Furaltadone Metabolite Research Articles

Original-structure-based hapten design strategy and development of a derivatization-free gold-based paper lateral-flow immunoassays for detecting furaltadone metabolite in shrimp

Veterinary drug residues in food-producing animals pose a threat to human health and public safety, and accurate measurement of these drug residues is necessary. However, detecting nitrofuran metabolite residues is challenging because of their low molecular weight and the long derivatisation time required for their indirect detection. Current antibodies produced against these metabolites have limited accuracy and sensitivity owing to the traditional and ineffective hapten design based on the reaction of 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) with nitrobenzaldehyde. In this study, a new antigen strategy was adopted using an original-structure-based hapten design combined with a new carrier protein with immunosynergistic effects to produce a monoclonal antibody that could recognise the metabolite of furaltadone (AMOZ) directly without derivatisation with excellent analytical performance. Finally, a gold-based paper lateral flow immunoassay for AMOZ without derivatisation was established with a visual limit of detection of 5 ng/mL, IC50 of 0.97 ng/mL and linear range (IC20∼IC80) of 0.45–2.07 ng/mL. The method was sensitive and accurate, and detection was completed within 10 min. This new antigen design strategy provides a theoretical basis and reference for antibody production of other ultrasmall molecules.

Preparation of Monoclonal Antibody and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Fluorescence-Linked Immunosorbent Assay for Detecting 3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in Edible Animal Tissue.

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1–105.3% and coefficients of variation of 4.7–9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples’ data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.

Massive engineering of spinel cobalt tin oxide/tin oxide-based electrocatalyst for the selective voltammetric determination of antibiotic drug furaltadone in water samples

Furaltadone (FT) is an antibiotic veterinary drug belonging to the nitrofuran group. FT and its metabolite have carcinogenic and mutagenic behavior, and the excreted metabolite of FT has contaminated the environment. Therefore, the determination of FT in the environment is essential. In view of this, we constructed a low-cost electrochemical sensor for the determination of FT based on cobalt tin oxide/tin oxide (Co2SnO4/SnO2, CTO/TO) nanoparticles. The one-pot synthesis of CTO/TO nanoparticles was prepared by a simple co-precipitation method and characterized by various analytical techniques such as PXRD, FT -IR, FE-SEM combined with EDX, HR-TEM and XPS. Subsequently, electrochemical techniques such as EIS, CV and DPV were performed for the detection and determination of FT using CTO/TO nanoparticles modified SPCE. Under optimal conditions, the designed sensor showed good sensitivity (0.66 µA µM-1 cm-2), wider linear ranges (0.1–73 µM and 91–1022 µM) and lower detection limit of 39 nM. The designed FT sensor has been successfully applied to real sample analysis in water samples and the recovery results obtained are very satisfactory. Based on these results, we believe that the as-prepared CTO/TO will be a potential material for FT sensors in environmental applications.

Signal amplified enzyme-linked immunosorbent assay with gold nanoparticles for sensitive detection of trace furaltadone metabolite

Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for detection of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). However, conventional ELISA bears drawback of unsatisfactory sensitivity. Hence, development of high-performance ELISA is of great significance for food safety. We report a signal amplified ELISA strategy for sensitive detection of AMOZ by incorporating gold nanoparticles (AuNPs) into conventional ELISA. In this system, AMOZ competes with bovine serum albumin-AMOZ (BSA-AMOZ) to bind to limited amount of monoclonal antibody; and a large amount of reporter antibody and horseradish peroxidase molecules are loaded onto AuNPs to amplify colorimetric signals. Under the optimized conditions, the signal amplified ELISA achieves a limit of detection of 10−2 ng/mL, which is 500-fold improvement than traditional ELISA in sensitivity. Moreover, pork liver samples spiked with different amount of AMOZ are successfully measured, suggesting this signal amplified ELISA can be used as a general signal enhancement strategy for determination of low-molecular weight contaminants in various foods.

Development of a gold immunochromatographic strip for the rapid detection of 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in catfish

ABSTRACT In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a gold immunochromatographic assay were developed to detect the furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The detection of AMOZ was based on the AMOZ derivative 3-([2-nitrophenyl] methyleneamino)-5-morpholinomethyl-2-oxazolidinone (2-NPAMOZ). The monoclonal anti-2-NPAMOZ antibody 4G11, was prepared and exhibited a 50%-inhibitory concentration (IC50) of 0.049 ng/mL. Its cross-reactivity with structurally related analogues was less than 3%. This immunochromatographic assay was assembled for the rapid screening of AMOZ in catfish samples. The strip assay had a cut-off value of 0.1 μg/kg and a limit of detection (LOD) of 0.009 μg/kg. Analysis of AMOZ in catfish indicated that the results from the strip assay were highly comparable to results obtained using ic-ELISA and LC-MS/MS. Therefore, the colloidal gold (CG) immunoassay is a sensitive screening method for the detection of AMOZ residues in catfish samples.

