The Study On Diagnosis Of Schistosomiasis Japonica With Recombinant SjP38 Antigen

Schistosomiasis remains one of the most serious public health diseases that have significant economic and public health consequences. It has been estimated that 200 million people are infected and 120 million are symptomatic. The major damage of the disease is due mainly to granulomatous reaction evoked by soluble egg antigens. Eggs retained in the gut wall induce the pathology of chronic disease, such as fibrosis, venous obstruction, portal hypertension, splenomegaly, and finally death. So it is important to diagnose the infection of S.japonicum as early as possible for schistosomiasis control.At present, parasitological and immunological tests are two main methods to diagnose schistosomiasis. Detection of excreted ova is the traditional diagnostic method for schistosome infections, and the infecting species can be identified by egg morphology. It has been widely and increasingly recognized that parasitological diagnostic methods have high specificity but low sensitivity. In addition, many of these methods are time-consuming and require trained personnel, which are difficult for routine diagnosis. It was anticipated that the difficulties associated with parasitological diagnosis might be overcome by adoption of immunological methods that provided direct evidence that an infection was present.However, to develop a convenient and rapid immunological diagnostic method, there are still many problems unresolved. First of all, stable and enough naturalantigen protein as diagnostic reagent is still unavailable. Secondly, for antigen detection methods, the low concentration of circulating antigens in patients' sera limits these antigens clinical application; on the other hand, to detect antibody it is hard to differentiate previous and current infections. Thirdly, to diagnose schistosomiasis at early infection stage is impossible yet.P38, a soluble antigen with molecular weight about 38 000, is one of the major proteins of S.m egg soluble antigen. Many experiments have proved that native and recombinant P38 protein can recognize circular antibody in infected sera at early stage, which indicates that this protein may be a good candidate for early diagnosis of schistosomiasis. In our previous study, the full length of S/P38 cDNA was cloned by PCR from the cercaria cDNA library of S.j. In present study, we further express this SJP38, prepared its monoclonal antibody, study its immune mechanism, and evaluate its' clinical diagnostic value. Objective:1. To identify the immune reaction of SJP38 proteins at early infection stage and assess its' capability as a diagnostic target antigen.2. To develop a colloidal gold immuno-chromatography assay (GICA) for detection of SJP38 antigen of Schistosomajaponicum.Methods:1. DNA fragment of 5/P38 was subcloned into expression vector pET-32a, and the recombinant plasmid was identified by restriction enzyme digestion and sequencing.2. The recombinant protein was induced by IPTG. The soluble rSjP38 was purified with Ni-NTA affinity column. After purification, immune activity of rSjP38 protein was identified by Western-blot and ELISA.3. S.j infected mouse model was prepared and sera at different infectionstages were collected. Mice sera were then determined by Western-blot with the recombinant protein.4. BALB/c mice were immunized with purified rSjP38 and hybridomas were generated with traditional hybridoma techniques. McAbs were screened by ELISA and limited dilution; and then purified by protein G chromatography. The subtype, specificity and affinity constant (Kaff) of McAbs was analyzed by subtype kit, Western-blot and ELISA respectively.5. All McAbs were labeled with HRP by sodium oxidation method. The McAb counterparts with high affinity and stability were found according to the OD450.6. SJP38 antigen and antibody in mice sera from pre-infection to post-infection (l-6w) were determined by time resolved fluoroimmunoassay (TRFIA). Sera of normal mice were used as parallel control.7 GICA recognizing rSjP38 antigen was developed with colloidal gold labeling and immuno-chromotagraphy techniques. Mice sera at different infection stages were tested with this GICA dipstick.Results:1 5/F38 gene was subcloned into pET-32a(+) plasmid, and its open read frame is proved to be correct by sequencing.2 The SJP38 was expressed in E.coli as a soluble fusion protein and the immunological activity of rSjP38 was confirmed by western-blot with infected rabbit sera.3 The rSjP38 antigen could react with circular antibody in mice sera from week 3 to 7 after infection, which indicated that rSjP38 might be potentialtarget protein for early diagnosis.4 Eight hybridoma cell lines secreting McAbs of anti-rSjP38 were obtained which have high affinity and reacted with native SJP38.5 Western boltting result showed that cercaria and SEA specifically reacted with the McAbs, but not with adult worm antigen.6 Five McAbs counterparts with high affinity and stability were found with sandwich ELISA.7 Two McAbs were selected according to the above counterpart experiments and the labeling and determine conditions were optimized. TRFIA was established to determine the SJP38 antigen and antibody. The best reaction model was: coating with 9G7 (3ug/ml) as capture-antibody and detecting with 1A6-EU3+(1:6O) as labeled-antibody.8 The SjP38 antigen and antibody infected mice sera at different infection time were analyzed by TRFIA. Statistical differences were found in general change trend of both SJP38 antigen and antibody between infected and normal groups(F= 6.353, P=0.016; F= 10.553, />=0.002). SJP38 antigen and antibody in infection sera could be determined from 3 to 6 weeks after infection, but not in control group. SJP38 antigen in infection group was found to increase with the infection time. However, the SjP38 antibody was found to fluctuate with the change of antigen.9. Two McAbs were selected according to the above counterpart experiments and the labeling and determine conditions were optimized. The best reaction model was: coating with 9G7 (2.5mg/ml) as capture-antibody and detecting with 1A6 (20ug 1A6 per milliliter gold colloidal) as labeled-antibody. According to above condition, colloidal gold immuno-chromatography assay (GICA) for detection of S. j wasdeveloped. 10. The GICA detection showed 40%, 50%, 60% and 80% positive for 3, 4, 5,and 6 weeks after infection. For negative controls, no positive was found. Conclusion:1. The 5/7*38 gene was subcloned and expressed as a soluble fusion protein in E. coli, and the SJP38 protein showed highly immune activity with Western-blot and ELISA. Positive reaction of rSjP38 with infected mice sera at early stage suggested that SJP38 might be a good candidate target protein for early-stage diagnosis.2. Eight McAbs of SJP38 were successfully obtained with traditional hybridoma technique. All McAbs reacted with native SJP38 with high affinity.3. SJP38 antigen and antibody in mice sera 3 weeks after infection were determined by double antibodies sandwich ELISA and indirect ELISA TRFIA. The immune reaction kinetics from pre-infection to 6 weeks post infection in mice sera showed significant difference between treatment and control, and also obvious difference following the infection stage.4. A simple and rapid colloidal gold immuno-chromatography assay (GICA) for detection of S. j was established, which could be further developed as a new diagnosis kit for S.j at early infection stage.