Development of an immuno-lectin-enzymatic assay for the detection of serum cancer-associated glycoproteins bearing Tn determinant

Abstract

We report the development of an immuno-lectin-enzymatic assay (CA83.4) with the purpose of quantifying serum glycoproteins bearing Tn determinant (GalNAc alpha-O-Ser/Thr). An anti-Tn monoclonal antibody (83D4) is bound to the solid phase in order to capture glycoproteins. After the addition of a test sample, we used biotinylated isolectin B4 from Vicia villosa and avidin-peroxydase to act as a detection system. The linear relationship between CA83.4 determinations and the serum dilutions, the reproducibility of the dosage in intra- and inter-assay, and the specificity of the test for the N-acetylgalactosamine residue in a-glycosidic O-linkages, demonstrated the reliability of this trial. Self-recognition of Vicia villosa B4 molecules (K-D: 0.73x10(-6) M determined using biosensor technology) could determine an additional step of signal amplification in this assay. Using 0.25 units/ml of CA83.4 antigen as the cut-off level, higher values were found in 25/49 patients with breast cancer, 8/13 with colorectal carcinoma, 3/11 with lung cancer, but in none of the 49 patients with non-malignant diseases nor in 97 healthy controls. This first report on soluble Tn-glycoprotein detection assays suggests that Tn-glycoproteins are specific serological tumor markers and we believe that they could represent a valuable tool in the diagnosis of cancer.