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Kong Y.,Chinese Academy of Agricultural Sciences | Zhang Q.,Chinese Academy of Agricultural Sciences | Zhang Q.,Quality Inspection and Test Center for Oilseeds and Products | Zhang W.,Chinese Academy of Agricultural Sciences | And 4 more authors.
Journal of Agricultural and Food Chemistry | Year: 2010

With a screening hemisolid stem-cell culture, four positive hybridomas 2B12, 2C1, 2F1, and 3D4 were screened and used to prepare four correspondent monoclonal antibodies (McAbs) against the pyrethroid insecticide deltamethrin. These McAbs showed I 50 values in a range of 17-94 ng mL-1, from among which the antibody 2B12 with the lowest I50 value was selected to develop an optimized enzyme-linked immunosorbent assay (ELISA). In the developed ELISA, the I50 of deltamethrin was 17.0 ± 3.3 ng mL-1 and the limit of detection (LOD) was 1.2 ± 1.3 ng mL -1. There seemed to be little or no cross-reactivity with other tested pyrethroids and their metabolites. For validation of the assay method, environmental water samples fortified with deltamethrin were analyzed with the ELISA and gas chromatography (GC) methods. The recoveries of the developed ELISA ranged from 82 to 117%, which were close to those of the GC method (94-103%). These results suggested that the developed ELISA based on the McAb 2B 12 could be used for the rapid and sensitive determination of deltamethrin in environmental water. © 2010 American Chemical Society. Source


Li X.,Chinese Academy of Agricultural Sciences | Li X.,Key Laboratory of Detection for Mycotoxins | Li X.,Chongqing University | Li P.,Chinese Academy of Agricultural Sciences | And 12 more authors.
RSC Advances | Year: 2013

This paper describes a simple strategy to obtain ultra-sensitive single-chain fragment variable antibodies (scFv) against aflatoxin by constructing a positive phage-display library. A series of variable regions of heavy and light chains in monoclonal antibodies were randomly recombined to set up the library. Nearly 20 hybridoma cell lines that generate monoclonal antibodies against aflatoxins were first used, and then a library of 3.5 × 105 members was synthesized. The library was highly functional because high-quality scFv antibodies against aflatoxins had been isolated from the library. In addition, because of the high specificity of the DNA fragments forming the library, the anti-aflatoxin scFv antibodies could be rapidly obtained without traditional panning procedures. Based on preliminary indirect enzyme-linked immunosorbent assay (ELISA) screening data, the best candidate scFv antibodies 1A7 and 2G7 were subcloned for soluble expression. Indirect competitive ELISA was used to evaluate the sensitivity and cross-reactivity of the expressed products. The IC50 value of 1A7 was 0.02 ng ml -1, which was specific to four major kinds of aflatoxins, whereas that of 2G7 was 0.01 ng ml-1, which was highly specific to aflatoxin B1. Compared with the anti-aflatoxin recombinant scFv antibodies found based on the most significant evaluation results in recent publications, 1A7 and 2G7 were found to be over 20 times more sensitive, as described in this study. This journal is © The Royal Society of Chemistry 2013. Source


Wang X.,Chinese Academy of Agricultural Sciences | Wang X.,Key Laboratory of Biology and Genetic Improvement of Oil Crops | Wang X.,Key Laboratory of Detection for Mycotoxins | Li P.,Chinese Academy of Agricultural Sciences | And 16 more authors.
Journal of Separation Science | Year: 2012

