Investigating monoclonal antibody against cytokeratin 19 tumor marker

Cytokeratin 19 (CK 19) is a member of the intermediate filaments that are widely expressed in thyroid, breast, colon, small and non-small cell lung, and prostate cancer cells. This paper aims to produce the monoclonal antibody against CK 19. Recombinant CK 19 was used to immunize of two 6–8 weeks old female Balb/c mice. For the first injection, recombinant antigen was mixed with the complete Freund′s Adjuvant, and for the second and third injections, mixed with the Incomplete Freund′s Adjuvant. Injections were performed at intervals of 15 days. Four days after the third injection, titration of mice serum was determined using indirect ELISA and better-immunized mice selected for fusion. Intravenous (IV) injection was performed 4 days before the fusion. The spleen cells of the mice were fused with sp2/0 cells using 50% v/v polyethylene glycol (PEG) and cells were suspended in HAT (Hypoxanthine-Aminopterin-Thymidine) medium. Supernatants of hybridoma cells were screened for antibody secretion by indirect ELISA. Two stable hybridomas were obtained among 78 hybridoma clones that reacted with the recombinant CK 19 by indirect ELISA. These hybridoma cells were monoclonal twice by limiting dilution. Besides, immunization by recombinant CK 19 led to the obtaining of two stable monoclonal antibodies against this antigen. Western blotting with HT-29 cell line demonstrated a band of upper than 43 kDa. Result of HepG2 indicated a band of lower than 45 kDa while MCF7 bands of 45 kDa and higher were observed for MCF7. A431 cell line was epidermoid carcinoma, as CK 19 did not exist in the epidermis, therefore no band was observed in A431 cell line.