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 arc 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 Z(AFP D2) showed high and specific binding affinity to AFP. An affibody dimer of Z(AFP D2) was created, named (Z(AFF D2))(2), expressed in E.coli and the purified (Z(AFP) (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 Z(AFP D2) or (Z(AFP D2))(2) and polyclonal antibody to detect AFP was developed, the detection limit of the immunoassay using (Z(AFP D2))(2) was 2 ng mL(-1) that was 4 times lower than Z(AFP D2), 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. (C) 2020 Elsevier B.V. All rights reserved.

Automated summary

The study aimed to develop a novel affibody-based ELISA method for detecting AFP. Through the creation of affibody dimer and the development of a two-site ELISA method, this approach may provide a new method for the detection of various cancer biomarkers.