Development of a triabody-based immunoassay against a pathogenic epitope in human ApoB100 for the diagnosis and monitoring of atherosclerosis

Abstract

Cardiovascular diseases are the number one deadly diseases worldwide, with atherosclerosis being their pathological basis. However, the development of atherosclerosis is chronic and asymtomatic, posing significant challenges for its early detection and timely treatment. Recently, a 3136–3155 amino acid sequence, p210, in the apolipoprotein B-100 (ApoB100) of low density lipid was found to be associated with the severity of atherosclerosis and the risk of cardiovascular events. To develop a precise and sensitive immunoassay for the early diagnosis of atherosclerosis, three phage libraries displaying single chain fragment variable (scFv) against the p210 peptide in human ApoB100 were constructed in the present study, and three high affinity scFvs obtained, which were then genetically engineered to constitute a triabody that significantly enhanced its functional affinity and antigen-capturing capability towards the pathogenic p210 peptides. Furthermore, a rapid and simple method was developed with the triabody for early monitoring of the disease progression in clinical settings. The linear range of the sandwich ELISA containing the triabody was 36–576 ng/mL, with a limit of detection of 28.5 ng/mL. In addition, the average recovery of intra- and inter-assay were 88.4 ± 3.0 % and 85.5 ± 5.2 %, respectively. The developed assay was highly sensitive and specific for the pathogenic epitope, and no cross-reactivity with other antigens in serum observed. Importantly, since the triabody developed with trimeric scFvs against the same epitope has 2.5 times higher affinity than that of normal antibody with monomeric scFv, its derived sandwich ELISA could measure the amounts of pathogenic epitopes at very low levels, thereby capable of detecting signals corresponding to the severity of the disease in patients at early stage of atherosclerosis. Collectively, the phage library-derived triabody were successfully translated into a quick immunoassay with higher affinity against a pathogenic epitope that allows not only early detection but also real-time monitoring of the disease progression to minimize the risk of atherosclerotic cardiovascular disease.