Yousheng Jiang

Development of a fast ELISA for the specific detection of both leucomalachite green and malachite green

Malachite green (MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms (LMG) that may reside in fish muscles for a long period, thus being harmful to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay (ELISA) to detect MG and LMG specifically. The monoclonal antibody (mAb) to LMG was generated using a hybridoma technique. The obtained mAb showed good cross-reactivity (CR) to malachite green (MG), but not to crystal violet (CV) and Brilliant Green (BG). The mAb was used to develop a fast detecting ELISA of MG and LMG in fish. By introducing the conjugation LMG-HRP, the detection capability was 0.37 ng mL−1 for MG and LMG. The mean recovery from spiked grass carp tissues ranged from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. The stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG.

Production and characterization of monoclonal antibodies against small hapten-ciprofloxacin

High affinity and specificity monoclonal antibody against small hapten-ciprofloxaicn (CPFX) was produced and was used for rapid CPFX immunoassay in food stuffs of animal origin. Firstly, two kinds of antigens for CPFX were made using carrier proteins, bovine serum albumin (BSA) and ovalbumin (OVA), by a modified carbodiimide method and then the hapten-protein conjugates were characterized by ultraviolet spectrophotometry to detect hapten density before being used for the immunization and detection purposes. The production of monoclonal antibodies (Mabs) was sought following the generation of appropriate CPFX-BSA conjugate. Spleen cells from Balb/c mice immunized with CPFXBSA conjugate were fused with SP2/0 myeloma cells, and hybridomas secreting antibodies against CPFX were selected and cloned. One Mab against CPFX was produced and the average affinity of the Mab was 2.88×10 9 L/mol. The number of the hybridoma chromosome was 90 to 105. Except for enrofloxacin, the Mab had not any cross-reactivity with other fluoroquinolones. In the optimized ELISA, the Mab gave a 50% inhibition of 7.77 ng/ml with a detection limit of 1.56 ng/ml.