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Talanta. 2016;146:394-400. doi: 10.1016/j.talanta.2015.08.049. Epub 2015 Sep 4.

Phage-free peptide ELISA for ochratoxin A detection based on biotinylated mimotope as a competing antigen.

Author information

1
State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.
2
Key Laboratory of Functional Small Organic Molecule (Ministry of Education), Jiangxi Normal University, Nanchang 330022, China.
3
State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Electronic address: yhxiongchen@163.com.

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.

KEYWORDS:

Mimotope; OTA; Peptide ELISA; Phage display random library

PMID:
26695281
DOI:
10.1016/j.talanta.2015.08.049
[Indexed for MEDLINE]

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