Characterization of monoclonal antibodies to serum galactosyltransferase.

Abstract

Monoclonal IgG and IgM antibodies (mAbs) directed against serum galactosyltransferase (GalTase) activities were prepared and characterized for their relative specificity for GalTase isoenzymes I and II (GalTase I and GalTase II). After immunization of mice with purified GalTase, 7 of 1680 fusion products screened were positive for anti-GalTase activity in a solid-phase assay; of these 7, 2 bound GalTase I in a somewhat selective manner while 1 (C6) was relatively specific for GalTase II. The Ka for anti-GalTase mAb ranged from 2.7 .times. 107 to 1.1 .times. 10-8 M-1. Enzymatically active GalTase could be recovered from an affinity column of C6 coupled to Sepharose 4B following application of a cell extract from a human colon cancer cell line [HCT-8], confirming that the mAb is directed against GalTase. A sandwich RIA [radioimmunoassay] was developed to measure soluble GalTase GalTase II in serum by using a combination of 2 isoenzyme nonspecific mAb (F5 and V10) coated on a solid-phase support followed by the addition of antigen and GalTase II-specific 125I-labeled C6. This assay was specific for GalTase II with a sensitivity of .apprx. 10 ng/ml. Evaluation of 240 sera demonstrated higher levels of GalTase II in patients with gastrointestinal cancer (48 ng/ml). In contrast to previous results with a radiochemical assay, some normal sera contained GalTase II (mean, 14 ng/ml) and some patients with liver disease had elevated levels (mean, 23 ng/ml). The studies demonstrate the production of moderate-affinity antibodies directed to serum GalTase isoenzymes and the development of an RIA useful in the study of GalTase.