title: Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.
abstract: Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China. Electronic address: heqinghua@ncu.edu.cn.
authors.forename: Qing-hua
authors.initials: QH
authors.lastname: He
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China. Electronic address: xuyang@ncu.edu.cn.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Jin-heng
authors.initials: JH
authors.lastname: Fu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Yan-ping
authors.initials: YP
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Zhui
authors.initials: Z
authors.lastname: Tu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Mei
authors.initials: M
authors.lastname: Shu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Yu-lou
authors.initials: YL
authors.lastname: Qiu
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Hong-wei
authors.initials: HW
authors.lastname: Yang
authors.affiliation: Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, PR China.
authors.forename: Yuan-yuan
authors.initials: YY
authors.lastname: Liu
mesh_terms: D000818:Animals; D002161:Camelids, New World; D061505:Cell Surface Display Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry; D061905:Single-Domain Antibodies / Q000276:immunology*; D000509:alpha-Fetoproteins / Q000032:analysis* / Q000276:immunology
chemical_list: D061905:Single-Domain Antibodies; D000509:alpha-Fetoproteins
keywords: Alpha-fetoprotein; Cancer biomarker; Immunoassay; Nanobody