Yu-Dong Shen

Jilin Agricultural University, Shengcheng, Guangdong, China

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Publications (31)74.63 Total impact

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    ABSTRACT: The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons enco4ding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L(-1) in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL(-1), respectively.
    Applied Microbiology and Biotechnology 11/2013; · 3.69 Impact Factor
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    ABSTRACT: A simple and fast homogeneous fluorescent polarization immunoassay (FPIA) was developed for the determination of furaltadone and its metabolite 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ). Monoclonal antibody with high cross-reactivity to furaltadone and the nitrophenyl derivative of AMOZ (NPAMOZ) were produced against a novel immunogen and the effects of several synthesized tracers on FPIA sensitivity studied. The proposed FPIA, using an optimum antibody and tracer pair, had an IC50 of 4.3 μg L-1 and limit of detection at 0.6 μg L-1 for furaltadone, and 2.7 μg L-1 and 0.3 μg L-1 for NPAMOZ. Recoveries of furaltadone from animal feeds by FPIA ranged from 79.6 to 87.7%, while recoveries of AMOZ from animal tissues ranged from 72.9 to 83.1%. Good correlation (R>0.99) between the results of this FPIA and a standard analytical method was obtained. The FPIA does not require separation or washing steps and the total time required for equilibrium of the antibody-tracer interaction is only 10 min. These results indicated that the proposed FPIA offers great potential and utility for the high-throughput screening of furaltadone residues in animal feed and its metabolite AMOZ residues in animal tissues.
    Combinatorial chemistry & high throughput screening 02/2013; · 2.46 Impact Factor
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    ABSTRACT: A heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) was developed. AMOZ was derivatised with 2-(4-formylphenoxy) acetic acid or 2-(3-formylphenoxy) acetic acid to obtain two novel immunizing haptens. The ability of these haptens in producing specific polyclonal antibodies against the nitrophenyl derivative of AMOZ (NPAMOZ) was compared with that of traditional immunizing haptens (derivatised AMOZ with 3-carboxybenzaldehyle or 4-carboxybenzaldehyle). The results indicated that the novel immunizing haptens were able to produce antibodies with almost a two-fold improvement in sensitivity of the ciELISA for NPAMOZ in comparison with the existing antibody based ELISAs. The differences in sensitivity were explained by the molecular modeling of the lowest energy conformations of NPAMOZ and the haptens. Another novel hapten, derivatised AMOZ with 2-oxoacetic acid, was synthesized and used as a heterologous coating hapten. The results showed that this strategy of using only a partial structure of the target molecule as the coating hapten was able to obtain a two to three-fold improvement in sensitivity. This study provided a modern approach for the development of an immunoassay with improved sensitivity for the metabolites of nitrofuran antibiotics.
    Talanta 01/2013; 103:306-13. · 3.50 Impact Factor
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    ABSTRACT: A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.
    PLoS ONE 01/2013; 8(8):e70451. · 3.73 Impact Factor
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    ABSTRACT: An indirect competitive chemiluminescence enzyme immonoassay (icCLEIA) for the quantitative analysis of furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone(AMOZ) in Metapenaeus ensis sample was established with the highly sensitive luminol-H2O2-HRP-4-iodophenol chemiluminescence reaction detection system. The icCLEIA method was based on a single-chain variable fragment (scFv) antibody against AMOZ derivative. The optimized assay conditions were as follows: 62.5 μg L−1 of coating antigen (5-morpholinomethyl-3-(glyoxalamino)-2-oxazolidone-ovalbumin, AMOZA-OVA) was used in the experiment, the dilute multiple of scFv antibody was 1:10, the immunoreaction time was 45 min, and the dilute multiple and incubation time of HRP-goat anti mouse IgG were 1:10000 and 50 min, respectively. The icCLEIA results showed that IC50 value and limit of detection (LOD) were 1.38 μg L−1 and 0.09 μg L−1, and linear range was in the range of 0.26–9.1 μg L−1. Inter-assay and intra-assay RSD were both below 15%. The scFv antibody showed high specificity. The average recoveries of four spiked level of AMOZ were 72.2%, 73.4%, 72.6% and 78.6%, respectively. In comparison with HPLC-MS/MS, there was a good correlation between the two methods (R2 = 0.9997). The established icCLEIA method could be further used for detecting AMOZ in aquatic products samples rapidly and efficiently.
    Chinese Journal of Analytical Chemistry. 12/2012; 40(12):1816–1821.
