title: Phage-free peptide ELISA for ochratoxin A detection based on biotinylated mimotope as a competing antigen.
abstract: To perform the biopanning of a mimotope peptide with reduced affinity to anti-ochratoxin A (OTA) monoclonal antibodies (mAbs), we executed two improved biopanning approaches with a commercial 7-mer peptide library. In the first approach, anti-mouse IgG antibodies were used to erect the anti-OTA mAbs; in the second approach, an ultralow OTA concentration (0.1 ng/mL) was used to perform the competitive elution of phage particles. After the fourth round of biopanning was completed, 30 identified clones were positive phage particles; of these phage particles, 16 exhibited strong competitive inhibition with a low OTA concentration of 0.1 ng/mL. DNA sequencing results revealed that the 16 phage particles represented six different peptide sequences. Among these particles, the phage particle with a peptide sequence of "GMVQTIF" showed the highest sensitivity to OTA detection. The biotinylated 12-mer peptide "GMVQTIF-GGGSK-biotin" was designed as a competing antigen to develop a competitive peptide ELISA. Under the optimal parameters, the proposed peptide ELISA with the biotinylated 12-mer peptide as a competing antigen exhibited good dynamic linear detection for OTA in the range of 0.005 ng/mL-0.2 ng/mL with a detection limit of 0.001 ng/mL. The median inhibition concentration of OTA was 0.024 ng/mL (n=6), which is approximately fivefold more efficient as a competing antigen than the OTA-HRP conjugates. Reaction kinetics revealed that the biotinylated 12-mer peptide exhibited lower affinity to anti-OTA mAbs than the conventional chemical OTA antigen. The practicality of the proposed peptide ELISA was compared with a conventional ELISA method. In summary, this study demonstrated a novel concept of the development of phage-free peptide ELISA for the detection of OTA by using a biotinylated mimotope peptide as a competing antigen. This novel strategy can be applied to sensitively detect other toxic small molecules during food safety monitoring.
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.
authors.forename: Xuqiang
authors.initials: X
authors.lastname: Zou
authors.affiliation: Key Laboratory of Functional Small Organic Molecule (Ministry of Education), Jiangxi Normal University, Nanchang 330022, China.
authors.forename: Chaochao
authors.initials: C
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Huang
authors.affiliation: Key Laboratory of Functional Small Organic Molecule (Ministry of Education), Jiangxi Normal University, Nanchang 330022, China.
authors.forename: Xuelan
authors.initials: X
authors.lastname: Chen
authors.affiliation: Key Laboratory of Functional Small Organic Molecule (Ministry of Education), Jiangxi Normal University, Nanchang 330022, China.
authors.forename: Lv
authors.initials: L
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Electronic address: yhxiongchen@163.com.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Xiong
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000276:immunology*; D007700:Kinetics; D057230:Limit of Detection; D009793:Ochratoxins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D009793:Ochratoxins; D010455:Peptides; C025589:ochratoxin A; D012710:Serum Albumin, Bovine
keywords: Mimotope; OTA; Peptide ELISA; Phage display random library