Novel conformation‐selective monoclonal antibodies against apoA‐I amyloid fibrils

The N‐terminal 1–83 fragment of human apolipoprotein A‐I (apoA‐I) carrying G26R mutation forms amyloid fibrils that potentially cause hereditary amyloidosis. We generated monoclonal antibodies that specifically bind to amyloid fibrils composed of not only apoA‐I fragment but also α‐synuclein. In silico modeling suggests that the antibodies might recognize the characteristic quaternary structures, constructed due to parallel stacking of VYV (in apoA‐I fragment) or LYV (in α‐synuclein) motifs. The Iowa (G26R) mutation in human apolipoprotein A‐I (apoA‐I), the major protein of plasma high‐density lipoprotein, is associated with systemic amyloidosis, and the N‐terminal 1–83 fragment of apoA‐I carrying this mutation has a strong propensity to form amyloid fibrils. Here, we generated and characterized novel monoclonal antibodies (mAbs) that display selective reactivity to apoA‐I amyloid fibrils. By immunizing BALB/c and A/J mice with apoA‐I 1‒83/G26R fibrils conjugated with hemocyanin and the hybridoma production, four IgM class mAbs were obtained. The generated mAbs exhibited strong reactivity to amyloid fibrils formed by the 1–83 fragment of apoA‐I, but not to the monomeric 1–83 fragment or full‐length apoA‐I. The apparent dissociation constant of the mAbs to apoA‐I fibrils was determined to be within the nM range. A time‐dependent aggregation assay demonstrated that the mAbs preferentially react with mature fibrils over non‐fibrillar aggregates formed by apoA‐I 1–83/G26R. In addition, dot blotting and ELISA using deletion or proline substituted variants of apoA‐I 1‒83/G26R suggest that the generated mAbs react to common structural features in apoA‐I amyloid fibrils. Indeed, the mAbs also recognized amyloid fibrils formed by α‐synuclein that has no sequence identity to apoA‐I. Thus, our newly generated anti‐apoA‐I fibril mAbs may be utilized for not only diagnosis of apoA‐I‐related amyloidosis but also structural analysis of amyloid fibrils as novel conformation‐selective antibodies.