ABSTRACT
To produce specific antibodies against malachite green ( MG) , one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay ( icELISA ) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value ( IC50 ) of 0. 96 μg/L, a linear range ( IC20-IC80 ) of 0. 1-8. 1 μg/L and a limit of detection ( LOD, IC10 ) of 0. 05 μg/L for determination of MG. The assay showed cross-reactivity of 18. 1%, 26. 5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0 . 1%) . The average recoveries of MG from spiked fish samples were from 87. 3% to 107. 3%. Good correlation (R2=0. 999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner.
ABSTRACT
To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone ( AMOZ ) in fish sample, an Eu3+ labeling time-resolved fluoroimmunoassay ( TRFIA ) was developed. The effects of experimental conditions including AMOZA-OVA concentration, dilution of antibody, and reaction time on the sensitivity of TRFIA were explored. The results showed that the optimized assay conditions were as follows:the AMOZA-OVA concentration was 0. 25 μg/mL; the antibody was diluted 5í104 folds, and the competitive reaction time was 50 min. Under optimal conditions, the method showed a detection limit of 0. 01 ng/mL, an IC50 of 0. 26 ng/mL and a linear range (IC20-IC80) of 0. 025-2. 83 ng/mL. The recoveries of AMOZ in fish at three spiked levels ranged from 78 . 0% to 86 . 0%, and the relative standard deviations were less than 15%. Good correlation between the ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suited for the determination of AMOZ residue in food samples.
ABSTRACT
4-Amino dimethyl phthalate as the hapten was coupled to carrier protein and then used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to dimethyl phthalate ( DMP) was thus obtained, and on the basis of this, an indirect competitive chemiluminescent enzyme-linked immunoassay ( icCLEIA ) was developed. The experimental parameters of icCLEIA were optimized as follows: the concentration of coating antigen was 50 μg/L, the primary antibody concentration was 92. 5 μg/L, the secondary antibody concentration was 1μg/mL, distilled water (pH 6. 0) was used as diluent solution and the competitive reaction time was 40 min. Under the optimal conditions, the icCLEIA exhibited a linear working range from 0. 74μg/L to 30. 32μg/L with the limit of detection of 0. 29μg/L. The cross-reactivity of thirteen structural analogues was lower than 5%. The recovery of DMP from spiked liquor and soy sauce samples ranged from 80 . 2% to 116 . 0% and the average RSD was less than 3 . 6%. The detection results of the spiked liquor and soy sauce samples were consistent with those by standard gas chromatography-mass spectrometry method. The developed icCLEIA method exhibited a practical potential for detecting DMP residue in food samples.
ABSTRACT
Due to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4-mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative ( hapten) was obtained. The antibody was labeled with horseradish peroxidase ( HRP) and used to develop a direct competitive enzyme-linked immunosorbent assay ( dc-ELISA) . The dc-ELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45. 49 μg/L, a limit of detection of 3. 0 μg/L and the linear range of 9. 2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83 . 6% to 112 . 7%. Good correlations between the results of dc-ELISA and standard HPLC-MS/MS were obtained. The proposed dc-ELISA is suitable for the determination of acrylamide in food samples.
ABSTRACT
The hapten of Flumequine(FLU) with four carbon atoms spacer arm(FLUABA) was synthesized and coupled to bovine serum albumin(BSA) as immunogen using activated ester method. Balb/c mice were immunized by the artificial immunogen and the splenocytes of immunized mice were fused with Sp2/0 cells to obtain the monoclonal antibody(McAbs). A hybridoma cell line(DB6-E7) secreting anti-flumequine McAbs was obtained by limited dilution method and screened by indirect enzyme-linked immunosorbent assay(ELISA) using heterogenous coating antigen. The results showed that the subtype of the McAb was IgG_1, and the affinity was 8.19×10~8 L/mol. The haptens of FLU, FLUABA and FLUACA, with different space arm, were separately linked to ovalbumin(OVA) for heterologous or homologous coating antigen. The results of indirect ELISA and indirect competitive ELISA(icELISA) indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. By heterologous coating antigen(FLU-OVA), the icELISA showed an IC_(50) value of 26.33 μg/L, LOD of 4.0 μg/L, and the workable range of 8-114 μg/L (IC_(20)-IC_(80)). 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 quinolones compounds or other structural similarity compounds. The developed icELISA for FLU can satisfy the detection criteria of flumequine in animal food-products.