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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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Standard curves of LMGin PBST and in sample extracts of differentdilution with or without solid-phase extraction (SPE) (n = 3). The matrix effect was studied by comparison of standard curvesobtained in PBST, in sample extracts cleaned up with SPE of 1:1, 1:5,1:10, 1:20 dilution, and in sample extracts without SPE.
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fig4: Standard curves of LMGin PBST and in sample extracts of differentdilution with or without solid-phase extraction (SPE) (n = 3). The matrix effect was studied by comparison of standard curvesobtained in PBST, in sample extracts cleaned up with SPE of 1:1, 1:5,1:10, 1:20 dilution, and in sample extracts without SPE.

Mentions: Due to the presence of the extractionagent, the sample component, and other ions, false positive or negativeresults from the matrix could be observed in the immunoassay. Forthe extraction of lipophilic LMG, it is necessary to minimize thematrix effect by using solid-phase extraction (SPE).23 In our study, the maximum absorption with 50 ng/mL LMGin 1:1, 1:5, 1:10, and 1:20 extract dilutions was 1.66 ± 0.02,1.76 ± 0.06, 1.79 ± 0.03, and 1.80 ± 0.05 (n = 3 per dilution), respectively, compared to 1.77 ±0.02 in PBST (Figure 4). The SC50 values were 8.09, 7.44, 7.64, and 7.66 ng/mL compared with 7.37ng/mL for PBST, indicating that the matrix effects of the three fishsamples were completely eliminated after pretreatment by PRS solidphase extraction. Although the 1:10 dilution showed less matrix effect,the higher limit of quantitation makes it unattractive. Thus, LMGwas extracted at a proportion of 1:5, and the extracts were diluted5-fold with PBST to minimize matrix effects.


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)

Standard curves of LMGin PBST and in sample extracts of differentdilution with or without solid-phase extraction (SPE) (n = 3). The matrix effect was studied by comparison of standard curvesobtained in PBST, in sample extracts cleaned up with SPE of 1:1, 1:5,1:10, 1:20 dilution, and in sample extracts without SPE.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Standard curves of LMGin PBST and in sample extracts of differentdilution with or without solid-phase extraction (SPE) (n = 3). The matrix effect was studied by comparison of standard curvesobtained in PBST, in sample extracts cleaned up with SPE of 1:1, 1:5,1:10, 1:20 dilution, and in sample extracts without SPE.
Mentions: Due to the presence of the extractionagent, the sample component, and other ions, false positive or negativeresults from the matrix could be observed in the immunoassay. Forthe extraction of lipophilic LMG, it is necessary to minimize thematrix effect by using solid-phase extraction (SPE).23 In our study, the maximum absorption with 50 ng/mL LMGin 1:1, 1:5, 1:10, and 1:20 extract dilutions was 1.66 ± 0.02,1.76 ± 0.06, 1.79 ± 0.03, and 1.80 ± 0.05 (n = 3 per dilution), respectively, compared to 1.77 ±0.02 in PBST (Figure 4). The SC50 values were 8.09, 7.44, 7.64, and 7.66 ng/mL compared with 7.37ng/mL for PBST, indicating that the matrix effects of the three fishsamples were completely eliminated after pretreatment by PRS solidphase extraction. Although the 1:10 dilution showed less matrix effect,the higher limit of quantitation makes it unattractive. Thus, LMGwas extracted at a proportion of 1:5, and the extracts were diluted5-fold with PBST to minimize matrix effects.

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