Cluster binding studies with two anti-Thomsen-Friedenreich (anti-core-1, CD176, TF) antibodies: evidence for a multiple TF epitope

Antibodies to carbohydrate epitopes are often of the IgM isotype and require multiple binding for sufficient avidity. Therefore clusters of epitopes are preferred antigenic sites in these cases. We have examined the type of clusters recognized by two anti-Thomsen-Friedenreich (TF, core-1, CD176) IgM antibodies, NM-TF1 and NM-TF2, using several different sets of TF-carrying synthetic glycoconjugates in ELISA experiments. To our surprise, the single most important factor determining binding strength was a close vicinity of several TF glycans at distances of ≤1 nm. Considering the known dimensions of IgM antibodies, our data strongly suggest that a cluster of up to four TF moieties, presenting as a "multiple epitope", is required to attach to a single combining site in order to result in adequate binding strength. This effect can also be achieved by "surrogate-multiple epitopes" consisting of separate TF-carrying molecules in close vicinity. In addition, it was found that serine-linked TFs are stronger bound than threonine-linked TFs by both antibodies. This peculiar type of cluster recognition may contribute to improved avidity and explicit tumor specificity