A fluorescent immunochromatographic strip using quantum dots for 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ) detection in edible animal tissues

ABSTRACTTo monitor the levels of furaltadone in animal tissues, an immunochromatographic strip (immunostrip) based on quantum dot was developed for rapid detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the metabolite of furaltadone. Four monoclonal antibodies (mAbs) against nitrophenyl derivative of AMOZ (2-NP-AMOZ) were generated from the hybridoma cell lines. Among them, the mAb 5C10/3A8 showed the highest sensitivity to 2-NP-AMOZ. Using an immunostrip reader, with the 2-NP-AMOZ as the target, the immunostrip had a liner range from 1 to 30 ng/mL, with 50% inhibition concentration of 5.22 ng/mL and limit of detection of 0.07 ng/mL. The recoveries for AMOZ ranged from 78.6% to 108.9%, with coefficients of variation below 12%. A good correlation (R2 = 0.9901) between the immunostrip and LC–MS/MS results in the tissues tested demonstrated the reliability of the immunostrip. These results indicate that proposed immunostrip is suitable for the rapid and quantitative detection of AMOZ residues in edible animal tissues.

Rapid and sensitive determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone by biotin–streptavidin-amplified time-resolved fluoroimmunoassay

A competitive time-resolved fluoroimmunoassay (TRFIA) using a biotin–streptavidin system was developed for the detection and quantification of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in aquatic tissues. AMOZ-bovine serum albumin was coated to a solid phase and then competed with a free nitrophenyl derivative of AMOZ (2-NP-AMOZ) in standards or samples for binding with biotin-anti-AMOZ-ovalbumin polyantibody. Later, the complex was recognized by europium-labelled streptavidin, which was used as a novel tracer to amplify the signal, and it also reacted faster than europium-labelled second antibody in traditional TRFIA. After optimizing the reaction conditions, the method showed high sensitivity and specification to 2-NP-AMOZ with a half maximal inhibitory concentration of 0.190 μg/l and a sensitivity of 0.019 μg/l with a working range from 0.025 to 10 μg/l. The data from fish and shrimp samples indicated that the limit of detection was 0.021 μg/kg, and the recovery rates were 84.1–107.0% and 80.9–98.4%, respectively, with an average RSD below 10%. High correlation rates were observed in TRFIA, high performance liquid chromatography and universal enzyme-linked immunosorbent assay methods. The experiment above confirms that biotin–streptavidin-amplified TRFIA could be an ultrasensitive and accurate tool for screening large numbers of aquatic products for the determination of AMOZ residues.

Hapten synthesis and production of specific antibody against 3-amino-5-morpholinomethyl-2-oxazolidone for immunoassay without derivatisation

ABSTRACT Immunoassay direct for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), the metabolite of furaltadone, based on a specific antibody was usually unavailable due to its small molecule weight. In this work, a hapten with a double bond and an active carboxyl group was designed and then conjugated to peptide dendrimer to obtain novel multiple haptens. The haptens and multiple haptens were coupled to bovine serum albumin as immunogens. Polyclonal antibodies showed binding to free AMOZ in a heterologous competitive indirect enzyme-linked immunosorbent assay were obtained. The assay showed IC50 (half maximal inhibitory concentration) of 4.1 μg/kg and limit of detection of 0.2 μg/kg for AMOZ. Recoveries of AMOZ from grass carp were tested from 83.1% to 117.0%, with the coefficient of variation below 12%. As no laborious derivatisation procedure was employed, the proposed assay should be useful for the screening of AMOZ for a large number of samples.

Bispecific Monoclonal Antibody-Based Multianalyte ELISA for Furaltadone Metabolite, Malachite Green, and Leucomalachite Green in Aquatic Products.

A new multianalyte immunoassay was designed to screen furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and leucomalachite green (LMG) in aquatic products using a bispecific monoclonal antibody (BsMAb). Gradient drug mutagenesis methods were separately used to prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient. BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid-hybridomas of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3 ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA, 3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced from MG by potassium borohydride was recognized by BsMAb. Recoveries of AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with HPLC analysis. Thus, the multianalyte ic-ELISA is suitable for rapid quantification of AMOZ, MG, and LMG in aquatic products.