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    ABSTRACT: Due to the large-scale use of organophosphorus pesticides (OPs), their contamination in the environment is widespread throughout the world, especially in developing countries. With increasing public concern, there is an urgent requirement for simple, rapid, high-throughput and highly sensitive analytical methods for the screening of OP residues in the environment. In this work, a monoclonal antibody-based sensitive one-step direct competitive time-resolved fluorescence immunoassay (TRFIA) with broad specificity to a class of OPs was developed. After optimization of the assay conditions, this method can detect thirteen OPs with a limit of detection below 10 ng mL−1. The recoveries of OPs from spiked environmental water samples ranged from 74.8% to 121.3%, with the coefficient of variation (CV) ranging from 6.4% to 15.1%. The correlation coefficient between the TRFIA results and standard HPLC-MS/MS results was 0.964. The proposed TRFIA was capable of high-throughput analysis of a large number of samples prior to chromatographic analysis, with good sensitivity, simplicity and rapidity.
    Analytical methods 09/2012; 4(10):3484-3490. · 1.86 Impact Factor
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    ABSTRACT: A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(L) genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25±0.03 and 0.02±0.004 ng mL(-1), respectively, and the linear response range extended from 0.05 to 1.45 ng mL(-1). The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between the data of dc-CLEIA and HPLC-MS (R(2)>0.99), indicating that the assay was an efficient analytical method for monitoring food safety.
    Analytica chimica acta 07/2012; 736:85-91. · 4.31 Impact Factor
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    ABSTRACT: An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC-MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC-MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.
    Journal of Agricultural and Food Chemistry 05/2012; · 2.91 Impact Factor
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    ABSTRACT: A single-chain variable fragment (scFv) linked alkaline phosphatase (AP) fusion protein for detection of O,O-diethyl organophosphorus pesticides (O,O-diethyl OPs) was produced and characterized. The scFv gene was prepared by cloning V(L) and V(H) genes from hybridoma cells secreting monoclonal antibody with broad specificity for O,O-diethyl OPs. The amplified V(L) and V(H) regions were assembled using a linker (Gly(4)Ser)(3) by means of splicing overlap extension polymerase chain reaction to obtain the scFv gene, which was cloned into the expression vector pLIP6/GN containing an AP gene to produce the scFv-AP fusion protein in Escherichia coli strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and competitive direct enzyme-linked immunosorbent assay (cdELISA). The fusion protein is bifunctional, retaining both antigen binding specificity and AP enzymatic activity. Analysis of spiked and blind river water and Chinese cabbage samples demonstrated that the fusion protein based cdELISA(FP) exhibited good sensitivity and reproducibility.
    Journal of Agricultural and Food Chemistry 04/2012; 60(20):5076-83. · 2.91 Impact Factor
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    ABSTRACT: A multi-analytes method for monitoring of organophosphorus pesticides (OPs) using a combination of broad-specificity direct competitive enzyme-linked immunosorbent assay (dcELISA) and high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed. The reaction formats of dcELISA as well as the matrix effects of vegetable samples by different treatments were studied. The dcELSIA based on horseradish peroxidase-labelled monoclonal antibody and solid-phase extraction can analyse 42 samples in duplicate simultaneously for 12 OPs with a limit of detection at 20μgL−1 within 40min, with good accuracy and reproducibility. For screening purpose, the dcELISA can distinguish positive samples from hundreds of negative samples at a rapid, high-throughput and low cost manner. The positive samples can be following confirmed by HPLC–MS/MS for the kinds and the relative amounts of OPs. The method is suitable for monitoring of OP contamination in vegetables samples with high-efficiency and low cost.
    Food Chemistry - FOOD CHEM. 04/2012;
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    ABSTRACT: Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.
    Journal of Agricultural and Food Chemistry 03/2012; 60(9):2069-75. · 2.91 Impact Factor
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    ABSTRACT: A simple, rapid and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) using a broad-specificity monoclonal antibody was developed. The effects of tracer structure, tracer concentration, antibody dilution, methanol content and matrix effect on FPIA performance were studied. The FPIA can detect 5 OPs simultaneously with a limit of detection below 10 ng mL(-1). The time required for the equilibrium of antibody-antigen interaction was less than 10 min. The recovery from spiked vegetable and environmental samples ranged from 71.3% to 126.8%, with the coefficient of variations ranging from 3.5% to 14.5%. The developed FPIA was applied to samples, followed by confirmation with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. The developed FPIA demonstrated good accuracy and reproducibility, and is suitable for rapid and high-throughput screening for OP contamination with high-efficiency and low cost.
