| Transmissible spongiform encephalopathies (TSE) are a group of rare, 100% fatality rate and transmissible neurodegenerative diseases. One of the known symptoms of TSE is mad cow disease, which is caused by abnormal form (PrPsc) of the normal host-encoded prion protein (PrPc). Since transmission of the prion pathogen from livestock to human has been found, the disease has soon become worldwide concern and a big threat to the world livestock trade. To prevent the human being and livestock from infection of the pathogen, it is critical to build sensitive and specific detection means. Two sandwich antibody chips has been developed to detect prion protein, using bovine prion (bPrPc) scFv fuse with streptavidin binding peptide (SBP), Fc fragment from human IgG1 and MAb KG9,. First, a thioredoxin-mature bovine prion fusion protein (TrxA-bPrPc) was constructed by attaching bPrP gene to pET32a (+) vector and expressed in E.coli AD494 (DE3). Second, the scFv was selected from an antibody phage display library constructed by immuning mice with the fusion protein TrxA-bPrPc. Finally, after five rounds of panning against recombinant bovine prion protein (rb-PrPc), Positive clones, named Z163, Z186 and Z1030, were obtained and showed relatively high affinity for rb-PrPc. Four recombinant scFv fusion proteins Z186-L-SBP, Z163-L-SBP, Z163-Fc, Z1030-Fc and mAb KG9 were selected for pair in sandwich ELISA. Results showed that only Z186-L-SBP/Z163-Fc and KG9/Z163-L-SBP pairs displayed obviously positive results. The KD for Z186-L-SBP, Z163, Z163-L-SBP and Z163-FC against rb-PrPc were 3.24×10-8M, 8.82×10-8, 4.11×10-8 and 8.10×10-9M, respectively by SPR analysis. Western blot showed that Z163-L-SBP, Z186-L-SBP, Z163-Fc and Z1030-Fc can specifically recognize recombinant bPrP27-30 linear epitope. It indicated Z163-L-SBP, Z186-L-SBP, Z163-Fc and Z1030-Fc could detect PrPsc.In one sandwich antibody chip, monoclonal antibody KG9 as capture probe was immobilized covalently on the on poly-L-lysine coated slides and Z163-L-SBP fusion protein as a recognition element for rb-PrPc and natural murine prion protein on poly-L-lysine coated slides. SA-modified Quantum Dot was used as detection probe. The detection limit was 1 pg/mL. In the second sandwich antibody chip, Z186-L-SBP...
|
PDF Full Text Request