Preparation And Application Of Epitopes’ Polyclonal Antibodies Aganist Pen A1, The Main Allergic Protein In Shrimp

Shrimp and other seafood are popular because they are delicious and rich nutrition, but shrimp isone kind of main allergens in food matrix, which will not only raise the health of consumers and thesafety of life, but also make negative effects on the quantity of our country’s marine products. For thisreason, more and more researchers pay close attention to food allergies. With the studies promoted stepby step, people have a good acknowledge on the identification of allergens epitopes, which are basiccondition for anaphylactic reactions. So there is necessary to make more meaningful researches ondetection and application using allergens epitopes. In my study, all the works are focus on the epitopesof Pen a1, which is one of the main allergic proteins in shrimp.The main contents and results are as follows:1. The five epitope peptides of Pen a1were chosen and synthesized using Fmoc Method, and thenconjugated to keyhole limpet （KLH） and bovine serum （BSA） by glutaric dialdehyde method to getartificial immunes and coating antigens, respectively. RE-HPLC was used to detect the purity of thepeptides:85.97%,88.85%,92.76%,95.44%and88.00%, respectively. Dot-immunobinding assaydetected the good bioactivity of the peptides. The binding ratios of artificial antigens were well, detectedby Ellman reagent.2. The New Zealand white rabbits were immunized with the five peptide-KLHs to get the specificantisera, and the titers of the five kinds of antisera all reached1.024×106. Severally taking the five kindsof peptides as competitive objects to conduct ic-ELISAs, the IC50of the five peptides to their antibodieswere0.1574μg mL^-1,0.4324μg mL^-1,0.2291μg mL^-1,0.1145μg mL^-1and0.3605μg mL^-1according tothe linear regression equations.3. One antibody was chosed to develop immunoaffinity column due to its specificity and acquiredquantity. Firstly, the selected antiserum was purified by Bitterness-Ammonium Sulfat and HiTraprProtein A FF, and coupled to CNBr-Activated Sepharose4B. The coupling rate was90.76%. Thecolumn capacity for1mL immunosorbents was2.84mg Pen a1. The recovery of the immunoaffinitycolumn was89.6%-93.6%. The service life of the immunoaffinity column was4cycles. The SDS-PAGEand Western Blot results displayed that the purified Pen a1had high immunogenicity.4. One of the obtained antibodies were used to detect the foods whether contained the allergicprotein Pen a1. The results showed that it was a quick method to estimate the Pen a1.5. The obtained antibodies were used to detect the immunogenicity of Pen a1, which wasirradiated at the dose of0and6.7kGy. The results of SDS-PAGE and MALDI-TOF MS showed thatnew protein bonds of20kD,40kD,60kD were detected. Western Bolt showed that the60kD proteinwas identified by all five kinds of epitopes antibodies.This study had a new try from epitopes of allergic protein to prepare corresponding antibodies,developed immunoaffinity column and detected the immunogenicity of allergic protein. The newreasearch method and results will be beneficial to the related researches about allergic proteins.