Development and characterization of three novel monoclonal antibodies against CA-125.

Cancer antigen 125 (CA-125) is the most widely used tumor marker for ovarian cancer. Thus, monoclonal antibodies (MAbs) against CA-125 are valuable reagents for the development of diagnostic tests and immunotherapy. We describe here the generation and characterization of three novel hybridoma cell lines producing MAbs against CA-125. CA-125 purified from culture supernatant of ovarian carcinoma cell line OVCAR-3 by affinity chromatography, was used for immunization of BALB/c mice. Three stable cell lines (3C8, 2B6, and 5A12) were selected for production of antibodies against CA-125 and were expanded in mass culture. All three antibodies were shown to recognize linear epitopes. Antibodies 2B6 and 5A12 were determined to recognize epitope cluster B (M 11-like); MAb 3C8 was classified as group A-epitope binders (OC 125-like). The antibodies produced may be used for the development and improvement of CA-125 immunoassays.

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase.

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).