Evaluation Of Recombinant 29 000 Extra Membranous Protein For The Immunodiagnosis Of Schistosomiasis Japonica And Preparation Of Monoclonal Antibody Against Metabolic Antigen Of Schistosoma Japonicum

Schistosomiasis remains one of the serious public health problems in China with a wide distribution. Immunodiagnosis plays an important role on prevention and control of Schistosomiasis, of which there are no effective methods for early diagnosis. The stool exam is the primary method to confirm the patients, but is time-consuming, laborious and apt to miss diagnosis early. So looking for more sensitive, more specific, more economic and more reliable diagnostic methods has become an important part of our basic research in schistosomiasis. Initially our laboratory had cloned, expressed and purified membrane protein Tetraspanin 2-A (rSjTsp2) and recombinant extramembranous protein 29KD (rSj29), demonstrating that rSj29 had good value of diagnosis to detect sera of acute schistosomiasis. Then we further expressed and purified proteins rSj29 and rSjTsp2. The preliminary experiment with 30 mixed positive sera and 30 negative sera suggested the best concentration of antigen and antibodies .The 394 sera and stool samples were collected from 5-80 years old inhabitants, from lower endemic areas (Wuhu city in Anhui Province). The stool samples were tested by nylon bag sedimentation/hatching method or modified Kato-Katz technique. The sera samples were tested by indirect hemagglutination test (IHA). The stool exam and IHA were tested by Anhui Provincial Institute of Schistosomiasis Control. Then the sera samples were tested by rSj29-ELISA and adult worm antigen of Schistosoma japonicum (AWA) ELISA. We use single blind method to test each sample. It was found that the positive detection rates of IHA, rsj29-ELISA, AWA-ELISA were 62.18%, 68.27%, 89.85% respectively, higher than the stool detection 4.82(x2=1259.7, P<0.05). The positive pieces both of IHA and AWA-ELISA were 227, the negative 22. Coincidence rate reached 63.20%. The positive pieces both of IHA and rSj29-ELISA were 219, the negative 99. Coincidence rate reached 80.71%, higher than that of IHA and AWA-ELISA. To compare with stool examination,the sensitivity of IHA, rSj29-ELISA and AWA-ELISA was 100%, 94.74%, 100%, the specificity was 39.73%, 33.07%, 10.67%. Coincidences with stool test were 42.64%, 36.04%, 14.97% respectively. There were also no statistically significant differences about the sensitivity and specificity with IHA or with rSj29-ELISA. The present study showed that the indirect ELISA with rSj29 antigen, easier to standardize yet with a simple preparation method had the same high level of sensitivity and specificity as IHA did for diagnosis of schistosomiasis.However, the specificity of ELISA and IHA method is low which makes it difficult to distinguish between antibodies past infections (including cured by chemotherapy) and the present infection, so the positive rate can not truly reflect the current patients. The detection of circulating antigen could reflect more accurately the efficacy of the host body, more reliably determine whether there are live worms to provide an accurate basis for treatment and epidemiological data. With the emergence and the improvement of monoclonal antibody technology, existing multi-use monoclonal antibody technology for detection of circulating antigen has been often used.For this we have prepared against Schistosoma japonicum metabolic antigen (Sj-MAg) monoclonal. A rabbit were infected with cercariaeum and killed after 42 days. Schistosoma japonicum was obtained by the portal vein perfusion. Then the metabolic antigen of Schistosoma japonicum (Sj-MAg) was prepared. Immune reaction, to identify schistosomiasis in human serum of Sj-MAg was confirmed. Determination titer of the female BALB/c mice immunized three times with rSjMAg is 1:32 000 or more after 6 weeks. Hybrid fusion cells with spleens of immunizes mice and SP2 / 0 myeloma cells filter by HAT selective medium, then subclone and expand culture. The supernatants secreted by the fusing cells were detected by ELISA . We establish stabile secretion of anti-Sj-MAg monoclonal antibody hybridoma cell lines. The monoclonal antibody immunoglobulin subclasses of two monoclonal antibody hybridoma cell lines both are IgG1 by Sigma antibody detection kit. Western-Blotting experiments identified two hybridoma cell lines. .