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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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Noncompetitive ELISAfor LMG performed with different phage-displayedpeptide and competitive ELISA based on mAb for LMG. Results are theaverage of three replicates. The full lines are the standard curvesof phage anti-immune complex assay. The dotted line is the inhibitorycurve determined by competitive model. The 50% saturation of the signal(SC50) and the half inhibitory concentration (IC50) was calculated using a logistic plot equation with OriginPro 8.5(OriginLab, Northampton, MA).
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fig3: Noncompetitive ELISAfor LMG performed with different phage-displayedpeptide and competitive ELISA based on mAb for LMG. Results are theaverage of three replicates. The full lines are the standard curvesof phage anti-immune complex assay. The dotted line is the inhibitorycurve determined by competitive model. The 50% saturation of the signal(SC50) and the half inhibitory concentration (IC50) was calculated using a logistic plot equation with OriginPro 8.5(OriginLab, Northampton, MA).

Mentions: The assay with cloneNCB1 demonstrated the highest sensitivity (SC50 = 7.02ng/mL), followed by clones NCB4, NCB13, NCB6, and NCB11 (SC50 values of 8.39, 10.58, 13.00, and 16.15 ng/mL, respectively). Foreach of the five clones, standard curves were estimated using theoptimized conditions. The noncompetitive standard curves are presented in Figure 3. The linear range of NCB1 was 1.35 to 21.56 ng/mL.By altering the traditional competitive assay to the noncompetitivemodel, we improved the sensitivity of the assay by 16-fold using thesame mAb. The sensitivity improvement is consistent with previouslyreported results.19


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)

Noncompetitive ELISAfor LMG performed with different phage-displayedpeptide and competitive ELISA based on mAb for LMG. Results are theaverage of three replicates. The full lines are the standard curvesof phage anti-immune complex assay. The dotted line is the inhibitorycurve determined by competitive model. The 50% saturation of the signal(SC50) and the half inhibitory concentration (IC50) was calculated using a logistic plot equation with OriginPro 8.5(OriginLab, Northampton, MA).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Noncompetitive ELISAfor LMG performed with different phage-displayedpeptide and competitive ELISA based on mAb for LMG. Results are theaverage of three replicates. The full lines are the standard curvesof phage anti-immune complex assay. The dotted line is the inhibitorycurve determined by competitive model. The 50% saturation of the signal(SC50) and the half inhibitory concentration (IC50) was calculated using a logistic plot equation with OriginPro 8.5(OriginLab, Northampton, MA).
Mentions: The assay with cloneNCB1 demonstrated the highest sensitivity (SC50 = 7.02ng/mL), followed by clones NCB4, NCB13, NCB6, and NCB11 (SC50 values of 8.39, 10.58, 13.00, and 16.15 ng/mL, respectively). Foreach of the five clones, standard curves were estimated using theoptimized conditions. The noncompetitive standard curves are presented in Figure 3. The linear range of NCB1 was 1.35 to 21.56 ng/mL.By altering the traditional competitive assay to the noncompetitivemodel, we improved the sensitivity of the assay by 16-fold using thesame mAb. The sensitivity improvement is consistent with previouslyreported results.19

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