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.

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.

An ultrasensitive competitive immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for direct detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in tissue and urine samples

3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) is the metabolite of furaltadone (FTD), one kind of nitrofuran antibiotics which have been banned as veterinary drugs due to the potential harmful effects on human health. AMOZ is stable in tissue matrices, and can be used as an indicator for illegal usage of FTD. In this study, a novel ultrasensitive competitive immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for direct detection of AMOZ in tissue and urine samples was developed. Monoclonal antibody (mAb) against AMOZ and Raman reporter 4-mercaptobenzoic acid (MBA) dual labelled colloidal GNPs (e.g. Ab-Au-MBA) was prepared, characterized and used as an immunoprobe. The specific Raman scattering intensity of MBA on the test line of the ICA strip was measured for quantitative determination of AMOZ. The assay was completed within 15min. The values of IC50 and limit of detection (LOD) of the assay for AMOZ were 0.04ngmL−1 and 0.28pgmL−1, respectively. There was no cross-reactivity (CR) of the assay with other three nitrofurans and five compounds. The ICA strips could be used for 7 weeks without loss of activity. The tissue and urine samples were spiked with AMOZ and the recoveries of AMOZ were in range of 87.8–105.9% with the relative standard deviations (RSD) of 1.4–9.0% (n=3). The results demonstrated that the proposed SERS-based ICA was able to directly detect AMOZ with high sensitivity, specificity, stability, precision and accuracy, and might be as a powerful method for the analysis of other analytes in different matrices.

Synthesis of furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) and novel haptens for the development of a sensitive enzyme-linked immunosorbent assay (ELISA)

A sensitive and specific monoclonal enzyme-linked immunosorbent assay (ELISA) for the determination of tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. Three different haptens of AMOZ were synthesized. The haptens (I, III) were derived from 4-carboxybenzaldehyde and ethyl 4-bromobutyrate, respectively. Hapten II was based on hapten I but a CN bond in the parent structure was reduced. Corresponding immunogens and coating antigens were prepared to improve the assay sensitivity. The hybridomas 4 × 108 secreting antibodies against CPAMOZ were obtained from immunogens I-BSA by monoclonal antibody (mAb) technology. The antibody indicated that: (1) the small molecular AMOZ cannot induce the specific response against AMOZ; (2) the weakened conjugation effect improved the recognition of AMOZ. Assessment of twelve coating antigen/antibody combinations consequently resulted in the development of an indirect competitive enzyme-linked immunosorbent assay (icELISA). The results showed that the sensitivity was highly improved about fifteen-fold for the novel heterologous coating antigen at low hapten density. The IC50 value of the ELISA for CPAMOZ was 0.13 ng mL−1. The cross reactivity values of the assay with NPAMOZ, AMOZ and FTD were 118%, 2.3% and 16.3%. Recoveries from AMOZ fortified animal tissues were in a range of 82.6–108.4% and the CV values were less than 12.5%. The immunoassay was further validated by a LC-MS/MS method and the two methods showed good correlation (r2 = 0.9908). The proposed icELISA could be used for an accurate monitoring of AMOZ in animal tissues.

A novel chemiluminescent ELISA for detecting furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) in fish, egg, honey and shrimp samples

In this study, an indirect competitive enzyme-linked immunosorbent assay with chemiluminescent (CLELISA) detection for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. A monoclonal antibody (MAb) against AMOZ was prepared through immunizing BALB/c mice with 4-carboxybenzaldehyle derivatized AMOZ (CPAMOZ), conjugated with bovine serum albumin (BSA) as antigen. The effects of the substrates luminol, p-iodophenol and urea peroxide on the performance of the assay were studied and optimized. In addition, the specificity of the MAb, estimated as the cross-reactivity values with 4-nitrobenzaldehyde derivatized AMOZ (NPAMOZ), CPAMOZ and AMOZ, was 100%, 27.45% and 0.18%, respectively. The sensitivity of the developed CLELISA was estimated as 50% inhibitory concentration (IC50) value (0.14μg/l) with a linear working range between 0.03 and 64μg/l, and a limit of detection of 0.01μg/l. The CLELISA described in this study was 5-fold more sensitive than the indirect competitive ELISA previously developed in our laboratory. Finally, this new CLELISA was compared with a commercial kit to detect NPAMOZ in spiked fish, shrimp, honey and egg samples. The recovery values from four spiked fish, shrimp, honey and egg samples with different concentrations of NPAMOZ in CLELISA were 92.1–107.7%. Thus, the immunoassay method described here has a broad detection range and high sensitivity and is a valid and cost-effective means for high throughput monitoring of residual AMOZ levels in fish, shrimps, honey and eggs with potential applications in other animal tissues.

