Generation And Characterization Of Monoclonal Antibody Against Nicotinamide N-methyltransferase

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines and other structural analogs. It is involved in the biotransformation of many drugs and xenobiotic compounds. NNMT is an S-adenosyl-L-methionine (Ado-Met)-dependent cytosolic enzyme.Recently, NNMT was suggested to be involved in the pathogenesis of Parkinson's disease. By examined the gene expression profiles of renal cell carcinomas (RCC) using a high-density oligonucleotide microarray, Yao M et al. found that NNMT gene was more than three times higher in clear-cell RCC than in chromphobe RCC and normal kidney tissue. Xu J et al. used DNA microarray to study the expression profiles of different human thyroid carcinoma cell lines, they found that a gene coding for NNMT was identified for being highly expressed in the papillary cell lines. By studying the expression of stress-related and DNA repair genes in bladder carcinoma cell lines, Kassem et al. found that NNMT might be linked to the response to ionizing radiation. The NNMT mRNA level in the human bladder carcinoma cell line that is resistant to radiation was higher than that in the sensitive subclone. The results indicate that NNMT may become a potential biomarker for clear-cell RCC, papillary thyroid carcinoma, and may has a potential role for predicting response to radiation in bladder cancer.The monoclonal antibodies against NNMT should prove to be beneficial to diagnosis and prognosis of clear cell RCC and papillary thyroid cancer.Objective: To establish the monoclonal antibody (McAb) against NNMT and study its characterization. Methods: NNMT-Glutathione S-transferase (GST) fusion protein and NNMT protein were prepared as antigen to immunize BALB/C mice. Using hybridoma technique, the hybridoma cells were obtained by fusing spleen cells of BALB/C mice to myeloma cells SP2/O and cultured in the HAT selective medium. The positive clones were screened by indirect ELISA and positive secretors were selected to reclone by the method of limited dilution. The McAb in peritonealfluid was purified by caprylic acid and saturated ammonium sulfate. McAb class and subclass were identified by HyCult biotechnology kit. Western-blot was done to verify specificity of the monoclonal antibodies. The relative affinities of antibodies were demonstrated by indirect ELISA. NNMT expression in normal human liver tissue, clear cell renal carcinoma tissue and papillary thyroid cancer tissue were characterized by immunohistochemistry. Results: Two stains of hybridoma cells (2F8, 1E7) secreting anti-human NNMT McAbs were obtained. McAbs secreted by both of strains have high specificity. The immunoglobulin subclasses of McAbs secreted from 2F8 and 1E7 hybridoma cell lines were IgG2bic and IgG2aK respectively. The relative affinity of 2F8 was greater than that of 1E7. Immunohistochemistry for NNMT showed strong tumour cell staining in clear cell RCC and papillary thyroid cancer. Conclusion: The McAbs with high specificity against human NNMT have been successfully prepared. The McAbs obtained by this strategy should prove to be beneficial to diagnosis and prognosis of clear cell RCC and papillary thyroid cancer.