Characterization of monoclonal antihuman small bowel and colon specific mucin antibodies.

ABSTRACT

Three human small bowel and colon mucosal specific monoclonal antibodies with distinct morphologic and electrophoretic characteristics were generated by fusion of immunized Balb/c spleen cells and murine plasmacytoma cells. Morphologic specificity by indirect immunofluorescence (IIF) revealed three antibody binding patterns corresponding to villus surface (TP-NG-43), goblet cell apical granules (TP-NG-2), and a combined surface/goblet cell apical granule antibody (TP-NG-20). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) produced three distinct electrophoretic migration patterns. These antibodies reacted with very high molecular weight determinants TP-NG-2, one band greater than 400 kD; TP-NG-20, two bands corresponding to 370-400 kD; and TP-NG-43, two bands in the 350-400-kD range with smaller bands in the 50-94-kD range. Cross-reactivity with various other human organ systems was evaluated by indirect immunofluorescence and SDS-PAGE electrophoresis with Western blotting. By IIF, all three monoclonal antibodies reacted very strongly with components of gastric mucosa. Weak cross-reactivity was seen with colon, rectum and mucin-producing adenocarcinoma of the colon. No cross-reactivity was observed by IIF with other mucin-containing and non-mucin-containing tissues. However, cross-reactivity with gastric mucin was not detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antibody reactivity with mucin was confirmed by purifying various regional gastrointestinal mucins and by subsequent testing by ELISA. Monoclonal antibody affinity columns were prepared and evaluated. The utility of these methods will allow for further definition of important goblet cell mucin glycoprotein characteristics and isolation of mucin subpopulations.