In this paper, we describe the development of an oil-absorbing matrix solid-phase dispersion extraction with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry suitable for screening of 68 pesticide residues (PRs) in peanut, soybean, rape seed, sesame, and sunflower seed. The 68 PRs include 27 kinds of organophosphorus, 23 organic chlorines, 11 synthetic pyrethroids, and 7 carbamates. Heptachlor epoxide was used as the internal standard. Aminopropyl silica was chosen as the dispersion sorbent of the oil-absorbing matrix solid-phase dispersion extraction and was applied to capture hydrophobic components from high oil samples. A 35-min orthogonal separation was performed by using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry with a nonpolar-polar column set. Identification of 68 PRs in the extract was finished by using the time-of-flight mass spectrometry in the assistance of an automated peak-find and spectral deconvolution software. A screening based on control design was introduced and explained. This screening method considerably reduced the cost for the quantitative and confirmatory analyses. The quality of present screening method was evaluated by the Document No. SANCO/10684/2009. The false positive rate and false negative rate provide a useful tool for the evaluation of screening performance. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Li X.,Chinese Academy of Agricultural Sciences | Li X.,Key Laboratory of Biology and Genetic Improvement of Oil Crops | Li X.,Key Laboratory of Detection for Mycotoxins | Li P.,Chinese Academy of Agricultural Sciences | And 16 more authors.
Analytical Chemistry | Year: 2012

We screened and established seven hybridoma cell lines that secrete anti-aflatoxin monoclonal antibodies with different sensitivities. Among these antibodies, 1C11 exhibited the highest sensitivity against all four major kinds of aflatoxins (AFB1, AFB2, AFG1, and AFG2) (IC 50 0.0012-0.018 ng mL -1 in the enzyme linked immunosorbent assay (ELISA) system, visual limit of detection of 0.03-0.25 ng mL -1). To better understand the interactions between these antibodies and aflatoxins, as well as to guide their potential sensitivity improvement in recombinant antibodies, we used multiple sequence alignment and molecular modeling combined with molecular docking to clarify the molecular mechanism of the highest sensitivity of 1C11 against aflatoxins. Our results show that hydrogen bond and hydrophobic interaction formed by Ser-H49 and Phe-H103 in the antibody with the hapten played the most important roles in determining the binding affinity. Further experiments performed on antibody mutants, designed on the basis of the computational models, supported the prediction of the interaction mode between the antibody and the hapten. Although the factors that influence antibody sensitivity are highly interdependent, our experimental and modeling studies clearly demonstrate how structural differences influence the binding properties of antibodies against the target hapten with different sensitivities. © 2012 American Chemical Society. Source


Wang X.,Chinese Academy of Agricultural Sciences | Wang X.,Key Laboratory of Oil Crop Biology of the Ministry of Agriculture | Wang X.,Quality Inspection and Test Center for Oilseeds and Products | Li P.,Chinese Academy of Agricultural Sciences | And 24 more authors.
Chinese Journal of Chromatography (Se Pu) | Year: 2011

The high performance liquid chromatography (HPLC)-triple quadrupole tandem mass spectrometry was applied for the determination of the aflatoxins: B 1 (AFB 1), B 2 (AFB 2), G 1 (AFG 1) and G 2 (AFG 2), in cereals and oils. The samples were first extracted by ultrasonic method. The optimized conditions of ultrasonic extraction were as follows: temperature of 50°C, extraction time of 3 min, methanol-water (containing 40 g/L NaCl) (80:20, v/v) solution as the medium and a ratio of sample to solvent of 1: 3 (g: mL). The extracts were then purified using an immunoaffinity column. The separation was performed on a C 18 column with mobile phases of 10 mmol/L ammonium acetate and methanol in gradient elution. The sensitive detection of the four AFT compounds by electrospray ionization mass spectrometry (ESI-MS) was carried out in selected reaction monitoring (SRM) mode with aflatoxin Ml (AFM 1) as the internal standard. Under the optimized conditions, the limits of detection of AFB 1, AFB 2, AFG 1 and AFG 2 were 0.002, 0.004, 0.004 and 0.012 μg/kg, respectively. The recoveries of aflatoxins in different spiked cereals and oils were in the range from 87% to 111%. The intra-day and inter-day precisions were not more than 6.7% and 5.6%, respectively. In comparison with the external standard method, this method can effectively inhibit the matrix effects and greatly improve the accuracy. Source

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