Application Of Monoclonal Antibody Against Recombinant Human 14-3-3 Zeta Isoform And Study On The Aberrant Methylation Of 14-3-3 Sigma Gene In Human Breast Cancers

Objective To prepare, purify, and identify the monoclonal antibody ( McAb ) against recombinant human 14-3-3 zeta isoform (rhl4-3-3zeta)secreted by hybridoma, to observe it's endogenous distribution in human tissues and to detect the 14-3-3 protein incerebrospinal fluid(CSF) of patients with central nervous system disorders. Methods. Hybridoma cell-suspension was injected into the peritoneal cavity of BALB/c mice and ascitic fluid was harvested. McAb was purified by salt precipitation and concentrated by Bradford method. The purified rhl4-3-3 zeta protein through electroelution was used to evaluate the titer and specificity of McAb. And the McAb subclass was tested. After examination of tissues distribution of human 14-3-3 with immunohistochemistry Western-blotting was performed to confirm the presence of 14-3-3 protein in CSF. Results The titer of purified McAb in ascitic fluid of mice was up to 1:10~6, with more than 96% of purity. Meanwhile, this McAb could recognize the specific endogenous 14-3-3 antigen. Western-blotting showed that 14-3-3 protein was consistently expressed in brain, kidney, lung, liver, stomach, ovary, breast, colon, and pancreas in human. No 14-3-3 zeta, however, was detactable in 280 CSF samples. Conclusion Monoclonal antibody to rh14-3-3 zeta was successfully generated and subjected to approach for the endogenous distribution of 14-3-3 protein in vivo. The present work may lay a foundation for clarification of the function and...