Objective: Traditional ochratoxin A(OTA) competitive antigen are high toxicity, high price and difficult preparation. Non-toxic and easy prepared OTA competitive antigen substitutes were expressed by recombinant filamentous phage which have OTA mimotope displayed on its p VI surface.
Methods: Recombinant phagemid pC89-COTA which contain OTA mimotope nucleotide sequence was constructed. Moreover, to increase the binding rate of OTA mimotope with antibody, Enterokinase cleavage sits were led into 5' terminal of OTA mimotope nucleotide sequence. The pC89-COTA was transformed into E. coli XL1-blue. The E. coli XL1-blue were infected by wild KM13 phage and recombinant phage with OTA mimotope displayed generated.
Results: OTA mimotope phage was successful expressed and OTA mimotope phage which digested by Enterokinase had significantly higher binding rate with antibody than phage which not digested by Enterokinase. Non-toxic OTA competitive ELISA was established by using this digested OTA mimotope phage, the detecting limitation was 100 microg/ml, the linear range of the inhibition curves was between 250 pg/ml and 1000 pg/ml. Spiked recoveries of the farina tritici blank samples, the recovery rate of OTA were 99.8% 112.3% and coefficients of variation were 8.19% 11.64%, then 16 commercially available samples were tested and the positive rate was 31.25%.
Conclusion: OTA mimotope phage were successfully expressed and non-toxic OTA competitive ELISA was established.
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Mimotope peptides for nanobodies: A nontoxic alternative to ochratoxin A and its application in chemiluminescence immunoassays for analysis of pepper samples.
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Article Synopsis
- ELISA is commonly used to detect mycotoxins in food, and this study introduces a new method that enhances sensitivity for ochratoxin A (OTA) detection using a biotinylated M13 bacteriophage as a competing antigen.
- The innovative system incorporates a high number of streptavidin-labeled horseradish peroxidases (HRPs) on M13, leading to a remarkable detection limit of 2.0 pg/mL for OTA, significantly outperforming traditional ELISA methods.
- The new method demonstrated low cross-reactivity with related mycotoxins and maintained accuracy in real corn sample testing, suggesting its potential for detecting various other analytes by adjusting peptide sequences.
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