Immunoenzymatic Tissue Chemistic Kit For Lung Cancer

Lung cancer is one of the most common malignant tumor in the world.In the resent years lung cancer's incidence and mortality have risen for the first.For such factors as air pollution increasing,lung cancer's incidence and mortality display a clear upward trend. Because of the imperfections of lung cancer diagnosis, many lung cancer suffers in clinic had already lost the best treatment time, so the diagnosis of lung cancer is especially important.At present H.E staining could commonly be used in clinical, but it could not be used to accurately diagnose the nature of the tumor. While immunohistochemistry can not only be used to accurately diagnose the nature of the tumor, but also can predict the biological behavior of tumor prognosis. In other areas, immunohistochemistry also can provided for guide chemotherapy of some certain tumors , its application prospect in determining the tumor's organization origin is unlimited.Therefore,developing immunoenzymatic tissue chemistic kit for lung cancer,that has great value and significance in practice for lung cancer's early diagnosis, prevention and treatment.In this topic, preparing the lung cance's monoclonal antibody ascites was producded in vivo with BALB / c mice. And lung cancer's monoclonal antibody in the ascites had been purificated by octanoic acid-ammonium sulfate. Purified monoclonal antibody'concentration was 3.0426mg/ml, purity was 80%-90%,it had reached the electrophoretically pure.The specific of lung cance's monoclonal antibody had been identified by immunohistochemistry.The result displayed that lung cance's monoclonal antibody was a specific antibody for the the large cell lung cancer.The associated protein of large cell lung cancer was analysed by Western blot ,it was shown that the large cell lung cancer's monoclonal antibody to the protein was about 90 KD protein of the NCIH460 cells.New Zealand white rabbits were injected with purified lung cancer's monoclonal antibody , after injection in four times, the titer of the antibody to McAb was above 1:1024000.The rabbits'serum was purificated by octanoic acid-ammonium sulfate. The concentration of purified rabbits'IgG was 28.5468mg/ml, its purity was 80% -90% which had reached the electrophoretically pure.The purified rabbit anti-monoclonal antibody had been marked in HPR,its titer and specific had been determined by direct-ELISA. The result displayed that the titer of HRP-antimonoclonal antibody was above 1:32000,it was specific for lung cance's monoclonal antibody. HRP's concentration is 1.584 mg/ml, IgG's concentrations is 2.5056mg/ml in HRP-antimonoclonal antibody, HRP /IgG ratio is about 1.60, combined rate is 31.68%, the titer is above 1:32000. HRP-antimonoclonal antibody was as the second antibody of the kit.Lung cancer diagnostic kit had been established. 3% hydrogen peroxide/ methanol was the best concentration of the diagnostic kit, and reaction time was 10 min at room temperature,.The best's temperature of the test was 37℃in 20 min in confining liquid. The effective range of lung cancer's monoclonal antibody was 1:50-1:500, the best dilution was 1:100, the reaction temperature were 37℃in 1h, the dosage was the 100ul. The effective range of HRP-antimonoclonal antibody was 1:50-1:800, the best dilution was 1:200, the reaction temperature was at 37℃in 30min, the best dosage was 100ul. The reaction time of DAB stain was between 30-50s, and the time of Hematoxylin stain is between 15-30s, the patho-organism in the paraffin section were just covered with the strain lique .