| Malachite green (MG) a dye of triphenyl methane had been extens ive ly used inaquaculture for prevention and treatment fungal or parasitic in fis h. MG is easilyrapid ly metabolized into leucoma lachite green (LMG).Use of MG in aquatic food ishighly restricted or banned in several countries because of toxicolo gicalcons iderations. However ille gal use of MG continues worldwide in aquaculture dueto its low cost and ready vailab ility. Surveilla nce of MG and LMG in aquacultureproducts is a necessary means to protect human hea lth. And the main techniq uesinclud ing Chinese standard for the quantitative analys is MG and LMG ishigh-performance liquid chromatography (HPLC), high-performance liquidchromatography-fluorescence deteaction (HPLC-FLD). These ways rely onexpensive instruments, operated by we ll-trained ana lysts，a nd prior preparation ofsamples that is time cons uming and is not a ideal for screening large number ofsamples. Alternative ly, enzyme-linked immunosorbent assay(ELISA) is a rapid，sensitive, sensitive method that is applicable to the one-site examination of a largenumber of samples.To our knowledge, to date, the ELISAs on the market had to be establishedseparately for detecting MG and LMG ind ivid ually in samp les， leadingtotheinconve nience of measuring the sum of MG and LMG. Using HPLC to analyse thesum of MG and LMG, a samp le pre-treatment step, e.g.oxidative ly converting LMGto MG before assay performance, is required. Accordingly, the aims o f this paperwere to adopt a nove l synthetic approach for the preparation of LMG derivatives, togenerate a polyclo na l antibody with high cross-activity with both LMG and MGand to deve lop a sensitive and group-specifc ELISA for detecting the sum of MGand LMG in samples.The LMG derivative bearing a carboxyl group or amino group on the phenylring was synthes ised and cova lently coupled to carrier proteins by the carbod iimide method and dia zobenzid ine method respective ly for preparatio n of the immunogenand coating antigen. The polyc lona l antibod ies against LMG were prepared byimmunis ing rabbits and used to establish an ind irect competitive ELISA. Theproposed ELISA was validated by a conventiona l HPLC method for the ana lys is ofLMG in fsh muscle and fshpond water samples.The antibodies aga inst LMG were obtained from rabbits. Based on theoptimized conditi ons exp lore the methods for establis hing the method. Standardcurve was constructed with concentrations o f0.02–81.92μg/L; titer o f the antibodyis1:64000. The IC50va lue was in the range1.072-15.36μg/L and the limit ofdetection at a signa l-to-no ise ratio was0.011μg/L. The cross-reactivity va lues ofthe LMG antibody with MG were91.2%, while less than0.2%cross-reactivity wasfound with other compounds. The correlation coeffcient of ELISA withhigh-performance liq uid chromatography (HPLC) was0.988(n=15) for LMG-fortifedcruc ian carp s amples, For (LMG+MG)-spiked fsh samples the results of ELISA weresimilar to the standards.These demonstrate that the ELISA has high veracity,sensitivity and high precis ion, developing a preliminary study for detecting the sumof MG and LMG in samp les of ELISA. Which provide important basis for theresearch and development the ELISA kits of the sum of MG and LMG. |
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