Purification Of Recombinant Treponema Pallidum Antigen TpN17 And Immunoassay For Treponema Pallidum Antibodies Using GoldMag Particles

Treponema pallidum (Tp), the pathogen of syphilis, is one of the most serious contagions that threatens human health. The predominant diagnoses Tp infection is to test the Tp antibodies in the serum samples using traditional methods such as venereal disease research laboratory (VDRL) and rapid plasma regain (RPR), fluorescent treponemal antibody-absorption test (FTA-ABS) and Treponema pallidum haemagglutination test (TPHA). Recently, enzyme-linked immunosorbent assay (ELISA) with microplates as carrier has been adopted to test IgM or IgG and widely used in syphilis screening and diagnosis. The content of this thesis includes two parts: 1. Expression and purification of Treponema pallidum antigen TpN17 in Pichia pastoris; 2. Immunoassay for Treponema pallidum (Tp) antibodies using GoldMag particles as a carrier.Recombinant Treponema pallidum antigen TpN17 was expressed in pichia pastoris by methanol induction, then the samples was precipitated with saturated ammonium sulfate and further purified using a cation exchange column. The effect of pH of loading buffer and the ion strength gradient of the elution buffer were investigated. The results indicated that the antigen TpN17 with a purity over 90% could be achieved using 20 mmol/L PB buffer (pH 6.9) as loading buffer and NaCl as elution buffer, the concentration of antigen was 0.66 mg/mL determined by Bradford method. The antigen was labeled by horseradish peroxidase (HRP) and the the titer of HRP-labled antigen was 1:3,200. The purified antigen and HRP-labled antigen could be used in immunoassay.GoldMag (Fe₃O₄/Au) particles are particle complexes composed of magnetic particles as the core component with gold nanoparticles chemically assembled on the magnetic core surface. They have advantages in both immobilizing protein through their gold nanoparticles and magnetic separation using the property of their magnetic core.An immunoassay for Treponema pallidum (Tp) antibodies using GoldMag particles as carrier was set up. The optimal amount of antigen coupled on GoldMag surface was 480 ng/tube, and 1:1,600 was the optimal dilution of HRP-labeled antigen. 194 serum samples from blood donors and 30 positive samples, which included the sera of patients with primary, secondary and terminal syphilis were determined in this method. 3 sera showed different results with commercial ELISA kits and this result showed that the sensitivety of the method using TpN17 as coated and labeled antigen needed improving.In order to establish an immunoassay for Treponema pallidum (Tp) antibodies with high sensitivity and precision, the commercial mixed recombinant Tp antigens (TpN47, TpN17 and TpN15) were used as coated and labeled antigens. The GoldMag particles coupled with these antigens, serum (or plasma) and HRP-labeled antigens were incubated simultaneously at 37℃for 30 min and the sandwich complex was formed on the surface of GoldMag particles. The 3, 3', 5, 5'-tetramethylbenzidene peroxidase substrate was introduced and the absorbance at 450 nm of solution was measured. The amount of antigens coupled on GoldMag particles and the dilution of HRP-labeled Tp antigens were optimized to be 420 ng/tube and 1:4,000 respectively. The Cutoff Value of this method was 0.105. The coefficient of variation (CV) of within- and between-batch was below 5% and 6.82% (n=5) respectively. The sensitivity of this testing system was at least ten times than the method using microplate as a carrier and the consistency between this new method and the conventional anti-Tp ELISA kits was 100% when testing 30 positive sera. The method enhanced the sensitivity compared to conventional immunoassay and it could be considered as an alternative to the conventional carrier.