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Wang Y.,Chinese Academy of Agricultural Sciences | Wang Y.,Key Laboratory of Biotoxin | Wang Y.,Quality Inspection and Test Center for Oilseeds Products | Zhang Q.,Chinese Academy of Agricultural Sciences | And 11 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2011

This paper describes the establishment of an immunoaffinity chromatography (IAC) for selective extraction of fenvalerate from vegetable samples. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against fenvalerate to CNBr-activated Sepharose 4B and packed into a cartridge. The extraction conditions were carefully optimized, including loading, washing and eluting solutions. Under the optimal conditions, the IAC column was able to capture fenvalerate with the maximum capacity of 4000ng. An average recovery of 94.5% and a RSD of 8.8% were obtained with six IAC columns prepared on six different days. Three vegetable samples spiked with fenvalerate at four different concentrations were extracted with IAC column and determined by gas chromatography with electron capture detection (GC-ECD). Chromatograms of final extracts were clean and fenvalerate could be easily detected without the interferences. The extraction recoveries and RSD were 74.7-96.5% and 2.5-5.2%, respectively, and the calculated limit of detection of the whole method was 0.008-0.012ngg -1. © 2011 Elsevier B.V. Source


Wang P.,Chinese Academy of Agricultural Sciences | Wan X.,Chinese Academy of Agricultural Sciences | Wan X.,Key Laboratory of Oil Crop Biology of the Ministry of Agriculture | Zhang Y.,Chinese Academy of Agricultural Sciences | And 2 more authors.
Biotechnology Letters | Year: 2011

A novel expression system was established in the oleaginous yeast, Lipomyces kononenkoae. The expression vector pLK-rhPHG of L. kononenkoae was constructed and using the hygromycin phosphotransferase gene and green fluorescent protein gene as reporter genes. A delta 6-fatty acid desaturase gene (D6DM) from Cunninghamella echinulata MIAN6 was then expressed in this strain. The recombinant strain accumulated about 1.2% γ-linolenic acid in the total fatty acids. © 2011 Springer Science+Business Media B.V. Source


Guo B.,Chinese Academy of Agricultural Sciences | Jiang M.,Chinese Academy of Agricultural Sciences | Jiang M.,Key Laboratory of Oil Crop Biology of the Ministry of Agriculture | Wan X.,Chinese Academy of Agricultural Sciences | And 4 more authors.
Journal of Microbiology and Biotechnology | Year: 2013

The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) that are important to human health. Here, we report a functional characterization of a &δ4-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops. © 2013 by The Korean Society for Microbiology and Biotechnology. Source


Jiang J.,Chinese Academy of Agricultural Sciences | Zhang D.,Chinese Academy of Agricultural Sciences | Zhang W.,Chinese Academy of Agricultural Sciences | Zhang W.,Quality Inspection and Test Center for Oilseeds | And 8 more authors.
Analytical Letters | Year: 2010

Monoclonal antibodies (MAbs) against pyrethroid insecticide fenvalerate were achieved, identified, and applied in environmental water. Mice were immunized with a novel synthesized hapten conjugated with bovine serum albumin (BSA). Three positive clones of MAbs were obtained after cell fusion and hybridoma selection, among them MAb-2 (5B10) showed the highest reactivity toward fenvalerate. The IC50 of MAb-2 was 94.5 ngmL-1; moreover, it showed lower cross-reactivity with other pyrethroids such as bifenthrin, tetramethrin, deltamethrin and beta-cypermethrin. Optimization of enzyme-linked immunosorbent assay (ELISA) was studied. The limit of detection (LOD) of the assay was 8.8 ngmL-1 and the detection range was 0.017-27.33 μgmL-1. For preliminary application, addition recovery experiments in water samples were performed. The mean recoveries of three kinds of samples varied from 90.6% to 108.7% and the coefficients of variation ranged from 0.5% to 5.3%. The results showed that MAb-2 could be used for the detection of fenvalerate contamination in environmental water. © Taylor & Francis Group, LLC. Source


Guan D.,Chinese Academy of Agricultural Sciences | Guan D.,Key Laboratory of Oil Crop Biology of the Ministry of Agriculture | Guan D.,Quality Inspection and Test Center for Oilseeds Products | Li P.,Chinese Academy of Agricultural Sciences | And 6 more authors.
Analytica Chimica Acta | Year: 2011

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M 1 (AFM 1) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM 1 surrogate, was generated by immunizing rabbits with F(ab′) 2 fragments from the anti-AFM 1 monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM 1 mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM 1 and the anti-Id antibody (y=31.91x-8.47, r=0.9997). The assay was applied to analyze AFM 1 in spiked milk samples. The IC 50 value of the surrogate calibrator curve was 2.4μgmL -1, and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y=0.81x+9.82, r=0.9922). © 2011 Elsevier B.V. Source

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