Engineering of a monoclonal antibody to a tumor associated NADH oxidase (tNOX), a putative cancer specific cell surface determinant
Abstract
Two monoclonal antibodies designated 12.1 and 12.5 were produced to tNOX (tumor-associated NADH oxidase) isolated from pooled sera of cancer patients. The properties of MAB 12.1 were as follows: (1) The inhibited the NADH oxidase of sera from cancer patients but not sera from healthy volunteers. (2) The mouse ascites slowed the growth of human cervical carcinoma (HeLa), sp-2 mouse myeloma and human mammary adenocarcinoma (BT-20) cells but did not have an effect on non cancerous MCF-10A human mammary epithelia. (3) MAB 12.1 reacted with tNOX in Western blots of pooled cancer sera. (4) In immunohistochemistry studies, ascites of MAB 12.1 stained the surface of HeLa cells. The DNA from the hybridoma cells was isolated and ScFv (single chain antibody variable fragment) were generated by recombinant DNA technology. ScFv are heterodimers consisting of variable region of antibody heavy and light chains tethered together by a polypeptide linker. The sequences of heavy and light chains of MAB 12.1 were obtained by PCR with degenerate primers. A linker sequence was incorporated into the specific primers and ScFv expression in E. coli was monitored with an anti E-tag monoclonal antibody. The properties of the anti tNOX ScFv were: (1) The ScFv blocked tNOX activity in pooled sera from cancer patients but had no effect on sera from healthy volunteers. (2) The ScFv fragments reacted with an antigenic determinant on the surface of HeLa cells. (3) The ScFv recognized bands present in sera from cancer patients associated with tNOX activity. The sera were from leukemia and lymphoma patients as well as patients with cancers of the lung, ovary, breast, prostate, endometrium, cervix and colon. The antibody provides evidence for a circulating form of the NOX protein that may distinguish sera of cancer patients from sera of healthy volunteers. Also the antisera may offer opportunities for future development of a pan cancer procedure with potential utility for the serum detection of metastatic cancer.
Degree
Ph.D.
Advisors
Morre, Purdue University.
Subject Area
Immunology|Pharmacology|Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.