ELISA detecting the anti-PEGylated FVIII antibodies. Antibodies (IgG on the left and IgM on the right) against the different FVIII molecules were detected by using each FVIII molecule as the capturing reagent. The bar colors indicate the used capturing molecule: red, octocog alfa; blue, rurioctocog alfa pegol; green, turoctocog alfa pegol; purple, damoctocog alfa pegol. Plasma samples were analyzed at different time points: 1, before SARS-CoV-2 vaccination; 2, before the PEGylated FVIII infusion (baseline); 3, after PEGylated FVIII infusion (30’); 4, 1 year after the second dose of the SARS-Co-V-2 vaccine. N indicates the pool of plasma from healthy subjects, and P indicates the pool of plasma from hemophilia A patients with anti-FVIII antibodies. Reactivity against the PEGylated FVIII products (higher with IgM than IgG) was found for both patients in plasma collected after the SARS-Co-V-2 vaccination but not in plasma obtained before. No significant signal against the non-PEGylated FVIII molecule was found. The pool of plasma containing anti-FVIII antibodies showed variable IgG reactivity against all molecules. No relevant signals were found with the pool plasma from healthy subjects.