Establishment And Clinical Application For The Typing Detection Of Syphilis Specific IgG?IgM Antibody

Syphilis is a kind of common transmission disease,which is caused by Treponema pallidum infection.It can involve various organs and clinical manifestations,which has as a strong invasive ability,serious harm to human health.Syphilis is the most serious sexually transmitted diseases after AIDS^[1].Syphilis infection pathogens of Treponema pallidum?TP?is difficult to be cultured in vitro.So it is hard to diagnose by pathogenic microorganism culture.Syphilis diagnosis is based on clinical manifestations and laboratory findings,Includingnonspecificlipidantigen?cardiolipin?detection anticardiolipin antibodies?reagin?specificity detection method and Treponema pallidum antibody as detection object^[2-4].The results of laboratory tests can only detect syphilis specific antigen,the results can not distinguish the types of antibodies detected.We can not only determine that the type of the antibody is IgG or IgM,but also cannot judge with previous infection or current infection.Direct detection syphilis specific antibody IgM methods in the international all need special commercial kit,operation steps,long detection time,batch detection method,so we can not ues in large-scale clinical application.The first part of this study:constructe recombinant plasmid of Treponema pallidum antigen TP47 gene,induce the expression of TP47protein and purify it.The purified TP47 protein is tested by WB to confirm recombinant protein.Result:The recombinant protein TP47 was able to react with the standard positive serum of syphilis,and did not react with normal human syphilis negative serum and Mixed sera from autoimmune diseases patients.The second part of this study:Design and manufacture a device which can realize semi-automated affinity chromatography for separating syphilis antibody IgG and IgM,and verify the stability of the device.Prepare the affinity column and the desalting column,and respectively arranged them into the affinity device according to the experimental order,and optimize affinity and desalting conditions.Result:affinity chromatography device has good stability,no leakage,no pollution,no specific adsorption and so on;after optimized the experimental conditions of the affinity column:?1?the sample of affinity column was determined to be 500ul;?2?The equilibrium solution of affinity column was 20mM containing 5g/l NaCL Tris buffer PH=7.5?4ml?;?3?The affinity column was eluted with KSCN of 3M 2ml.At the same time,the affinity filler can be fully combined with the specific syphilis antibody,and the balance liquid can wash away the residue of non-specific protein after 4 ml;after optimized the experimental conditions of the desalting column:?1?10.the sample of the column was 200ul affinity column eluent;?2?The equilibrium solution of the desalting column is 20mM containing 5g/l NaCL Tris buffer PH=7.5?1ml?;?3?The elution of the desalting column was 0.2M NaOH,2ml.At this time,the collected eluent had no influence on the subsequent detection of SCN^-ions,which was convenient for subsequent sample detection;the samples collected from the desalination column can be detected by IgG and IgM gold labled strip.The third part of this study:230 cases of syphilis antibody positive samples were detected by affinity chromatography,and 40 cases of them was detected by WB.The results were statistically analyzed.Result:40 cases detected by affinity device compared with the results by the WB method,the statistical results showed that two kinds of detection method results have no statistical difference?SPSS11.0 software,paired X2 test,p>0.05?.This paper through affinity chromatography device independently developed,wrap the existing Treponema pallidum specific antigen T1and TP58,TP56 in affinity chromatography,specifically separate IgG and IgM antibody sample in the sample by IgG?IgM gold labled strip for syphilis antibody detection,which provides basis for clinical diagnosis of syphilis infection in patients.At the same time,the recombinant expression protein of TP47 was constructed by genetic engineering technology and validated by clinic,which was used for reducing the cost of manufacture and laying the foundation for a one-time use.