Zhen-Lin Xu

Determination of Furaltadone Metabolite in Fish by Chemiluminescence Enzyme Immunoassay

Abstract An indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) detection for the furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ) was established. The effects of the coating antigen concentration, antibody dilution, immunoreaction time, reaction buffer and its ion concentration on the performance of the assay were studied and optimized. The results showed that the optimized assay conditions were as follows: the coating antigen concentration was 10 ng mL−1, the polyclonal antibody was diluted 60000 times, immunoreaction time was 50 min and the buffer solution was 0.01 M phosphate buffer solution (PBS, pH 7.4). Under the optimized conditions, the linear detection range of the developed icCLEIA was 0.026–3.52 μg L−1, the half inhibitory concentration (IC50) was 0.29 μg L−1 and the limit of detection (IC10) was 0.012 μg L−1. The average recoveries of AMOZ of spiked fish ranged from 101.0% to 115.5%. In conclusion, the icCLEIA is applicable for detection of trace AMOZ in fish samples.

Indirect Competitive Chemiluminescence Enzyme Immunoassay for Furaltadone Metabolite in Metapenaeus Ensis

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-H 2 O 2 -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 IC 50 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 ( R 2 = 0.9997). The established icCLEIA method could be further used for detecting AMOZ in aquatic products samples rapidly and efficiently.

Preparation of Carbon Dots and Their Application in Food Analysis as Signal Probe

Abstract As a new class of fluorescent nanoparticles, carbon dots have potential to replace semiconductor quantum dots due to their excellent optical properties, good biocompatibility, low toxicity, facile synthesis, low cost and easy functionalization. By virtue of characteristics mentioned above, carbon dots-based fluorescent probes showed great potential in the field of food analysis. In this paper, we reviewed the production of carbon dots, their properties and applications in food analysis such as food additives detection, metal ion monitoring, food borne pathogens detection, organic pollutants and food nutritional components analysis. The challenges and perspectives of carbon dots in this exciting and promising field were also discussed.

Indirect Competitive Chemiluminescent Enzyme Immunoassay Method for Determination of Dimethyl Phthalate in Soy Sauce and Liquor

Abstract 4-Amino dimethyl phthalate (ADMP) as hapten was coupled to carrier protein and subsequently used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to dimethyl phthalate (DMP) was obtained, and on the basis of this, an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. The experimental parameters for icCLEIA were optimized as follows: the concentration of coating antigen was 50 μg L −1 , the primary antibody concentration was 92.5 μg L −1 , the secondary antibody concentration was 1 μg mL −1 , ultrapure water (pH 6.0) was used as dilution solution and the time for competitive reaction was 40 min. Under the optimal conditions, the icCLEIA exhibited a linear working range from 0.74 μg L −1 to 30.32 μg L −1 , with the limit of detection of 0.29 μg L −1 . The cross-reactivity with thirteen structural analogues was less than 5%. The recoveries of DMP from spiked liquor and soy sauce samples were from 80.2% to 116% and the average RSD was less than 3.6%. The detection results of spiked liquor and soy sauce by icCLEIA were consistent with those by standard GC-MS method. The developed icCLEIA method exhibited a practical potential for detecting DMP residue in food samples.

Application of Time-resolved Fluroimmunoassay for Determination of Furaltadone Metabolite 3-Amino-5-morpholinomethyl-2-oxazolidinone

Abstract To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in fish sample, a Eu3+ labeling time-resolved fluoroimmunoassay (TRFIA) was developed. The experimental conditions were optimized as follows: the AMOZA-OVA concentration was 0.25 μg mL−1; the antibody was diluted by 50000 folds, and the competitive reaction time was 50 min. Under the optimal conditions, the method showed a detection limit of 0.01 ng mL−1, an IC50 of 0.26 ng mL−1 and a linear range (IC20−IC80) of 0.025−2.83 ng mL−1. The spiked 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 ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suitable for the determination of AMOZ residue in food samples.

Development of an Indirect Competitive Chemiluminescence Enzyme-linked Immunoassay for Parathion

Polyclonal antibody(PcAb) against parathion was raised and used to develop an indirect competitive chemiluminescence enzyme-linked immunoassay(icCLEIA).The hapten was prepared from thiophosphoryl chloride after a three-step substitution reaction.The hapten was coupled to bovine serum albumin(BSA) and ovalbumin(OVA) as immounogen and coating antigen respectively by active ester method.New Zealand rabbits were immunized by the immunogen to obtain anti-parathion polyclonal antibody.Several parameters that might affect icCLEIA performance were carefully optimized.Under the optimum conditions,the linear range of the developed icCLEIA was 0.24-15.83 μg/L,the IC50 was 1.14 μg/L and the limit of detection was 0.09 μg/L.The average recovery of parathion from spiked vegetables and water samples ranged from 74.6% to 121.0%.In conclusion,the icCLEIA is a practical method for trace detection of parathion in real samples.

Displacement Fluorescence Polarization Immunoassay for Herbicide Butachlor

A displacement fluorescence polarization immunoassay(DFPIA) was developed for the detection of herbicide butachlor.The influence of the tracer structure on the assay performance was examined and an immunocomplex of tracer with longest arm was screened.The displacement reaction curves showed a kinetic nature of second-order reaction.The limit of detection(LOD) was 22.6 μg/L with 321.3 μg/L of IC50 and 59.6-1472.1 μg/L of working range.The detection time was only 12.5 min,and the recovery ranged from 88.1% to 112.5%.The immunocomplex was stable for more than one week at 4 ℃.The proposed DFPIA was suitable as a surveillance screening method for the detection of the herbicide butachlor in environmental water samples.

Development of an Enzyme-linked Immunosorbent Assay Method for Detection of Tenuazonic Acid: Development of an Enzyme-linked Immunosorbent Assay Method for Detection of Tenuazonic Acid

To develop an immunoassay method for tenuazonic acid (TeA), two haptens, 2-(2-(1-(5-(sec-butyl)-2-oxo-2,5-dihydro-1H-pyrrol-3-yl) ethylidene)-hydrazinyl) acetic acid (TeAHGA) and 5-(sec-butyl)-3-(1-hydrazonoethyl-4-hydroxy-1H-pyrrol-2 (5H)-one (TeAH) derived from TeA by hydrazine hydrate and glyoxylic acid were synthesized. Then TeAHGA-BSA conjugate was used as immunogen and the specific polyclonal antibody against TeAH was prepared. Furthermore, an indirect competitive ELISA (icELISA) method for TeA with TeAH as target analyte was established. The optimized assay conditions were 0.156 µg/L of heterologous coating antigen (TeAH-OVA, ovalbumin), with PBS as assay buffer, the incubation times for the primary antibody and the secondary antibody were 40 and 20 min, respectively. The icELISA results showed IC50 value, limit of detection (LOD) and linear range as 1.61 ng/mL, 0.08 µg/L and 0.19-12.89 µg/L, respectively. The average recovery rates for standard addition of TeA from tomato and flour samples were 67.2%-89.8% and 74.8%-93.7%, respectively.

Production and Identification of Monoclonal Antibody Against Flumequine and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay

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.