Accessibility of antigenic sites recognized by AUA1, HMFG1 and HMFG2 monoclonal antibodies: its influence on antibody binding of live cells.

We assessed the immunoreactivity of live and alcohol-fixed monolayers of HRA-19, a rectal adenocarcinoma cell line, to the monoclonal antibodies AUA1, HMFG1 and HMFG2. Differences in staining patterns between live and alcohol-fixed colonies were found. The well-polarized cells forming the centers of the monolayer colonies showed strong membrane staining when the cells were alcohol-fixed prior to AUA1 incubation, but showed no staining when the cells were alive during the incubation. When AUA1 incubation was done both before and after alcohol fixation, membrane staining was again seen, ruling out the possibility of antigenic modulation. Incubation of live cells with AUA1 together with EDTA showed strong staining of dissociating cells. It is concluded that AUA1 antigenic sites, which on polarized cells are basolateral in location, are inaccessible to the antibody-containing culture fluid, which bathes the apical aspects of the cells, but they become accessible after alcohol fixation, or treatment with EDTA. HMFG1 antigenic sites are located on the apical cell membrane, and accordingly, no differences were seen between incubation of live and alcohol-fixed cells when incubated with HMFG1. The antigenic sites of HMFG2 are partly intracellular, and in our monolayer model, the staining of live cells was weaker and more scarce than on alcohol-fixed cells. It is concluded that immunostaining of cytological and histological material of tumours may not adequately predict antibody binding on live cells, and thus, these findings are of importance in the context of selection of monoclonal antibodies for clinical radio-immunotargeting.