Aptamers for nanobodies: A nontoxic alternative to toxic ochratoxin A in immunoassays

Yingyu Hou; Xixia Liu; Yongshu Li; Jianjun Hou; Huan Liu; Qin Wu; Juewen Liu

doi:10.1016/j.bios.2023.115995

Aptamers for nanobodies: A nontoxic alternative to toxic ochratoxin A in immunoassays

Biosens Bioelectron. 2024 Mar 15:248:115995. doi: 10.1016/j.bios.2023.115995. Epub 2023 Dec 30.

Authors

Yingyu Hou¹, Xixia Liu², Yongshu Li¹, Jianjun Hou¹, Huan Liu¹, Qin Wu¹, Juewen Liu³

Affiliations

¹ Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Huangshi, Hubei province, 435002, China.
² Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Huangshi, Hubei province, 435002, China. Electronic address: liuxixia@hbnu.edu.cn.
³ Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON, N2L 3G1, Canada. Electronic address: liujw@uwaterloo.ca.

PMID: 38176255
DOI: 10.1016/j.bios.2023.115995

Abstract

To measure toxins using immunoassays, hazardous toxin standards need to be added for quantification. To solve this problem, we propose to use aptamers as competitors to replace toxin standards. In this work, aptamers specific for ochratoxin A (OTA) nanobodies were selected using a DNA library containing a 36 nucleotide random region. The obtained sequences were highly aligned and the best competitor was identified to be a sequence named apt2-OT based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). The K_d of apt2-OT was measured to be 2.86 μM using local surface plasmon resonance spectroscopy. The optimal apt2-OT was identified to substitute the OTA standard with a concentration needed for 50% inhibition of binding (IC₅₀) of 3.26 μM based on a nontoxic direct competitive ELISA. The equivalence relationship between the aptamer and OTA was established in a flour sample, and a recovery experiment was performed. The detection limit for this method was 0.23 ng/mL, with a linear range from 0.25 to 10.50 ng/mL. The recovery rate was 97.5%-115.5%. This study provides a low-cost, rapid and environmentally friendly alternative to the development of immunoassays for toxins.

Keywords: Aptamers; ELISA; Nanobody; Ochratoxin A; SELEX.

MeSH terms

Aptamers, Nucleotide* / chemistry
Biosensing Techniques* / methods
Immunoassay
Limit of Detection
Ochratoxins* / analysis
Ochratoxins* / toxicity
Single-Domain Antibodies*

Substances

ochratoxin A
Single-Domain Antibodies
Aptamers, Nucleotide
Ochratoxins