Enhanced sensitive immunoassay: Noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green

Jie Xian Dong, Chao Xu, Hong Wang, Zhi Li Xiao, Shirley J. Gee, Zhen Feng Li, Feng Wang, Wei Jian Wu, Yu Dong Shen, Jin Yi Yang, Yuan Ming Sun, Bruce D. Hammock

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG-mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R2 LMG = 0.9841; R 2 MG = 0.993; R2 Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

Original languageEnglish (US)
Pages (from-to)8752-8758
Number of pages7
JournalJournal of Agricultural and Food Chemistry
Volume62
Issue number34
DOIs
StatePublished - 2014

Keywords

  • leucomalachite Green
  • malachite green
  • phage anti-immunocomplex assay

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Chemistry(all)

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    Dong, J. X., Xu, C., Wang, H., Xiao, Z. L., Gee, S. J., Li, Z. F., Wang, F., Wu, W. J., Shen, Y. D., Yang, J. Y., Sun, Y. M., & Hammock, B. D. (2014). Enhanced sensitive immunoassay: Noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green. Journal of Agricultural and Food Chemistry, 62(34), 8752-8758. https://doi.org/10.1021/jf5019824