Identification And Extraction Of The Effective Components From Shrimp And Its Application In Detection For SIgE

ObjectiveThe coated antigen, as the key factor, would affect the accuracy and sensitivity of the test result directly. Feneropenaeus Chinensis has been known as an important source of food allergy in Tianjin China. In this experiment, we investigated Fenneorpenaeus Chinensis to analyze major allergenic components in protein extracts of shrimp which had reacted with the serum IgE of shrimp allergic patients. In this research, the major allergen were separated and evaluated the detection sensitivity as the coating antigen by Dot-bot immunoassay and indirect ELIAS test. In a word, the purpose of this study was to increase the sensitivity of detection for serum specific IgE(sIgE) by improving the quality of coated antigen from shrimp.Method1. To identification the protein components and the major allergens of shrimpTo analyse the protein components, the muscle proteins from fresh Feneropenaeus Chinensis were separeated by SDS-PAGE. The major allergic proteins were identficaed by immunoblot analysis by serum allergen-sepcific immunoglobulin (Ig) E to Feneropenaeus Chinensis.2. To separate and identificate the major allergic proteins of shrimpThe purpose proteins were separated by two methods. We used both SDS-PAGE and immunoblot inhibition to analyse the compennts of separation proteins, and comparated the effects of separation of two kinds of methods. And then, we separated a large amounts of purpose proteins for next experiment by the optimal method.3. To check and compare the sensitivity of the proteins as the coated allergen mentioned aboveThe sensitivity of detection of the separated components were assessed by indirect ELIAS. Result1. Identification the protein components and the major allergens of shrimpThe crude extract of shrimp proteins was separated by SDS-PAGE as shown in the result. At least15protein bands were clearly detected with molecular masses140.130.120.110.98.90.85.65.53.37.35.33.29.19and11kDa, respectively. There were mang bands above55kD with at low concentration. While, there were a few bands below55kD with at high concentration. The greatest intensity corresponded to molecular mass of50kDa.39kDa and19kDa, knowed as high concentration. Among20sera from patients with shrimp allergy patients and1serum from healthy volunteer were tested. Results are showed that the high frequency resonse binds’ moelcular weight were34.55.72.100.130.140.170and above170kDa. To statistical analyze of20patients’ serum samples exhibited IgE binding to proteins of shrimp showed that above55kDa components were major allergens.2. To separate and identificate the major allergic proteins of shrimpPurified shrimp was extract by Gel Filtration Chromatography and Ultracentrifugation. The result of the purified shrimp proteins were separeted by SDS-PAGE. Protein binds of peakl-6B sample which separated by Gel Filtration Chromatography were mainly above55kDa, reducing the proteins of50.39and19kDa. The protein of19kDa was separated by Ultracentrifugation, but the proteins of39and50kDa were residued more than peakl-6B. A large amounts of major allergic proteins were separated by Gel Filtration Chromatography, and the Immunoblot inhibition experiments showed that the inhibitor of major allergic proteins could effectively inhibit response of the shrimp proteins and IgE antibodies of20sera from patients with shrimp allergy.3. The major allergic proteins of shrimp applicated in detection for sIgE as coating antigens Dot-blot experiment was performed to compare reactivity with sIgE between two kinds of coating antigens. All grey level of each dot was analysis by Image J software, the result was showed the samples with lower sIgE titer have more significant increased grey level. The result of indirect ELISA showed that in14cases of shrimp allergy patients, except2patients,12patients’ peakl-6B OD values of purified protein group was significantly higher than shrimp raw extracts group.Conclusions1. The crude extract of shrimp proteins was separeted by SDS-PAGE, and at least15protein bands were clearly detected.2. The studies had showed that IgE antibody of20srea bound to the above55kDa major allergen. The component peoteins of peakl-6B were collected by Gel Filtration Chromatography and peakl-6B was immuno-competent.3. Purification of peakl-6B as the coating antigen could effectively improved sensitivity of detection at situation of low sIgE level.