The Research Of Diagnostic Strip Detecting Urine Kit Of Anti CK20 Monoclonalantibody Labeled With Colloidal Gold And Its Clinical Application

Objective Its objective is to create colloidal gold diagnostic strip of antiCK20 monoclonal antibody (abbreviated as CK20 Strip).CK20 strip is to used to screen bladder cancer(BC) by detecting directly urine of patients,which can provide clinical doctors with objective diagnostic proof in order to diagnose BC earlier. Methods The research includes three parts: purification of CK20 antigen and identification,study of the monoclonal antibody(McAb) and identification ,designsing CK20 Strip and clinical test.The first part is purification of CK20 antigen and identification. Purification is the key point because it is related closely to the whole research. Isolation of CK20 antigen from human T24 cell line is hard because the amount is less in culturing cells. We took advantages of anion exchange chromatography to separate CK20 accordding to its chemical features.SDS-PAGE and Western-blot were used to identify it. The second part is the study of antiCK20 monoclonal antibody(McAb) and identification. CK20 McAb obtained by hybridoma technique. Lymphoctyes from the spleens of mouse being immuned fused myeloma cells by PEG4000. ELISA was used to select positive cells excreting McAb and cloning cells was got by limiting dilution. And we have inoculated cloning cells into abdominal cavitys of mice to let them produce ascitic fluid containing monoclonal antiCK20 antibody. Finally, affinity chromatography of SPA-Sepharose CL-4B was employed to purify monoclonal antiCK20 antibody from ascitic fluid, and its specificity, titre, isoforms were affirmed by Western-blotting,ELISA and two directions diffusion of agar.The third part is designing CK20 Strip and clinical test .We have used nitric fibrous membrane to absorb antiCK20 monoclonal antibody labeled with colloidal gold, and fixed the membrane on plastic strip as CK20 strip .We have detected 51 hematuria samples of BC and 19 urines of non-tumor's patients and 20 urines of normal humen with the CK20 strip, UBC (Urinary bladder cancer) and BTA (bladder tumor antigen) at same time.The clinical value of CK20 strip was evalued through comparing its detecting results with that of UBC and BTA.Rsults We have got about 1.44mg CK20 antigen. SDS-PAGE showed that the molecular weight was 46kD and the Western-blot showed that there was specific reaction between the antigen and antiCK20 monoclonal antibody. One hybridoma cell strain excreting antiCK20 monoclonal antibody was got, and its chromosome's modal number was 101(99-103), subset was IgG1.The titer of antibody was 106 There was specific reaction between the monoclonal antibody and T24 cell. We use CK20 strip detected urine of patients and the results were compared with that of UBC and BTA.The results show that the sensitivity, specificity, positive likelyhood ratio and negative likelyhood ratio of CK20 strip were similar with that of BTA, but better a little than that of UBC.Conclusion we believe that CK20 strip owns high clinical application value in screening BC .It is the first product in China, and cheaper than that of the abroad reagent related. The detecting method is so simple ,fast and the detecting result is so stable ,credible that can be carried out in any class Lab.