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.

Development of a Monoclonal Antibody-Based Competitive Indirect Enzyme-Linked Immunosorbent Assay for Furaltadone Metabolite AMOZ in Fish and Shrimp Samples

A monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ELISA) with improved sensitivity and specificity for the determination of furaltadone metabolite 5-methylamorpholino-3-amino-2-oxazolidone (AMOZ) was described. AMOZ was derivatized with 2-(3-formylphenoxy)acetic acid and coupled with bovine serum albumin to form a novel immunogen. BABL/c mice were immunized and monoclonal antibody specific to the nitrophenyl derivative of AMOZ (NP-AMOZ) was produced and characterized. Four other haptens with different heterology to the immunizing hapten were synthesized and coupled to ovalbumin as coating antigens to study the effect of heterologous coating on assay sensitivity. Under the optimized heterologous coating format, the competitive indirect ELISA showed very high sensitivity to NP-AMOZ, with an IC(50) of 0.14 μg/L and limit of detection of 0.01 μg/L. The assay showed high specificity toward NP-AMOZ, and negligible cross-reactivity with analogous compounds was observed. The average recoveries of AMOZ from spiked fish and shrimp samples were estimated to range from 81.0 to 104.0%, with coefficients of variation below 20%. Good correlation was obtained between the results of ELISA analysis and of standard liquid chromatography-tandem mass spectrometry analysis. These results indicated that the proposed ELISA is ideally suited as a monitoring method for AMOZ residues at trace level.

Novel hapten synthesis for antibody production and development of an enzyme-linked immunosorbent assay for determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

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.

Direct determination of furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidinone, in meats by a simple immunoassay

Furaltadone is a banned drug for use in food producing animals and the marker residue of furaltadone in edible tissues is its metabolite, 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). In this study, a novel polyclonal antibody of furaltadone was produced by using of the conjugate of furaltadone-bovine serum albumin as immunogen. The obtained antibody showed good specificity and sensitivity toward AMOZ with an IC50 value of 2.3 ng/mL. Then, an indirect competitive immunoassay (ELISA) with heterologous coating antigen was developed to directly determine AMOZ in animal meat with a simple sample preparation. The limit of detection for AMOZ in meat was 0.4 ng/g. At three fortification levels, the intra- and inter-assay recoveries from blank samples were in a range of 85.0%–103%. The ELISA method was further confirmed with a HPLC method in which AMOZ was determined after derivatization with 2-naphthaldehyde. The two methods showed good agreement from the analysis of incurred pork samples.

Direct detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in food samples without derivatisation step by a sensitive and specific monoclonal antibody based ELISA

AMOZ (3-amino-5-methylmorpholino-2-oxazolidinone) is the metabolite of furaltadone (FTD) which has been banned as a veterinary drug. In this study, three AMOZ derivatives (CPAMOZ) were synthesised and monoclonal antibody against o-nitrobenzaldehyde derivatived AMOZ (NPAMOZ) was produced. The IC50 value of the ELISA for NPAMOZ was 0.17ngml−1. The cross reactivity values of the assay with NPAMOZ, CPAMOZ, AMOZ and FTA were 100%, 61.7%, 14.8% and 10.4%, respectively. As no NPAMOZ or CPAMOZ exist if no derivitizing reagent added and FTA is quite unstable, therefore, 14.8% of cross reactivity with AMOZ makes the ELISA capable of directly detecting AMOZ without derivatisation. AMOZ spiked food samples were analysed by ELISA. Recoveries of 72.6–121.2% and intra-assay coefficients of variation of 6.1–17.7% were obtained. The proposed ELISA which also confirmed by HPLC with a good correlation provides an alternative sensitive and fast quantitative method for directly detecting AMOZ in food samples.

Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)1Abbreviations used: scFv, single-chain variable fragment; AMOZ, 3-amino-5-morpholinomethyl-2- oxazolidone; NPAMOZ, 2-nitrobenzaldehyde derivative of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidone; VH, heavy chain variable region; VL, light chain variable region; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ni-NTA, nickel nitrilotriacetic acid; icCLEIA, indirect competitive chemiluminescence enzyme immunoassay; PAb, polyclonal antibody; MAb, monoclonal antibody; RAb, recombinant antibody; PCR, polymerase chain reaction; IPTG, isopropyl β-d-1-thiogalactopyranoside; RLU, relative light units.1. Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC50 value of 1.46±0.01ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.