title: Enhanced sensitive immunoassay: noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green.
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.
authors: Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University , Guangzhou 510642, China.
authors: Jie-Xian
authors: JX
authors: Dong
authors: Chao
authors: C
authors: Xu
authors: Hong
authors: H
authors: Wang
authors: Zhi-Li
authors: ZL
authors: Xiao
authors: Shirley J
authors: SJ
authors: Gee
authors: Zhen-Feng
authors: ZF
authors: Li
authors: Feng
authors: F
authors: Wang
authors: Wei-Jian
authors: WJ
authors: Wu
authors: Yu-Dong
authors: YD
authors: Shen
authors: Jin-Yi
authors: JY
authors: Yang
authors: Yuan-Ming
authors: YM
authors: Sun
authors: Bruce D
authors: BD
authors: Hammock
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000032:analysis / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D019151:Peptide Library; D012394:Rosaniline Dyes / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D019151:Peptide Library; D012394:Rosaniline Dyes; C005095:malachite green; C015320:leucomalachite green