establishment Of A New Method To Detect Food Allergen Specific IgE And It's Clinical Application

Objective To prepare a detective membrane strip of serum food allergen-specific IgE and estimate it's clinical application value in allergic diseases,in order to provide an effective and economic technique for the clinical detection of food allergen-specific IgE.Methods 1.Food allergen crude extracts of egg white,milk,wheat,peanut,soybean, cashew nut,crab,shrimp were prepared,determined the protein concentration of different allergens and condensed them.The proteins of the food allergen extracts were separated by means of SDS-PAGE.2.Chose nitrocellulose membrane as the solid support,optimized the coating and the detecting conditions.Used positive serum to detect,normal serum and fetal bovine serum as negative control.The conditions under which the coloration of positive reaction was clear and the background of negative reaction was clean were considered to be optimal.3.Prepared the membrane strip which could detect eight kinds of food allergens simultaneously, and evaluated it's sensitivity,reproducibility and stability.4.The membrane strip and the German Allergyscreen-testing system Were compared by the detecting results of 210 patients with allergic diseases.Results The optimal coating and detecting conditions of the food allergens were same,but the allergen concentration for spotting were different.The optimal spotting concentration were as follows:egg white:0.25mg/ml,milk:1mg/ml,wheat:1mg/ml, peanut:1mg/ml,soybean:1mg/ml,cashew nut:1mg/ml,crab:0.25mg/ml,shrimp:0.25 mg/ml.The optimal experimental condition and procedure were:The NC membrane was adopted as the solid support.After spotting once,the food allergens were incubated for 2 hours at room temperature,2%PVA blocking for 1 hour.Then added 1:10 diluted serum and incubated for 2 hours on the rocker at room temperature, washed the membrane strip and incubated with 2μg/ml AP-Labeled Anti-IgE Ab for 30 minutes at room temperature.Then washed the membrane strip and added substrate solution,developed the color for about ten minutes.Finally,washed the membrane strip and judged the results after the membrane strip was totally dried.The membrane strip had good sensitivity,reproducibility and stability.Compared with the German Allergyscreen-testing system,the membrane strip had no difference in detecting the majority of food allergens such as egg white,milk,peanut,soybean, crab and shrimp(P＞0.05),but the detection rate of wheat and cashew nut had difference(P＜0.05).Their positive detectical coincidence was 63.6%,negative detectical coincidence was 94.6%,the total coincidence was 70.5%.The membrane strip's detecting results of 30 normal serum were all negative.Conclusion The advantages of the membrane strip for detecting serum food allergen-specific IgE were little serum needed,short cycle time,multiple detective allergens,simple manipulation,easy to observe the results and low cost.The whole detecting process was less than four hours.This method had obvious clinical application value,it shoud be a novel detection method for the allergic diseases with broad perspectives.