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

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 alpha-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.