Xianle Yang

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.

Transcriptional responses in the hepatopancreas of Eriocheir sinensis exposed to deltamethrin

Deltamethrin is an important pesticide widely used against ectoparasites. Deltamethrin contamination has resulted in a threat to the healthy breeding of the Chinese mitten crab, Eriocheir sinensis. In this study, we investigated transcriptional responses in the hepatopancreas of E. sinensis exposed to deltamethrin. We obtained 99,087,448, 89,086,478, and 100,117,958 raw sequence reads from control 1, control 2, and control 3 groups, and 92,094,972, 92,883,894, and 92,500,828 raw sequence reads from test 1, test 2, and test 3 groups, respectively. After filtering and quality checking of the raw sequence reads, our analysis yielded 79,228,354, 72,336,470, 81,859,826, 77,649,400, 77,194,276, and 75,697,016 clean reads with a mean length of 150 bp from the control and test groups. After deltamethrin treatment, a total of 160 and 167 genes were significantly upregulated and downregulated, respectively. Gene ontology terms “biological process,” “cellular component,” and “molecular function” were enriched with respect to cell killing, cellular process, other organism part, cell part, binding, and catalytic. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes showed that the metabolic pathways were significantly enriched. We found that the CYP450 enzyme system, carboxylesterase, glutathione-S-transferase, and material (including carbohydrate, lipid, protein, and other substances) metabolism played important roles in the metabolism of deltamethrin in the hepatopancreas of E. sinensis. This study revealed differentially expressed genes related to insecticide metabolism and detoxification in E. sinensis for the first time and will help in understanding the toxicity and molecular metabolic mechanisms of deltamethrin in E. sinensis.