Citation: WANG Yu, YANG Jin-Yi, XU Zhen-Lin, QI Ping, SHEN Yu-Dong. Synthesis of Novel Hapten and Development of Monoclonal Antibody-based Enzyme-Linked Immunosorbent Assay for Malachite Green in Fish Samples[J]. Chinese Journal of Analytical Chemistry, ;2016, 44(9): 1385-1393. doi: 10.11895/j.issn.0253-3820.160360 shu

Synthesis of Novel Hapten and Development of Monoclonal Antibody-based Enzyme-Linked Immunosorbent Assay for Malachite Green in Fish Samples

WANG Yu ^1, ,
YANG Jin-Yi ² ,
XU Zhen-Lin ² ,
QI Ping ¹ ,
SHEN Yu-Dong ²

1.
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China
2.
Guangzhou Institute for Food Control, Guangzhou 510410, China

Received Date: 11 March 2016
Revised Date: 6 June 2016

Fund Project: This work was supported by the National Natural Science Foundation of China (No. 31371769) and the Natural Science Foundation of Guangdong Province, China (Nos. 2013030013338, 2014A030306026)

Figures(7)

To produce specific antibodies against malachite green (MG), one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay (icELISA) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value (IC₅₀) of 0.96 μg/L, a linear range (IC₂₀-IC₈₀) of 0.1-8.1 μg/L and a limit of detection (LOD, IC₁₀) of 0.05 μg/L for determination of MG. The assay showed cross-reactivity of 18.1%, 26.5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0.1%). The average recoveries of MG from spiked fish samples were from 87.3% to 107.3%. Good correlation (R²=0.999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner.
- Malachite green,
- Hapten,
- Monoclonal antibody,
- Antigenic-determinant,
- Enzyme-linked immunoassay
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