P-20 - Immunodetecting lipoxidation: epitope mapping of HNE-modified human serum albumin using two different anti-HNE pAbs

Abstract

Lipids are susceptible to damage by reactive species, and many lipid peroxidation products can be formed. 4-hydroxynonenal (HNE) is one of the most abundant and is known to form covalent adducts with proteins. Here we describe for the first time the use of peptide arrays to evaluate the effect of oxidative post translation modifications on epitope binding of antibodies (Abs). Peptide arrays covering the sequence of human serum albumin (HSA) or ovalbumin (OVA) were used to assess the binding of two anti-HNE polyclonal antibodies (pAbs) by comparing HNE-treated membranes with non-treated membranes. The pAbs were generated against HNE treated HSA and HNE treated KLH, each. The OVA peptide array was used as a control to investigate HNE specific epitopes. The HNE treatment of the membranes induced pAbs binding to several peptides, and also changed the binding pattern, suggesting that HNE determines the epitope recognition of the pAbs. For the HNE treated HSA membrane, different epitopes were detected by both pAbs but the strongest recognition for both was the peptide 365DPHECYAKVFDEFKPLV381. For the HNE treated OVA membrane, four epitopes showed very clear binding by both pAbs, suggesting that despite being polyclonal, the Abs recognize the same HNE adducts, regardless of the protein.