Evaluation of oxidized alpha-1-antitrypsin in blood as an oxidative stress marker using anti-oxidative α1-AT monoclonal antibody

Abstract

Background: α1-AT is a 52-kDa acute-phase protein and a typical serine proteinase inhibitor, which is present in human serum. In vivo, the inhibitor prevents tissue damage by inactivating proteinases, such as elastase, that are released from activated neutrophils in the presence of inflammation. Methods: We obtained a monoclonal antibody against oxidized α1-AT(3F4) using chloramine T-oxidized α1-AT as the antigen. Results: This antibody did not react with either the native α1-AT or the elastase–α1-AT complex. However, it reacted with α1-AT oxidized by various oxidants and peroxide lipid. The oxidized α1-AT is a polymer with a molecular mass of 100–200 kDa in addition to the 52-kDa protein that corresponds to the native α1-AT in sera. In vitro evaluations reveal that fatty acids are involved in the polymerization. Furthermore, the concentrations of oxidized α1-AT in the sera of patients with inflammatory and rheumatoid diseases were higher than those in healthy subjects. Conclusions: We considered that 3F4 is an effective antibody that can specifically recognize oxidized α1-AT, a marker of oxidative stress.