Establishment Of Visual Antibody Microarray In Food Allergen Detection

As the incidence of food allergy increased year by year, more and more attentions are paid for it. FAO reported that more than 90% of food allergies were caused by 8 major food allergens. Up to date, there is no effective treatment for food allergy, the only effective way is to avoid the intake of foods containing some certain food allergens. Since trace amount of allergen can cause serious allergic reactions, thus, developing an accurate, sensitive and quick detection of food allergens is much more significant to improve the level of food safety and protect people's health.There is a wide range of allergens, and a variety of food can cause allergy of suffers, how to detect the"hidden"allergen in foods quickly becomes the main focus of consumers. Protein microarray technology has drawn more and more attentions of scientific reaearchers for its advantage of high specificity, sensitivity and high-throughput. In this research, Penaeus Vannamei, Ruditapes philippinarum, Scomberomorus niphonius, and Arachis hypogaea L. were choosed as materials, a visual antibody microarray method was established based on optimization of key factors such as microarray slide supporters and application conditions of visual antibody microarray; the detection conditions of visual antibody microarray were determined by orthogonal test, performance of the method, standard addition experiment and detection of allergen in processed foods were studied. Main conclusions are listed as following:1,3D slides modified with agarose are confirmed as perfect supportes of visual antibody microarray by comparing the immobilization effect of the 2D slides and 3D slides, and the optimum concentration of agarose is 1.2%.2,The best conditions of visual antibody microarray for allergen detection of shrimp, clam, mackerel and peanut were determined through the orthogonal test. The results showed that the detection limit of shrimp allergen by visual antibody microarray was 100.0ng/mL with linear range of 100ng/mL-100μg/mL, the detection limit of clams allergen was 10.0ng/mL with linear detection range of 100ng/mL-100μg/mL, the detection limit of mackerel allergen was 100.0ng/mL with linear detection range of 1μg/mL-lmg/mL, the detection limit of peanut allergen was 100.0ng/mL with linear detection range of 1μg/mL-1mg/mL.3,Performance test results of of visual antibody microarray show that:the method has good precision:the intra-assay coefficient of variation was<10%and inter-assay CV was<15%. The activity of antibody microarray remained stable for 120 days during storage at 4℃in the sealed wet box. The method has good specificity, cross-reaction only took place among the proteins with high homology; and the results of standard addition experiment show that the recovery of different spiked samples ranged from 70% to 105%.4. The efficiency of visual antibody microarray in detection of allergen in processed food was further validated with double-antibody sandwich ELISA, the positive agreement between the visual antibody microarray and double-antibody sandwich ELISA occupied 77.78%. With characters like high throughput, low cost, simple operation and visible results, this method has shown great potential in the massive parallel analysis of processed food allergens simultaneously without precision instruments.Currently, the studies of the immunoassay for food allergens are mainly focused on the traditional enzyme immunoassay, few studies about antibody microarray detection was carried out, research of food allergen detection with visual antibody microarray has not been reported. In this study, visual antibody microarray with good stability and precision for detection of food allergen is developed for the first time based on improved double-antibody sandwich ELISA. The established methods offered a more effective technology for food screening detection and showed great practical significance in enhancing food security and protecting the health of sensitive populations.