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Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage. Siti Salwa Hasmoni, Khatijah Yusoff, and Wen Siang Tan* J. Gen. Appl. Microbiol., 51, 125-131(2005) (Received June 23, 2004; Accepted January 12, 2005). 91394025 甘博介. Introduction-Phage ELISA.

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Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

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  1. Detection and precipitation of hepatitis B core antigenusing a fusion bacteriophage Siti Salwa Hasmoni, Khatijah Yusoff, and Wen Siang Tan* J. Gen. Appl. Microbiol., 51, 125-131(2005) (Received June 23, 2004; Accepted January 12, 2005) 91394025 甘博介

  2. Introduction-Phage ELISA • The phage enzyme-linked immunosorbent assay (phage-ELISA) is a modified ELISA-based technique with the primary antibody replaced with the fusion phage. • M13 phage has been chosen in this experiment, use E. coli ER2738 to prepare

  3. The Life Cycle of M13 F-pilus

  4. Phage Display Technology • 利用轉殖將一段短片段的peptide gene附於噬菌體中，進而在不影響其感染力的情況下產生所需要的peptide於phage表面蛋白上以供利用 • Use anti-HBcAg monoclonal antibody to select which phage has the highest interaction

  5. 將目標peptide表現於phage的表面蛋白上

  6. Peptide-WSFFSNI • A short peptide which is selected from phage peptide library • Has high affinity interaction with HBcAg(Hepatitis B core antigen) • Phage bearing the sequence WSFFSNI has the potential to be further developed as an immunodiagnostic marker

  7. Results-Detection of HBcAg with fusion phage via phage/mAb ELISA. HBcAg + Fusion Phage HBcAg + mAb 1:5000=1.6mg/ml

  8. Results-Detection of HBcAg with dot blot assay. Fusion phage Monoclonal antibody

  9. Results-Detection of HBcAg in HBV positive serum samples using phage ELISA

  10. Results-Detection of HBcAg in HBV positive serum samples with fusion phage via phage-dot blot assay 1x1012pfu/ml

  11. Discussion • HBcAg has been one of the most important markers for HBV infections. • Production of monoclonal or antibodies raised in cell lines or animals are costly and time consuming, which usually takes months • the phage bearing the WSFFSNI sequence interacts tightly to HBcAg with a dissociation constant (KDrel) less than 25 nM

  12. Conclusion • Recombinant M13 phage which is cheaper in terms of production and can be rapidly propagated. • The purification of the filamentous phage involves lesser steps compared to that of monoclonal antibody.

  13. The Advantage of Phage-ELISA • This type of phage-based diagnostic reagent can save time, cost and energy required to produce monoclonal antibodies. • Higher sensibility than general amount of mAB (monoclonal antibody)

  14. THE END

  15. The type of phage display system

  16. ELISA From http://juang.bst.ntu.edu.tw/ →莊榮輝老師網頁

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