    Analytica chimica acta 12/2011; 708(1-2):123-9. · 4.31 Impact Factor
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    ABSTRACT: The use of chlorpromazine (CPZ) as a sedative for livestock has been prohibited in the European Union and many other countries. In this study, a new hapten7-amino-chlorpromazine sulfoxide (hapten 1) against CPZ was synthesized and coupled to ovalbumin (OVA) as an immunogen to produce polyclonal antibody (PAb) specific for CPZ. An heterologous hapten7-(4-carboxyl-phenylazo)-chlorpromazine (hapten 2) was synthesized and coupled to bovine serum albumin (BSA) as coating antigen to improve the assay sensitivity. The results showed that the hapten-heterologous systems exhibited 20 times higher sensitivity than the hapten-homologous one. Based on the hapten-heterologous system, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for CPZ was developed, the IC50 value was 0.58 ng mL−1 and the limit of detection was 0.03 ng mL−1. The assay showed no cross-reaction with the CPZ analogues. The average recoveries of CPZ from spiked samples were estimated to range from 77.1% to 98.6%, with a coefficient of variation (CV) of less than 10.9%. Linear regression analysis showed a good correlation between the CPZ concentrations obtained from ELISA and HPLC analysis, which suggested that the ELISA is a convenient supplementary analytical tool for monitoring CPZ.
    Analytical methods 12/2011; 3(12):2797-2803. · 1.86 Impact Factor
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    ABSTRACT: An immunizing hapten (4-(carboxymethoxy)phenyl)bis(4-(diethylamino)phenyl)methylium for brilliant green (BG), a triphenylmethane dye with a potential illegal use in fish feeding, was synthesized and used to produce polyclonal antibody (PcAb) against BG. Unexpectedly, the obtained PcAb showed high cross-reactivity (CR) to malachite green (MG) and crystal violet (CV) in an indirect competitive enzyme-linked immunosorbent assay (icELISA). After screening against three heterologous coating antigens, the icELISA exhibited good sensitivity and uniform response to BG (IC(50) of 1.98 ng mL(-1) and CR of 100%), MG (IC(50) of 1.61 ng mL(-1) and CR of 105%) and CV (IC(50) of 1.34 ng mL(-1) and CR of 142%) when using (4-(carboxymethoxy)phenyl)bis(4-(dimethylamino)phenyl)methylium as the coating hapten. Therefore, a broad-specificity icELISA for simultaneous determination of BG, MG and CV was developed. The recoveries of single analyte and mixture of three analytes from spiked grass carp tissues were estimated ranging from 74.94% to 110.39%. A statistically significant correlation of results was obtained between the developed icELISA and previously established HPLC approaches with the food-relevant three triphenylmethane dyes concentration range 1.83-200 ng mL(-1) (R(2)=0.9224), indicating good accuracy of the icELISA and suitability for the broad-specific detection of the three triphenylmethane dyes in grass carp tissues.
    Analytica chimica acta 11/2011; 707(1-2):148-54. · 4.31 Impact Factor
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    ABSTRACT: The development of easy-to-use and rapid-monitoring immunoassay methods for organic environmental pollutants in a class-selective manner is a topic of considerable environmental interest. In this work, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) based on a monoclonal antibody (MAb) with broad-specificity for organophosphorus pesticides (OPs) was applied to the detection of O,O-diethyl and O,O-dimethyl OPs in water samples. The ciELISA conditions were carefully optimized to obtain a three to five-fold improvement of sensitivity for most OPs, and thirteen OPs were determined at concentrations ranging from 0.017 to 30 ng mL(-1). The determination of spiked environmental water samples showed average recoveries from 81.5% to 115.1%, with the coefficient of variation (CV) ranging from 6.1% to 20.9%, which showed satisfactory reproducibility of the developed ciELISA. To overcome the negative aspect of broad-specificity immunoassays not providing qualitative and quantitative analysis of individual OPs in blind samples, we used "percent inhibition rate" to make the developed ciELISA a semi-quantitative method, which allows the monitoring of positive samples from hundreds of negative samples. The determination of OPs in blind water samples by the developed ELISA with confirmation by HPLC-MS/MS analysis demonstrated that the assay is ideally suited as a screening method for OP residues prior to chromatographic analysis.
