Development of active affibody aggregates induced by a self-assembling peptide for high sensitive detection of alpha-fetoprotein

Abstract

Alpha-fetoprotein (AFP) is one of the most important biomarkers associated with primary liver cancer and the development of accurate and sensitive methods for detecting the serum level of AFP is of great importance in cancer diagnosis. In this study, three self-assembling peptides (SAPs) tagged AFP specific affibody dimer (ZAFP D2)2 recombinant protein, (ZAFP D2)2-ELK16, (ZAFP D2)2-L6KD and (ZAFP D2)2-R18A were successfully produced and characterized. The (ZAFP D2)2-ELK16 affibody aggregates showed higher and more specific binding affinity to AFP compared to (ZAFP D2)2 and other two SAP-tagged (ZAFP D2)2 and in addition displayed enhanced AFP-detection sensitivity and improved stability. A sensitive two-site enzyme-linked immunosorbent assay for AFP using (ZAFP D2)2-ELK16 as the detection reagent was also established. The AFP detection limit and linear range using (ZAFP D2)2-ELK16 based two-site ELISA were 9.7 ng mL−1 and 20–600 ng mL−1, which were significantly improved compared to those of the (ZAFP D2)2 based two-site ELISA for AFP. To detect AFP in serum samples, the average recovery ranged from 95.02% to 110.41% and the relative standard deviation was in the range of 2.45%-3.97%, which meet the standard clinical testing requirements. This proposed strategy provides a high yield, easy purification and green biosynthesis method to prepare SAP-based affibody aggregates for enhanced detection sensitivity by improving the immunoassay signal amplification activity.