Preparation And Identification Of C-myc Monoclonal Antibody

Oncogene c-myc, located on chromosome 8q24.12-8q24.13, is an important member of the myc family, as a similar objects of the myeloid leukemia virous oncogene v-myc, encoding a 62kD protein molecular.The function of gene c-myc are various, not only as a translocation gene, but also as a regulated gene acting on many Human tumors via promoting cell proliferation and division to immortalization, under regulation of many subjects.In normal cells, proto-oncogene c-myc once was activated, converting to cancer gene, activating transcription and translation, the c-myc mRNA and c-myc protein expression would be abnormally high, making the cells far from the regulation constraints of normal growth to highly proliferative potential, turning into the transformation of phenotype and the inhibition of cell differentiation, and promoting cell malignant transformation to cancer.In view of the overexpression of c-myc was positively correlated tumor, the anti-serum of c-myc can be used for diagnosis. To explore the methods of producing monoclonal antibody with using prokaryotic expressed c-myc protein, we designed a method of immunization. The c-myc protein expressed in E.coli strain BL21 was identified by Western blot. Three BALB/c mice were immunized with purified c-myc protein for 3 times. Then the serum was picked up, and the titer of antibody was measured by ELISA (Enzyme-linked immunosorbnent assay). In the present study, we obtained two monoclones followed by clone selection, referred to SHH-1H11 and SHH-2A9. The titers of antisera, 1H11 and 2A9 were 1:10240 and 1:5120 in cell media, and 1:64000 and 1:32000 in the ascites, respectively. The subtypes of antibodies, 1H11 and 2A9 were IgG1 and IgM, respectively. The BCA Protein Assay Reagent Kit tests showed that the antibody concentrations of hybridoma antisera and ascites, SHH-1H11 were 0.47mg/ml and 2.51mg/ml, SHH-2A9 were 0.49mg/ml and 2.45mg/ml. No cross-reaction of the monocolonal antibodies was found between c-myc and the other proteins. The monoclonal antibody specific for c-myc prepared showed high-titer and high-affinity, laying a foundation for producing a diagnostic method for cancer using the monoclonal antibody prepared.