Facile Generation of Neutralizing Antibodies on Tyrosine Phosphorylated IRS1 by Epitope-Directed Elicitation

Generating antibodies specific to the functional epitope containing phosphotyrosine remains highly challenging. Here, we create an "epitope-directed immunogen" by incorporating fluorosulfate-l-tyrosine (FSY) with cross-linking activities into a specific tyrosine phosphorylation site of insulin receptor substrate 1 (IRS1) and immunizing mice to elicit site-specific antibody responses. By taking advantage of antibody clonal selection and evolution in vivo, we efficiently identified antibodies that target the IRS1 Y612 epitope and are capable of neutralizing the binding interactions between IRS1 and p85α mediated by the phosphorylation of Y612. This epitope-directed antibody elicitation by encoding the cross-linking reactivity in the immunogen potentially enables a general method for facile generation of neutralizing antibodies to protein tyrosine phosphorylation sites.