Selection and characterization of a novel affibody peptide and its application in a two-site ELISA for the detection of cancer biomarker alpha-fetoprotein.

Alpha-fetoprotein (AFP) is one of the most important biomarkers associated with primary liver cancer, and the main approaches for diagnosis are based on immunoassay. Affibody is a 58 amino acids peptide derived from the Z domain of staphylococcal protein A and generally applied in imaging diagnosis, clinical therapeutics and biotechnology research. The aim of this study was therefore to develop a novel affibody-based ELISA for detection of AFP. After three rounds of biopanning, six AFP-binding affibody peptides were selected using phage display technology, among them affibody ZAFPD2 showed high and specific binding affinity to AFP. An affibody dimer of ZAFPD2 was created, named (ZAFP D2)2, expressed in E.coli and the purified (ZAFP D2)2 recombinant protein showed higher binding affinity to AFP, as well as high thermal stability. A novel affibody-based two-site ELISA method using ZAFPD2 or (ZAFP D2)2 and polyclonal antibody to detect AFP was developed, the detection limit of the immunoassay using (ZAFP D2)2 was 2 ng mL-1 that was 4 times lower than ZAFPD2, which meets the requirements for practical application. Therefore, this concept of affibody-based ELISA may provide a new method for the detection of various cancer biomarkers.