Abstract
The present study was aimed to develop a novel antibody-aptamer based hybrid detection strategy for specific and sensitive detection of aflatoxin B1 (AFB1) from contaminated food grains. The study comprises generation of ssDNA aptamers and anti-AFB1 IgG against AFB1 toxin. The generated bio-probes (aptamers and antibodies) were further characterized for their specificity and sensitivity using indirect ELISA. The generated aptamers namely AFB1a and AFB1b showed prominent reactivity and selectivity against AFB1 toxin. These aptamers were further characterized for their secondary structures and dG values were determined as −4.6 and −2.75 Kcal/mol, respectively. The detection limit (LOD) of AFB1a and anti-AFB1 IgG was determined as 5 and 10 ng/mL, respectively. The characterized aptamers and antibodies against AFB1 were used to develop the sandwich immunoassay. Anti AFB1 IgG was used as a capturing antibody whereas anti-AFB1a aptamer was used as its revealing partner in the assay. The limit of detection (LOD) of the immunoassay was determined to be 5 ng/mL of AFB1 standard toxin and showed no cross-reactivity with closely related mycotoxins. To assess the reliability of the developed method, several field samples contaminated with aflatoxin B1 was included in the study and results were validated with commercial AFB1-ELISA Kit. Additionally, the spiking studies were also carried out to demonstrate the consistency and dependability of the developed hybrid sandwich immunoassay wherein the toxins recovered were found to be ranging between 73 and 98.80% with the LOD at 5 ng/mL. In conclusion, the developed method may find the better utility in routine food testing laboratories for assessment of AFB1.
Highlights
Mycotoxins are low molecular weight (MW ∼700 Da) toxic secondary metabolites produced by filamentous fungi
Generation of AFB1 Aptamers Aptamers specific to Aflatoxin B1 was generated by immunoaffinity column based Systematic Evolution of Ligands by Exponential enrichment (SELEX) with five and three rounds of SELEX and counter SELEX respectively
Amplified aptamer pools were cloned into a pGMET cloning vector, transformed positive colonies for target insert were recovered and plasmids were purified and sequenced for target Aptamer (AFB1a and b) sequences (Table 1)
Summary
Mycotoxins are low molecular weight (MW ∼700 Da) toxic secondary metabolites produced by filamentous fungi. Significant groups of mycotoxins present in food are aflatoxins (AF) (Aspergillus sp.,), ochratoxin A (OTA) (Aspergillus sp., and Penicillium sp.,), trichothecenes, zearalenone and fumonisins B1 and B2 (FM) (Fusarium sp.,) (Kalagatur et al, 2017). Aflatoxins are well known toxins produced by A. flavus and A. paraciticus. Aflatoxin B1 (AFB1) has been placed as major Class I human carcinogen by IARC (International Agency for Research on Cancer) and is approximately 3-fold toxic than the other mycotoxins. Aflatoxin contamination in crops occurs due to high temperatures (27–38◦C) and relative humidity (85%) that favors Aspergillus growth in the field. The post-harvest contamination by aflatoxin occurs due to the prevalence of moisture content favoring mold growth and inappropriate agricultural practices (Torres et al, 2014; Gacem and El Hadj-Khelil, 2016; Kalagatur et al, 2018)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
