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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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Schematic model of phageanti-immune complex assay (PHAIA). Thepeptide displayed on phage clones recognizes the immune complex toform the sandwich-model immunoassay. The detectable signal is amplifiedwith antiphage antibody conjugated horseradish peroxidase(HRP), whichcatalyzes 3,3′,5,5′-tetramethylbenzidine(TMB)to produce a blue colored solution.
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fig1: Schematic model of phageanti-immune complex assay (PHAIA). Thepeptide displayed on phage clones recognizes the immune complex toform the sandwich-model immunoassay. The detectable signal is amplifiedwith antiphage antibody conjugated horseradish peroxidase(HRP), whichcatalyzes 3,3′,5,5′-tetramethylbenzidine(TMB)to produce a blue colored solution.

Mentions: So far, all of the immunoassays reported previously for MG andLMG are based on conventional competitive immunoassays.23−27 To expand the sensitive immunoassay for MG and LMG, we developedthe noncompetitive PHAIA with the anti-LMG monoclonal antibody (mAb),as shown in Figure 1. The phage-displayed peptidesthat were selective for the LMG-immunocomplex were chosen from thecircular random eight-amino-acid library. One of the phage cloneswas used to establish noncompetitive immunoassays for quantitativeestimation of MG and LMG in fish samples.


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)

Schematic model of phageanti-immune complex assay (PHAIA). Thepeptide displayed on phage clones recognizes the immune complex toform the sandwich-model immunoassay. The detectable signal is amplifiedwith antiphage antibody conjugated horseradish peroxidase(HRP), whichcatalyzes 3,3′,5,5′-tetramethylbenzidine(TMB)to produce a blue colored solution.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematic model of phageanti-immune complex assay (PHAIA). Thepeptide displayed on phage clones recognizes the immune complex toform the sandwich-model immunoassay. The detectable signal is amplifiedwith antiphage antibody conjugated horseradish peroxidase(HRP), whichcatalyzes 3,3′,5,5′-tetramethylbenzidine(TMB)to produce a blue colored solution.
Mentions: So far, all of the immunoassays reported previously for MG andLMG are based on conventional competitive immunoassays.23−27 To expand the sensitive immunoassay for MG and LMG, we developedthe noncompetitive PHAIA with the anti-LMG monoclonal antibody (mAb),as shown in Figure 1. The phage-displayed peptidesthat were selective for the LMG-immunocomplex were chosen from thecircular random eight-amino-acid library. One of the phage cloneswas used to establish noncompetitive immunoassays for quantitativeestimation of MG and LMG in fish samples.

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