The Characterization of Cross-reactive Antibodies to Thomsen- Friedenreich a/b and Related Glycan-conjugates with Polyacrylamide Carriers in Patients with Gastrointestinal Cancer

Abstract

The level of IgG and IgM antibodies (Abs) to the tumor-associated Thomsen-Friedenreich antigen (TF, Galβ1- 3GalNAcα) in the serum of patients with gastrointestinal cancer is reduced and the elevated anti-TF IgG level is positively associated with survival of patients with gastric cancer as shown earlier using ELISA with the TFpolyacrylamide (TF-pAA, an amide-type conjugate). The reactivity of Abs to the standard conjugate TF-PAA is low. To characterize the specificity of Abs, they were affinity-isolated from sera of patients by using different TF-sorbents. These were: 1) IgG populations that differed in the reactivity and cross-reactivity to TF, TFβ (Galβ1-3GalNAcβ), GA1 and Gb5tri (the Gb5 trisaccharide, Gal1-3GalNAcβ1-3Gal) conjugates. However, all the populations showed a crossreactivity to the pAA-carrier. 2) The pAA-carrier-independent cross-reactive IgG Abs to TF, TFβ, GA1 and Gb5tri glycans, where TFβ and its cross-reactive TF were minimal ligands to Abs. 3) The pAA-non-reactive IgM Abs whose profile of reactivity was similar to that of population 2 but their specificity to TFβ was lower. In the most samples the Abs were more specific to TFβ than TF conjugates. The terminal Galβ residue was essential for antibody binding. IC50 of glycoconjugates was in the range of from 3 × 10-8 to 5 × 10-6 M. GA1-PAA-reactive Abs bound the GA1 glycolipid and weakly bound GM1. No or weak binding of the IgG antibodies to the unrelated antigens used in the determination of polyreactivity was observed. Thus, the antibody populations varied in reactivity and cross-reactivity to TF, TFβ, GA1 and Gb5tri . The cross-reactivity of Abs to the pAA-carrier with unsubstituted amide groups may be explained by its spatial similarity to these glycans. The determination of antibody populations using TFβ, GA1 or Gb5tri conjugates instead of TF-pAA may be more informative for diagnostic purposes and monitoring of patients with cancer.