A 2-SITE LECTINOENZYMATIC ASSAY FOR DETERMINATION OF TUMOR-MARKER GLYCOPROTEINS IN RECTAL SECRETIONS

A method is described for a titre-tray based two-site lectinoenzymatic assay of glycoproteins. WGA lectin, reacting with the core-part of gl ycans, was combined with lectins PNA and DBA, the latter two reacting with terminal parts of glycans. A standard curve was obtained with bov ine submaxillary gland asialomucin, and measurements of human rectal s ecretion were calibrated against this curve. The assay showed an intra -assay reproducibility of 2.4-7.5%, and inter-assay reproducibility of 3.9-20.8%. Recovery tests showed a linearity close to predicted value s. The selected standard was ideal as inhibition of lectin binding by monosaccharides showed similar inhibition profiles for human rectal se cretion and for asialomucin standard. Neuraminidase treatment dramatic ally increased the PNA binding to human rectal secretion immobilized o n WGA. Western blotting of human rectal secretion demonstrated a large range of lectin-reactive glycoproteins, the main fraction reacting wi th all lectins being approximately 250 kDa. The assay described is wel l suited for studies of the glycan part of tumour marker glycoproteins , and changes occurring in these. It has a high sensitivity by ignorin g that the glycans may be present on different molecules. Examination of rectal secretions from various cancer patients showed significantly increased PNA binding, as well as an increased PNA/DBA binding ratio, in patients with colorectal cancer (p<3x10(-3)) and, unexpectedly, in patients with other cancers (p<5x10(-3)).