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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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Correlation analysis between the PHAIAand HPLC (n = 3). (A) Samples spiked with LMG. (B)Samples spiked with MG. (C)Sample spiked with a mixture LMG and MG (1:1). All the sample extractedby the same procedure were divided into two equal parts and testedby PHAIA and HPLC.
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fig5: Correlation analysis between the PHAIAand HPLC (n = 3). (A) Samples spiked with LMG. (B)Samples spiked with MG. (C)Sample spiked with a mixture LMG and MG (1:1). All the sample extractedby the same procedure were divided into two equal parts and testedby PHAIA and HPLC.

Mentions: The accuracyand precision of the PHAIA format were evaluated by determinationof MG and LMG in spiked fish samples as well as by spiking with themixtures of the two analytes. The MG was reduced to LMG by potassiumborohydride so that it could be detected by the specific anti-LMGmAb. As shown in Table 3, the mean recoveryrate measured using the LMG-PHAIA standard curves were 75.33–88.00%for LMG, 80.67–84.45% for MG, and 75.60–83.93% for theLMG and MG mixture. The comparison of results with HPLC shown in Figure 5 indicated that good correlations were obtainedat the 5, 10, and 20 ng/mL spiked levels.


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)

Correlation analysis between the PHAIAand HPLC (n = 3). (A) Samples spiked with LMG. (B)Samples spiked with MG. (C)Sample spiked with a mixture LMG and MG (1:1). All the sample extractedby the same procedure were divided into two equal parts and testedby PHAIA and HPLC.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Correlation analysis between the PHAIAand HPLC (n = 3). (A) Samples spiked with LMG. (B)Samples spiked with MG. (C)Sample spiked with a mixture LMG and MG (1:1). All the sample extractedby the same procedure were divided into two equal parts and testedby PHAIA and HPLC.
Mentions: The accuracyand precision of the PHAIA format were evaluated by determinationof MG and LMG in spiked fish samples as well as by spiking with themixtures of the two analytes. The MG was reduced to LMG by potassiumborohydride so that it could be detected by the specific anti-LMGmAb. As shown in Table 3, the mean recoveryrate measured using the LMG-PHAIA standard curves were 75.33–88.00%for LMG, 80.67–84.45% for MG, and 75.60–83.93% for theLMG and MG mixture. The comparison of results with HPLC shown in Figure 5 indicated that good correlations were obtainedat the 5, 10, and 20 ng/mL spiked levels.

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