Detecting lipoxidation: diagnostic tools for the translation and commercial application

Oxidative stress is strongly associated with the pathophysiology of inflammation diseases such as sepsis, diabetes and cardiovascular diseases. The oxidants formed in inflammation can cause oxidative damage to lipids, producing short chain electrophilic oxidized lipid products that can react with nucleophilic amino acid residues of proteins, a process known as lipoxidation [1]. 4-hydroxy-2-nonenal (HNE) is one of the quantitatively most important products of lipid peroxidation and can react by Michael addition or form Schiff base adducts with proteins, leading to cellular dysfunction. As lipoxidation products have potential as biomarkers of oxidative stress and disease, development of rapid assays for them is required. The aim of this work was to produce antibodies that recognize HNE-modified human serum albumin for future use in diagnostic assays of plasma. Human serum albumin (HSA), the most abundant protein of the human sera, was reacted with HNE at a 1:10 ratio. Analysis of intact HSA-HNE by direct infusion ESI-MS suggested the presence of (on average) 4.7 molecules of HNE/HSA and MALDI-TOF analysis showed an increase in mass corresponding to the addition of 3 molecules of HNE/HSA. Proteomic analysis of tryptic digests gave with confident identification only of HNE Michael addition, on the histidine of peptide VH²⁴²TECCHGDLLECADDR. To complement the results, an ELISA assay was performed with a commercial anti-HNE antibody and specific binding to HSA-HNE was observed, in contrast to low binding of un-modified HSA. The HSA-HNE adducts were subsequently immunized into sheep for generation of polyclonal antibodies and ELISA assays performed with enriched antibodies showed specific binding to HNE-modified proteins. In parallel, a potential reactive peptide sequence (LQQC³⁴PFE; m/z= 862.3) from HSA was synthesised and reacted with HNE. Using ESI-MS, HNE Michael adducts (m/z= 1018.4) on the cysteine residue were confirmed and Schiff base adducts (m/z= 1000.4), probably on the free N-terminal group, were also observed. These adducts were purified by reverse phase HPLC and immunized in rabbit and sheep for generation of antibodies anti-HNE-modified LQQCPFE. In conclusion, HNE adducts on HSA were confirmed and used for the generation of polyclonal antibodies. Enriched antibodies showed binding to HNE-modified HSA, but some cross-reactivity to HSA. Future work includes the improvement of the antibody purification process and testing anti-sera from rabbit and sheep immunized with LQQC(HNE)PFE.