Development and characterization of an immunosensor for the determination of oxidized low density lipoprotein (ox-LDL)

Abstract

Purpose/Objective: The oxidized lipoproteins, especially the oxidized low density lipoprotein (ox-LDL) are present in the plasma of patients with atherosclerosis and have been attributed an important role in illness development. Therefore, the determination of ox-LDL in plasma is essential not only to investigate its relevance for the atherosclerotic diseases, but also to contribute to disease diagnosis. Thus, this work aims to couple monoclonal antibody to biosensing technology, and produce an immunosensor capable of fast and accurate detection/quantification of ox-LDL in point-of-care. Materials and methods: oxLDL was prepared and employed to produce anti-oxLDL monoclonal antibodies by hybridomas cells (previously obtained by our group), which were cultured in DMEM with fetal calf serum, glutamine and gentamicin. The monoclonal antibodies were purified by affinity chromatography using Protein G. The immunosensor was set-up by self-assembling cysteamine (Cys) on the gold (Au) layer (4 mm diameter) of a disposable screen-printed electrode. Antibodies were let react with N-Hydroxysuccinimide and ethyl (dimethylaminopropyl) carbodiimide, and subsequently incubated in the Au/Cys. All these steps were followed by electrochemical techniques. Results: Three monoclonal antibodies showed the ability of detecting exclusively ox-LDL antigen. All these structures are to be involved in the construction of the immunosensing platform. Employing Cys self-assembling on gold is a successful way of binding the antibody to a physical support. One of the difficulties around this process is to avoid non-oriented binding of the antibody, which could prevent antigen access to the Fab region. So, the *COOH groups were previously activated, ensuring that mostly the Fb region is involved in connecting the antibody. After optimization of all chemical and physical variables, this sensor will be tested in patients plasma of hypercholesterolemic, with or without atherosclerosis, and normocolestolemics. Conclusions: The use of monoclonal antibodies on a biosensing platform seems to be a very attractive approach and is expected to provide very important information, correlating the level of ox-LDL fraction with the atherosclerosis disease, in a simple, fast and cheap way. After complete optimization, this device has the potential to improve the current diagnosis of atherosclerosis.