The immunochemical characterisation, epitope analysis and diagnostic applications of neo-antigens formed by lipoxidation of human serum albumin

Abstract

Lipid oxidation has long been linked to the generation of toxic products seen to trigger many different anti-inflammatory responses. Products such as 4-hydroxynonenal (HNE) and acrolein can react with nucleophilic groups in proteins to form lipoxidation adducts, that have been detected in diseases like diabetes, cancer and cardiovascular diseases and, hence, are considered powerful biomarkers of oxidative stress in vivo. In this work, the reactivity of HNE towards human serum albumin (HSA), the major protein of human serum, was studied and the lipoxidised protein was used as an immunogen to derive anti-HSA-HNE antibodies (Ab) for subsequent characterisation and as a basis for development of a rapid diagnostic test. Mass spectrometry (MS) analysis confirmed the formation of HNE adducts on 15 HSA peptides, and one of the HSA-HNE immunised sheep sera was shown to contain specific antibodies to these adducts. Peptide arrays covering the HSA sequence and treated post peptide synthesis with HNE were used in a novel approach to characterise the binding patterns of the in-house anti-HNE Ab, and the results were in agreement with those obtained by MS. Nevertheless, the antibody showed cross-reactivity to HSA, which suggested some of the epitopes included not only the HNE moiety of the adducts, but also regions of HSA. Despite the cross-reactivity of this generated Ab, its potential for diagnostic applications was assessed by its deployment in a lateral flow assay together with a commercial anti-HNE Ab. The assay was able to distinguish HNE-modified proteins from non-modified ones, although further optimization is required. Finally, RNA from B lymphocytes of the HSA-HNE immunized sheep was extracted for construction of recombinant Fab and scFv phage display libraries, for the selection of monoclonal antibodies, but biopanning of anti-HSA-HNE clones was shown to be challenging due to the preponderance of anti-HSA specific clones present in the libraries