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

Dong JX, Xu C, Wang H, Xiao ZL, Gee SJ, Li ZF, Wang F, Wu WJ, Shen YD, Yang JY, Sun YM, Hammock BD - J. Agric. Food Chem. (2014)

Bottom Line: After three rounds of panning-elution, five peptides that bound the LMG-mAb immunocomplex were obtained.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.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University , Guangzhou 510642, China.

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 (R(2)LMG = 0.9841; R(2)MG = 0.993; R(2)Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

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Screening of positiveclones by noncompetitive phage ELISA. Clones1, 4, 6, 11, 13, 17, 18, 19, 20 reacting specifically with the leucomalachitegreen-monoclonal antibody (LMG-mAb) immunocomplex showed little ornegligible signal with the bovine serum albumin (BSA) or uncombinedmAb.
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fig2: Screening of positiveclones by noncompetitive phage ELISA. Clones1, 4, 6, 11, 13, 17, 18, 19, 20 reacting specifically with the leucomalachitegreen-monoclonal antibody (LMG-mAb) immunocomplex showed little ornegligible signal with the bovine serum albumin (BSA) or uncombinedmAb.

Mentions: In this study,we used the circular random eight-amino-acid library which was displayedon the pIII coat protein of filamentous phage. Compared with the othermajor coat protein, pVIII, pIII can tolerate the insertion of longerpeptides because pIII is the longest coat protein and only 3–5peptides copies are displayed.30 We believedthat the space between each peptide makes the immunocomplex more accessiblefor binding. The principle of noncompetitive immunoassay requiresthe phage-displayed peptides to recognize the antibody–analytecomplex rather than any other part of antibody. After three roundsof panning, 32 clones were picked randomly and tested for bindingto the anti-LMG mAb-coated wells in the presence or absence of LMG.Nine of 32 clones showed negligible binding to the uncombined antibody,although bounding specifically to the immunocomplex in the presenceof 50 ng/mL LMG (Figure 2).


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

Dong JX, Xu C, Wang H, Xiao ZL, Gee SJ, Li ZF, Wang F, Wu WJ, Shen YD, Yang JY, Sun YM, Hammock BD - J. Agric. Food Chem. (2014)

Screening of positiveclones by noncompetitive phage ELISA. Clones1, 4, 6, 11, 13, 17, 18, 19, 20 reacting specifically with the leucomalachitegreen-monoclonal antibody (LMG-mAb) immunocomplex showed little ornegligible signal with the bovine serum albumin (BSA) or uncombinedmAb.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150606&req=5

fig2: Screening of positiveclones by noncompetitive phage ELISA. Clones1, 4, 6, 11, 13, 17, 18, 19, 20 reacting specifically with the leucomalachitegreen-monoclonal antibody (LMG-mAb) immunocomplex showed little ornegligible signal with the bovine serum albumin (BSA) or uncombinedmAb.
Mentions: In this study,we used the circular random eight-amino-acid library which was displayedon the pIII coat protein of filamentous phage. Compared with the othermajor coat protein, pVIII, pIII can tolerate the insertion of longerpeptides because pIII is the longest coat protein and only 3–5peptides copies are displayed.30 We believedthat the space between each peptide makes the immunocomplex more accessiblefor binding. The principle of noncompetitive immunoassay requiresthe phage-displayed peptides to recognize the antibody–analytecomplex rather than any other part of antibody. After three roundsof panning, 32 clones were picked randomly and tested for bindingto the anti-LMG mAb-coated wells in the presence or absence of LMG.Nine of 32 clones showed negligible binding to the uncombined antibody,although bounding specifically to the immunocomplex in the presenceof 50 ng/mL LMG (Figure 2).

Bottom Line: After three rounds of panning-elution, five peptides that bound the LMG-mAb immunocomplex were obtained.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.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University , Guangzhou 510642, China.

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 (R(2)LMG = 0.9841; R(2)MG = 0.993; R(2)Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

Show MeSH