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Separation, purification, and identification of flagellin, and preparation of its antisera

Author(s): M.D. Hu1, G.S. Wang1, J. Xu1, W. Yao1, B.F. He1, Y. Yang1, M. Mao2, Q. Wang3 and J.C. Xu1

The aim of this study was to separate, purify, and identify Salmonella paratyphi A flagellin, and to prepare its antisera. Primary flagellin was isolated from S. paratyphi A using the acid lysis method. The flagellin was purified with weak anion exchange chromatography and the protein was identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and negative staining with phosphotungstic acid with scanning electron microscopy (SEM). The production of the obtained flagellin was then quantified. New Zealand white rabbits were then immunized with the isolated flagellin, the presence of serum anti-flagellin antibodies was assessed with the immunoblot test, and its potency was determined with the double immunodiffusion test. The results of SDS-PAGE showed that the molecular weight (m.w.) of the purified flagellin was 52 x 103. The immunoblot test also showed a band at 52 x 103 m.w.