The Study Of Establishment And The Accuracy Of An Aquatic Product Allergen Detecting Method

The issue of food allergy has caused numbers of international organizations andcountries to pay great attention, but the diagnosis of food allergens is still a challenge.Due to the safety of the in vivo experiments, in vitro serum-specific IgE test in foodallergens diagnosis is being more and more concerned. Currently, there are a lot of invitro commercial serum-specific IgE testing equipments and kits available, in whichthe Immuno CAP is the method that has been most extensively investigated in thecontext of food allergy while it is not widely use because of the high cost. In addition,the types food allergens are of much distinction due to the regional differences, whilethe most food allergen detecting kits are imported overseas and the coated allergenscouldn’t meet our nation’s demands, which lead to a poor detecting accuracy andreproducibility. This study based on the above issues, aims to come up with a foodallergen detecting methods for Chinese allergic patients with the advantages of highaccuracy, convenience, fast and low cost.In this study, fish were chosen for the subject investigated as the major foodallergens for Chinese people. To explore the selection criteria of the allergens used tobe tested, allergen identification, cross-reactivity research and study of the effects ofprocessing on allergen immune activity have been carried out; the visible foodallergen detecting method based on dot-blot is established and its performance hasbeen measured. Finally, the method is compared with the other detecting methods toverify its accuracy. The main results are as follows:1. The allergens of7commonly edible fish: Alaska pollock(Theragrachatcogramma）, red snapper(Pagrosomus Major), salmon(Salmo salar), spanishmackerel(Scomberomrus niphonius), turbot(Scophthatmus maximus), large yellowcroaker(Larimichthys crocea) and carp(Cyprinus carpio) were determined, results showed that the major fish allergens were35~42kD、48~51kD、28~30kD and~17kDproteins to Qingdao residents and the major allergen was a41kD protein, which wasdistinct from the common consensus that parvalbumin was the major allergen in fish.Meanwhile, the study of the cross-reactivity of the7fish also indicated there was aquite strong cross-reactivity between different species of fish proteins. Therefore, theallergen detecting results of one fish could also represent for the other kinds.2. After thermal processing, the turbot fish protein fractions of the larger molecularweight were degradated in high degree, the immunocompetence was almost loss.Thermal processing could reduce the immunocompetence of fish allergen protein.Steamed for5min and30min, the immunocompetence was reduced by67.9%and89.2%respectively. However parvalbumin and its immunocompetence was notinfluenced, showed a good heat resisitance property. Meanwhile, there was a newallergenic protein of~18kD molecular weight generated after steaming. The resultsshowed that thermal processing brought large changes in fish allergenimmunocompetence. That means the allergic reactions caused by fresh fish may notnecessarily happened when it is processed, which supply some guiding significance inallergen detection, that is to say, it is necessary to distinguish more meticulous inallergen detection such as the fresh state and the processed state of the same food toget more accurate results.3. Nitrocellulose membrane was used as the carrier of food allergen, the visiblefood allergen detecting method is established, the conditions are: the allergen coatedconcentration is1mg/mL, human surem is diluted10folds, incubated at37℃for1h,the secondary antibody HRP-goat anti human IgE is diluted1000folds, incubated at37℃for45min, the chromogenic reagent TMB is covered for15min in darkness. Performance test results of visible food allergen detecting method show that there is agood stability with the intra-assay coefficient of variation was1.9%~6.7%，andinter-assay CV was3.3%~8.9%.4. The visible food allergen detecting method was compared with the skin prick test and western-blot, the results showed that there was no significant difference amongthe three methods. The consistency of visible food allergen detecting method betweenwestern-blot and SPT was82%and62%respectively. Besides, when compared withanother commercial food allergen detecting kit, the test results of visible food allergendetecting method was more closer to SPT results owe to the occurrence of falsenegative reactions was reduced.In sum, this study established a visible food allergen detecting method, which isconvenient in operation with an entire process of only2.5h; low-cost, specialequipment was not required for results interpretation; is able to be used as a highthroughput detection chip, can provide accurate test result, has good value of practicalapplication. In addition, the conception of ‘allergen meticulous screen’ was proposedfrom the perspective of processed foods for the first time, which put forward a newidea for the development of food allergen detection.