Preparation Of Monoclonal Antibodies Against Cancer/Testis Antigens PAGE-4, LAGE-1 And NXF-2 And Their Distribution In Normal And Malignant Tissues

The malignant tumor is still the leading disease threatening the human life and health at present. As the investigation of Cancer/Testis Antigens (CTAs) is deepened constantly, the importance of CTAs has been attached to the basic research, diagnosis and therapy of tumor gradually. Unfortunately the research on CT antigens mostly remains in DNA and mRNA level at the present time, and the investigation of CTAs on protein level is barely limited to immunogenic analysis. The main reason for the obstruction is lacking specific monoclonal antibodies (mAbs) against CT antigen.To analyze the distribution of PAGE-4, LAGE-1 and NXF-2 in normal tissues and tumor and investigate the relation of their expression at mRNA and protein levels with the grade and stage of cancer, the preparation of mAbs against PAGE-4, LAGE-1 and NXF-2 with high specificity and sensitivity is a precondition.The monoclonal antibodies against PAGE-4, LAGE-1 and NXF-2 were prepared with the lymphocyte hybridoma technology, and the correspondingmAbs with high specificity and sensitivity which recognize native molecules were selected from the originally obtained mAbs. The optimal working concentrations of the mAbs LX-PAGE4. 2, LX-LAGE1. 9 and LX-NXF2 in immunohistochemistry staining is 0. 5 g/ml, 2. 5 g/ml, 5. 0 g/ml respectively. Using the obtained mAbs and the methodology of DNA and mRNA research, the expression of PAGE-4, LAGE-1 and NXF-2 at mRNA and protein levels in the normal tissue and tumor was investigated. We found that PAGE-4 was strongly expressed in spermatogonia and some of primary spermatocytes and distributed in cytoplasm and cell nucleus, while LAGE-1 was localized in the cell nucleus of spermatogonia and some primary spermatocytes and NXF-2 was also expressed in the cytoplasm and cell nucleus of spermatogonia and primary spermatocytes, but mainly distributed in the nucleus. Furthermore normal prostate, uterus and pancreas expressed PAGE-4 protein which was distributed in the cytoplasm. In the case of LAGE-1, this molecule was detected in the cytoplasm of the normal heart, kidney, pancreas, stomach, small intestine, liver and esophagus. In addition we found that normal brain tissue also expressed LAGE-1 which localized in the cell nucleus differently. NXF-2 was detected in the cytoplasm of the prostate mainly and in the nucleus of brain at high expression level. Finally, we detected the expression of these CTAs in tumor tissue and found that the expression of PAGE-4 molecule in prostate adenocarcinoma and uterus adenocarcinoma was observed and the protein localized in the cytoplasm. And LAGE-1 protein was strongly expressed in prostate adenocarcinoma, bladder cancer, liver cancer, lung cancer and breast cancer and distributed in the cytoplasm of cancer cells mainly. NXF-2 was found to be expressed in...