| Treponema pallidum (TP) is the pathogen of syphilis which is a kind of sexually transmitted diseases. The incidence of syphilis ascends gradually in China last decade and it is necessary to establish a rapid, sensitive method to detect specific antibody/antigen or nucleic acid for syphilis in molecular level. The predominant diagnostic method in TP infection is to test the TP antibodies in serum including venereal disease research laboratory (VDRL), rapid plasma regain (RPR), fluorescent treponemal antibody-absorption test (FTA-ABS). Treponema pallidum haemagglutination test (TPHA) and enzyme-linked immunosorbent assay (ELISA).With the developing of TP recombinant antigen expression technology, microplate-based sandwiches ELISA has been widely used in IgM or IgG detection for syphilis screening and clinical diagnosis. However, the sensitivity is limited. Nowadays, the sensitive method based on nucleic acid has not yet been widely used in TP detection. Therefore, it is of importance to develop a new sensitive and simple method for detection of TP antibody.We have synthesized the Fe3O4/Au composite particles which have advantages both magnetic particles and colloid gold. These composite particles were named as GoldMag? and commercialized. A sensitive method using GoldMag? particles for detection of TP antibodies was established, including:1. Preparation of TP antigens immunomagnetic particles;2.Optimization of the immunoassay system; 3.Optimization of the chemiluminescence immunoassay system;4. Evaluation of chemiluminescence enzyme immunoassay system.The principle of detection method is as following: the GoldMag?particles coated with mixed TP antigens (TP47, TP44.5, TP17 and TP15) was reacted with serum sample and then the HRP-labeled TP antigens was added to formed a sandwich complex on magnetic particle surface. Then, the chemiluminescence system (Luminol-H2O2-PIP) catalyzed by HRP was introduced and the intensity of chemiluminescence were read to determine the TP antibody in serum. The optimal coupling amount was 5μg antigens on 1 mg particles and 30μg particles were used as carrier for one test. The optimal dilution of HRP-labeled antigens was 1:3000 and 30 min was the optimal time for the immunoreaction to form the sandwich complexes. For chemiluminescence detection system, the concentration of luminol, H2O2 and PIP were selected as 1×10-3 mol/L,1×10-3 mL/L and 3×10-3 mol/L in 1×PBS buffer (pH 8.0), respectively. A calibration curve was plotted by relative chemiluminescence intensity vs the dilution of a TP-positive serum (R2=0.9988). The detection limitation for TP was 16 times lower than that of the commercial EL1SA kit based on microplates. The relative standard deviation (RSD) was 5.5 % in 11 times measurement of standard positive serum.The result indicates that the GoldMag? particles based chemiluminescence immunoassay for TP antibody is more sensitive, reliable, and easier method without radioactive pollution. The detection system provides a premising method in syphilis screening and clinical diagnosis.
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