Simultaneous determination of malachite green, brilliant green and crystal violet in grass carp tissues by a broad-specificity indirect competitive enzyme-linked immunosorbent assay
Graphical abstract
Highlights
► A broad-specificity ELISA for three triphenylmethane dyes was developed. ► The effect of hapten structures on assay performance was studied. ► The developed ELISA showed good sensitivity and uniform response to three analytes. ► Good accuracy and reproducibility were obtained in spiked fishes samples.
Introduction
Malachite green (MG, Fig. 1), a triphenylmethane dye [1], has been illegally used as bacteriostasis agent to treat aquacultured fish. Due to its potential animal mutagenicity and teratogenicity properties [2], [3], [4], MG has been banned in aquaculture industry in European Commission and U.S, and also not approved in China (Ministry of Agriculture Bulletin No. 193). The minimum required performance limit (MRPL) was set for 2 μg kg−1 in fish muscles in these countries. However, owing to triphenylmethane dyes’ low cost and ready availability, some MG analogs, such as brilliant green (BG, Fig. 1) and crystal violet (CV, Fig. 1), are probably used as the substitutes of MG. BG and CV had been found to have anti-bacterial and anti-fungal properties similar to MG [5], [6]. According to the chemical similarity, some people concluded that BG, CV and their residues are toxic to humans after effectively being absorbed by fish [7]. Therefore, it is very significant to monitor these residues of triphenylmethane dyes in aquatic products.
The conventional methods for quantitative analysis triphenylmethane dyes and their leuco-metabolites mainly include liquid chromatography with visible and/or fluorescence detection [8], [9], [10], liquid chromatography–mass spectrometry [11], [12], capillary electrophoresis [13], [14]. These approaches require expensive instrumentation, tedious pretreatment processes and are time consuming. Immunoassays as rapid, low cost and higher sample throughput are alternative quantitative methods compared to conventional methods and have received an increasing attention in the detection of harmful residues [15], [16].
However, efficient immunoassay methods for the detection of triphenylmethane dyes could be seldom found. Until now, there was one paper in which two indirect competitive enzyme-linked immunosorbent assay (icELISA) kits constructed with the MG and leucomalachite green (LMG, Fig. 1) antibodies were developed to determine MG and LMG respectively in fish and fishpond water [17]. The limit of detection (LOD) was 0.05 μg kg−1 for both MG and LMG in that work, and the MG antibody showed 100% cross-reactivity (CR) to CV. Another paper described an icELISA format using group-specific antibody to simultaneously detect LMG and MG in water and fish samples. The LOD for LMG was 0.02–0.10 ng mL−1 and the CR values of LMG antibody for MG, CV and BG were 95%, 29% and less than 1% respectively [18]. Noticeably, yet no report on an ELISA method for determination of BG and CV was found.
As part of our ongoing research for immunoassay for triphenylmethane dyes [19], [20], the development of an ELISA method with a good specificity to BG was proposed as the initial goal. An immunizing hapten (4-(carboxymethoxy)phenyl)bis(4-(diethylamino)phenyl)methylium for BG was synthesized and coupled to bovine serum albumin (BSA) for the immunogen. New Zealand white rabbit was immunized to produce polyclonal antibodies (PcAb) against BG. Interestingly, the obtained PcAb showed high CRs to MG and CV in an icELISA. Therefore, in the following attempt, we changed the strategy to develop a broad-specificity icELISA [21] for simultaneous determination of BG, MG and CV in one single test. Several heterologous coating haptens were synthesized and used to improve the sensitivity and uniform response to the analytes. An icELISA with high sensitivity and uniform response for the three analytes was finally developed. The icELISA was applied to determine spiked grass carp tissues, and the results were compared with that of high performance liquid chromatography (HPLC). To the best of our knowledge, it could be the first report on the development of ELISA for simultaneous determination of BG, MG and CV with good sensitivity and uniform response.
Section snippets
Reagents and instruments
MG (98% purity), CV (98% purity), 4-hydroxybenzaldehyde, sodium chloroacetate, N,N-diethyl aniline, N,N-dimethyl aniline, Amberlyst 15 Resin were obtained from Alfa Aesar Company (Augsburg, Germany). BG (99% purity stated by EA), bovine serum albumin, dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), ovalbumin (OVA), 3,3′,5,5′-tetramethylbenzidine (TMB), complete and incomplete Freund's adjuvants were received from Sigma–Aldrich (St. Louis, USA). Horseradish peroxidase-conjugated goat
Hapten design and synthesis
Hapten design is a key step for the successful preparation of an immunoassay antibody against low molecular weight organic molecules [27], [28]. A suitable hapten for immunization should not only be consistent with the target molecules in common chemical structure but also have active functional group for coupling with carrier protein [29]. Initially, we designed an immunizing hapten (DBG) bearing carboxymethoxy directly on the phenyl ring for BG. Accidentally, we discovered the corresponding
Conclusions
Two immunogens and four coating antigens against triphenylmethane dye compounds were prepared. After assessment of eight coating antigen/PcAb combinations, a best broad-specific icELISA for detecting MG, BG and CV was exploited. According to the CR or IC50 values, we investigated the effects of the hapten structure on the specificity and sensitivity of icELISA, and provided further evidence for the general hapten design. In addition, recoveries for single analyte residues and mixtures of three
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (30901005), Guangdong Provincial Municipal Science and Technology Project (2010A080403005), Science and Technology Planning Project of Guangdong Province (2010A090200084, 2009B011300005).
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