A specific monoclonal antibody targeting C-36 peptide of alpha1-antitrypsin as a tool in respiratory research

C-terminal fragments of alpha1-antitrypsin (AAT) occur in human biological fluids and tissues, including normal and emphysema lungs. We developed novel mouse monoclonal antibody (IgM, C42 mAb) against C-36 peptide resulting from elastase-associated cleavage of AAT. We addressed the usefulness of the C42 mAb in dot and western blots, immunomicroscopy and flow cytometry. According to the dot-blots, C42 mAb detect C-36 peptide at the range of 0.1-0.05 µg and show no cross-reactivity with native, polymeric or oxidized full-length AAT, AAT-elastase complex and shorter C-terminal fragments of AAT. The epitope recognized by the C42 mAb is unknown but it becomes unavailable after peptide denaturation and separation by electrophoresis following blotting. Because many antibodies towards conformational epitopes did not bind their target proteins in the western blot assays, we think that the C42 mAb is conformational structure-dependent. On the other hand, C42 mAb, unconjugated or conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays specifically detected C-36 peptide in human blood cells. The distributions of the C-36 peptide and the full-length AAT protein in human neutrophils and adherent PBMCs we assessed by laser scanning confocal microscopy. The 3D reconstruction of the confocal sections revealed only a partial co-localization between C-36 and AAT signals. The AAT we detected throughout the cell but mostly at the cell periphery, whereas C-36 peptide was predominantly present in perinuclear areas. Hence, C42 mAb may be a useful tool in respiratory research studies associated with health and disease.