Magnetic Enzyme Chemiluminescence Immunoassay For Detection Of Treponema Pallidum Antibodies

Syphilis is a kind of sexually disease caused by Treponema pallidum. It has a chronic bacterial infection and remains a worldwide public health concern. The overall rate of incidence of the syphilis increase gradually. The main laboratory diagnostic tool of syphilis is to detect specific antibody responsed to Treponema pallidum, including Nontreponemal Test and Treponemal Test. With the development of recombinant specific Treponema pallidum antigen, a sandwiches ELISA method has already established to detect IgM or IgG in serum or plasma using a microplate as a carrier. However, the sensitivity of these methods is very limited. The chemiluminescence immunoassays using magnetic particles as a carrier shows several advantages such as high sensitivity and specificity. Therefore, Magnetic Enzyme Chemiluminescence Immunoassay (MECLIA) has drawn scientists's attention in the detection of infectious diseases.GoldMag(?)composite particles was used as a carrier to set up a MECLIA method in the detection of TP antibodies. The GoldMag(?) particles were coated with mixed Treponema pallidum antigens (TP15,TP17,TP44.5 TP47), then the serum (or standard) sample and HRP-labeled Treponema pallidum antigens were added (HRP-TP15,HRP-TP17,HRP-TP44.5,HRP-TP47) to the system, After incubation, a sandwich complex formed on GoldMag(?) particles surface. Finally, the substrate of chemiluminescence system was introduced and the result was read by a chemiluminescence instrument. The conditions for each step were optimized and the final protocol was evaluated, the sensitivity was also compared with commercialized kit using microplate as a carrier.The optimal coupling amount was 7μg on 1 mg GoldMag(?), the optimal dilution solution of HRP-labeled antigen was 1%BSA in PBST and optimal dilution solution of serum (or standard) sample was 0.1%BSA in PBS. The rate of HRP-labeled antigen was 1:3000. The results showed that the system were stable at 37℃for 6 days. The CV was between 2.86- 5% in intra-assay and 5.1-9.7% in inter-assay. The result of 10 times measurement of standard positive serum showed the relative standard deviation was 5.42%. A calibration curve was determined and the linear range of TP antibody was 0.25-4 NCU/mL, with the correlation coefficient 0.9980. The method was used to detect 120 TP negative sera (including 80 TP negative sera,20 HBV positive sera and 20 HCV positive sera) and the results showed that the specificity was 100%. Compared with commercial CLIA kit using microplate as a carrier, the sensitivity had a 4 times higher.The results show that the method of detection of TP antibody using GoldMag(?) particle combined chemiluminescence detection system is a more reliable method. It has a high sensitivity and specificity. Besides, it has a good precision and stability. Therefore, it will be a promising method for the detection of TP antibody in clinical diagnosis and for the screening of blood donors in the near future.