    Journal of Environmental Monitoring 09/2011; 13(11):3040-8. · 2.09 Impact Factor
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    ABSTRACT: The type of hapten linkage to a carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon and a carboxamido spacer). These haptens were conjugated to bovine serum albumin (BSA) and used as immunogens to produce broad-specificity monoclonal antibodies (MAbs) to organophosphorus pesticides (OPs). Three-dimensional (3D) structures of hapten-lysine conjugates were optimized using molecular modeling (MM) to mimic conformations of hapten-BSA conjugates. The results from MM studies revealed a change of the 3D conformation and electrostatic potential of hapten 1 when the monocarboxylic acid linker was coupled to lysine. This result was consistent with the observed high-cross-reactivity of the corresponding MAb-H1 for the OPs. The competitive indirect enzyme-linked immunosorbent assay based on MAb-H1 is ideally suited to be used as a screening method for OP contaminants.
    The Analyst 06/2011; 136(12):2512-20. · 4.23 Impact Factor
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    ABSTRACT: A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3',6'-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL(-1) and the IC(50) (50% inhibition) value was 164.7 ng mL(-1). The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography-mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure.
    Analytical and Bioanalytical Chemistry 02/2011; 399(6):2275-84. · 3.66 Impact Factor
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    ABSTRACT: A sensitive and specific polyclonal antibody (PcAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for sodium saccharin is described. 6-Amino saccharin was coupled to carrier protein for artificial antigen by diazotisation. New Zealand white rabbits were immunised to obtain anti-sodium saccharin PcAb and then icELISA was developed. The assay showed high sensitivity and specificity to sodium saccharin, with the 50% inhibition value (IC50) of 0.243μgmL−1, workable range (IC30–IC70) of 0.050–12.8μgmL−1 and limit of detection (LOD, IC20) of 0.021μgmL−1. The average recoveries of sodium saccharin in spiked food samples were estimated ranging from 70.7% to 98.8%. A statistically significant correlation of results was obtained between this new ELISA and previously established HPLC approaches with the food-relevant sodium saccharin concentration range 0–320μgmL−1 (R2=0.9887–0.9975). These results indicated that the established ELISA was a potential and useful analytical tool for rapid determination of sodium saccharin residue in food samples.
    Food Chemistry - FOOD CHEM. 01/2011; 126(2):815-820.
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    ABSTRACT: A monoclonal antibody (mAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was used to model two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationships (QSARs) to study antibody recognition. On the basis of insights obtained from the QSAR models, two heterologous coating haptens, 4-(diethoxyphosphorothioylamino)butanoic acid (hapten 2) and 4-(diethoxyphosphorothioyloxy)-2-methylbenzoic acid (hapten 3) were designed, synthesized, and used to develop heterologous ciELISAs with significantly improved sensitivity. The heterologous ciELISA using hapten 2 as the coating hapten showed good sensitivity in a broad-specific manner for eight O,O-diethyl OPs and may be used as a screening method for the determination of these OPs. Our studies demonstrated that molecular modeling can provide insights into the spatial and electronic effects of molecular structures that are important for antibody activity, which can then be used to improve immunoassay sensitivity.
    Analytical Chemistry 10/2010; 82(22):9314-21. · 5.70 Impact Factor
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    ABSTRACT: The hapten of Flumequine (FLU) with four carbon atoms spacer arm (FLUABA) was synthesized and coupled to bovine serum albumin (BSA) for immunogen by the activated ester method. BALB/C mice were immunized with the artificial immunogen and the splenocytes of immunized mice were fused with SP2/0 cells to obtain the monoclonal antibody (McAb). A hybridoma cell line (named DB6-E7) secreting anti-FLU McAbs was obtained by limited dilution method and screened by heterologous enzyme-linked immunosorbent assay (ELISA). The results showed that the subtype of the obtained McAb was IgG1, and the affinity was about 8.19 × 108 M−1. The haptens of FLU, FLUABA, and FLUACA with different space arms were linked to ovalbumin (OVA) for heterologous or homologous coating antigens. The results of indirect ELISA and indirect competitive ELISA indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. Using FLU-OVA as coating antigen, the ELISA showed an IC50 value of 26.33 μg L−1, a limit of detection (LOD) of 4.0 μg L−1, and the workable range of 8–114 μg L−1 (IC20–IC80). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity (< 0.1%) was detected between the obtained McAbs and the several major quinolones compounds or other structural analogs. The developed ELISA can satisfy the detection criteria of FLU in animal food-products.
    Chinese Journal of Analytical Chemistry - CHINESE J ANAL CHEM. 01/2010; 38(3):313-317.