Establishment of double-resistant sandwich system of advanced oxidized protein products

Objective An immunological method for the detection of advanced oxidized protein products (AOPPs) was established.Methods Monoclonal antibody was prepared by oxidation modification of albumin with hypochlorous acid (HOCl) as immunogen. The specificity of monoclonal antibodies was detected by indirect, competitive ELISA and Western Blot. The stability of double antibody sandwich ELISA system was compared with that of chloramine T method.Results The monoclonal antibody (AP-4C5) reacts specifically with HOCl-oxidized proteins of different species (mouse, bovine, human, rabbit) and different species (serum albumin, serum fibrinogen, and low density lipoprotein), but does not react with unoxidized proteins. The results of Western Blot showed that AP-4C5 could bind specifically to albumin and fibrinogen with different oxidation degrees, and the bands deepened with the increase of oxidation degree. In sandwich ELISA system of AP-4C5 and 3F2, AOPPs can be specifically detected in the range of 0.25-2μg/ml (R2=0.9918). AP-4C5 and goat anti-HSA sandwich ELISA system could specifically detect 1.5 - 25μg/ml AOPP (R2=0.96875). Compared with chloramine T method, it is more suitable for detecting low concentration AOPPs.Conclusion A double-antibody sandwich ELISA method based on anti-AOPPS monoclonal antibody was developed.