The Technique Of Quick Detecting Multiple Food Allergens In Vitro

Objective: To establish a novel method based on detecting of histamine release ratiofrom sensitized mast cells to assess and identify the food allergens, and to develop a method of dot-ELISA which can be used to detect specific IgE for multiple food allergens simultaneously.Methods: The BALB/c mice were immunized with extract of shrimp proteins, HEW(hen egg white proteins) and peanut proteins with the adjuvant of aluminum hydroxide. The spefic IgE of food allergens was measured by indirect ELISA. The peritoneal mast cells were collected, stimulated by allergen in vitro. Under electronic microscopy, the morphological difference of mast cells was analyzed before and after degranulation. The histamine release ratio was detected by fluorescence assay. The main allergen components in food allergen extract were analyzed by Western blot, and purified by anion exchange chromatography. The specific IgE of food allergens was detected by dot-ELISA.Results: The levels of sIgE and histamine release ratio in allergic mice model weremuch higher than those in control. The sIgE was the highest at 14th day after challenge, reached 1/6400, 1/6400 and 1/3200 in shrimp, peanut and HEW group respectively. The histamine release ratio of HEW group was 60.75% and the control was 37.19%; that of shrimp groups were 71.53% and 88.48% respectively by intraperitoneal injection (i.p) or hypodermic injection (i.h), but the control was 43.76%. The degranulated mast cells had some vacuoles and less compact granules in cell plasma. The main component, OVA of HEW, which molecular weight is 40 kD, and Pen m 2 of shrimp, which molecular weight is 36 kD, had been identified by Western blot analysis. In dot-ELISA, the optimal coating amount of various allergen extracts were 2 uL (about 10 μg proteins), and the suitable dilution of serum to detect was 1:80. The types of sIgE could be determined by the dots on the NC membrane coated with the specific allergens. There was no cross reactivity among peanut,shrimp and egg white. The same results could be repeated at different time, and the coated NC membrane could be stored stably at 4°C for 3 months.Conclusion: The results revealed that the method of detection of histamine releasemay become a prospective technique to assess and identify food allergens. Dot-ELISA could be used to detect multiple food allergens simultaneously, which was specific, stable and well-repeatable, so as to be applied in clinic allergies diagnosis.