MONOCLONAL-ANTIBODIES REACTING WITH THE MUC2 MUCIN CORE PROTEIN

This study sought to produce monoclonal antibodies (MAbs) which reacte d with the MUC2 core protein. Two MAbs [3A2 (IgG1) and 4F1 (IgM)] were produced by immunising female BALB/c mice with gel-formed mucin from the LS174T colon cancer cell line followed by a KLH conjugate of a 29 amino acid synthetic peptide whose sequence was derived from the varia ble number of tandem repeats (VNTR) region of a MUC2 cDNA clone. The M Abs reacted with synthetic MUC2 VNTR peptides but not synthetic MUC1 o r MUC3 VNTR peptides, and showed specific reactivity in Western blotti ng with a high molecular weight protein produced by the LS174T colon c arcinoma cell line. The use of shorter peptides indicated that the min imum peptide epitopes for these MAbs were different. Mab 3A2 reacted w ith amino acids 5-19 of the MUC2 VNTR by inhibition ELISA but not by d irect ELISA, while 4F1 reacted with this peptide in both assays. Furth ermore, 4F1 reacted in direct ELISA when a larger (29 amino acid) MUC2 -derived peptide was coated onto the assay plate by incubating in carb onate buffer or by drying the peptide onto the assay plate, while 3A2 only reacted when this peptide was coated in carbonate buffer. The dif ferent specificity of the MAbs was also illustrated by the reactivity of 4F1 but not 3A2 with partially deglycosylated cystic fibrosis mucin . Immunohistochemical analysis with these MAbs revealed a strong react ivity with lung, gastric and colon tumours relative to normal tissue, with some breast and ovarian tumours also reacting. Both MAbs stained some normal goblet cells in the perinuclear region but not the mucin d roplet or secreted mucin, indicating a reaction with immature (poorly glycosylated) mucin in the endoplasmic reticulum and/or golgi, but not with mature (fully glycosylated) mucin. In contrast, tumours showed s trong diffuse cytoplasmic staining. 4F1 also showed weak apical cytopl asmic staining in some goblet cells and stained some tumours which sho wed no reactivity with 3A2. These antibodies should prove useful in th e study of MUC2 structure and function, and in the diagnosis of some t umours.