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| Purification of Pen a 1 Using Immunoaffinity Column of Polyepitope Antibody Against Pen a 1 |
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| DOI：10.3969/j.issn.1004-4957.年份.月份 |
| KeyWord:Pen a 1 polyepitope antibody immunoaffinity column ELISA |
| Author | Institution |
| 赵杰,高美须,潘家荣,兰丽平,睢珂,牟慧,许舒婷 |
1.中国农业科学院农产品加工研究所;2.中国计量学院生命科学院 |
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| Abstract: |
| A new method for the purification of shrimp allergenic protein Pen a 1 was developed.The epitope peptide（aa 247 to 284） of the Pen a 1 was synthesized by standard Fmoc and conjugated to KLH and BSA to get the artificial immune antigen（peptide-KLH） and the coating antigen（peptide-BSA），respectively.New Zealand white rabbits were immunized with immunogen peptide-KLH to produce antisera and the antisera were purified by bitterness-ammonium sulfat and HiTrap rProtein A FF.The pure antiserum was coupled to CNBr-Activated Sepharose 4B.The titer of purified antibody was 2.05×106 by indirect ELISA and the IC50 was 0.21 mg/L by indirect competitive ELISA method.There was no cross-reactivity between the antibody and the proteins from shrimp except for Pen a 1 by indirect competitive ELISA method.The coupling efficiency of the antibody with CNBr-Activated Sepharose 4B was 90.76% and the column capacity for 1 mL immunosorbents was 2.84 mg Pen a 1.The recoveries of the immunoaffinity column were in the range of 89.6%-93.6%.The service life of the immunoaffinity column was 4 cycles.The SDS-PAGE and Western Blot results displayed that the purified Pen a 1 had high immunogenicity. |
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