Binding characteristics of an anti-Sia alpha 2-6GalNAc alpha-Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132)

To determine the epitopic structure for an anti-Sia alpha 2-6GalNAc alpha-S er/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mu cin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. F rom tryptic digest, a major glycopeptide designated as T3 was obtained as a n immunoaffinity column-bound fraction. On solid-phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treat ment with thermolysin, T3 was converted into about 50 fragments, as found o n fractionation by HPLC. Several of them were strongly immunoreactive and h ad the same amino ac id sequence, i.e. Phe-Ser*-Gly-Glu-Thr*-Ser*-Thr*-Thr* -Val-Ile-Ser*-Gly-Thr*-Asn-Val, where asterisks denote the sites of attachm ent of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of th e carbohydrate attached in these glycopeptides led us to postulate that a c luster composed of four sialyl Tn antigens is the essential epitopic struct ure for MLS 132.