Tao Le

Dual-label quantum dot-based immunoassay for simultaneous determination of Carbadox and Olaquindox metabolites in animal tissues

A novel and reliable dual-label direct competitive fluorescence-linked immunosorbent assay (dc-FLISA) based on quantum dots (QDs) was developed for the simultaneous determination of the major metabolites of Carbadox and Olaquindox residues in animal tissues, using anti-QCA monoclonal antibodies and anti-MQCA polyclonal antibodies labeled with QD520 and QD635, respectively. The limits of detection for QCA and MQCA were 0.05 and 0.07ng/ml, respectively. The method was used to analyze fortified samples and analyte recoveries ranged from 81.5% to 98.2% (QCA) and 84.2% to 95.7% (MQCA). Good correlations between the dc-FLISA method and HPLC were demonstrated for the determination of QCA and MQCA residues in swine tissue samples, confirming the reliability of the proposed method.

Development of an immunochromatographic assay for detection of tylosin and tilmicosin in muscle, liver, fish and eggs

An immunochromatographic assay (ICA) was developed and applied for the residue determination of tylosin and tilmicosin in muscle, liver, fish and eggs. The assay is based on a competitive format, and its sensitivity is improved by using a novel criterion to screen the optimal amount of nanogold-labelled monoclonal antibody. Owing to the novel nanogold particle labelling method, 10 min was sufficient to perform the ICA, the visual detection limits were 10 and 20 ng/g for tylosin and tilmicosin, respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 0.1 to 100 ng/mL, with IC50 values of 2.9 and 4.1 ng/mL for tylosin and tilmicosin, respectively. The recovery rates ranged from 71.5 to 103.2%, with coefficient of variation <14.1%, when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 10–100 ng/g. The results of ICA were consistent with those of ELISA kit and high-performance liquid chromatography in the analysis of tylosin in the incurred tissues, demonstrating the practical applicability of the developed assay in real samples. Over all, to our knowledge, this is the first report of qualitative and semi-quantitative residue determination for tylosin and tilmicosin by ICA.

Development and validation of an immunochromatographic test strip for rapid detection of doxycycline residues in swine muscle and liver

A one-step lateral flow immunochromatographic technique using colloidal gold-labelled monoclonal antibody (Mab) was developed for the rapid detection of doxycycline (DOX) residues in swine tissues. For this purpose, a Mab against DOX named as 2.3/3A6 with low cross-reactivity (CR) to other tetracyclines (TCs) was produced. The sensitivity of the test strip was as low as 7 ng/mL for DOX and the half maximal inhibitory concentration (IC50) was calculated to be 22±2 ng/mL by relative optical density. The test can be completed within 10 min and the detection limit was 20 ng/mL in unaided visual assessment. Recoveries in samples spiked with DOX at concentrations of 20, 40 and 80 ng/g, were demonstrated to be from 81% to 95% for muscle samples and from 81% to 92% for liver samples. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 3% to 6% and from 4% to 8%, respectively. Comparing with high performance liquid chromatography (HPLC) method in sensitivity and accuracy for the detection of DOX in swine tissues, the test strip showed good agreement. Therefore, the test strip is suitable for rapid and reliable determination of DOX residues in swine tissues semi-quantitatively or qualitatively.

Development of a Time-Resolved Fluoroimmunoassay for the Rapid Detection of Methyl-3-Quinoxaline-2-Carboxylic Acid in Porcine Tissues

A time-resolved fluoroimmunoassay for the specific determination of methyl-3-quinoxaline-2-carboxylic acid in animal tissues, a marker residue of olaquindox, was developed. The IC50 of the assay was found to be 1.46 ± 0.19 ng/mL of methyl-3-quinoxaline-2-carboxylic acid in phosphate-buffered saline samples and the detection limit was 0.16 ± 0.03 ng/mL. For porcine liver and muscle samples spiked with 5, 10, and 15 ng/g, the recovery ranges were 95.7–112.3% and 98.5–116.2% and the coefficients of variation were 9.3–11.5% and 8.9–14.2%, respectively. The time-resolved fluoroimmunoassay results correlated well with high performance liquid chromatography results (correlation coefficients of 0.991 for liver and 0.988 for muscle). This study suggests that this method is simple, fast, and sensitive for the high-throughput determination of methyl-3-quinoxaline-2-carboxylic acid in animal tissues.

Development and validation of an enzyme-linked immunosorbent assay for rapid detection of multi-residues of five quinoxaline-1,4-dioxides in animal feeds

In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for the simultaneous quantitative determination of five quinoxaline-1,4-dioxides (olaquindox, carbadox, mequindox, quinocetone and cyadox) in porcine and chicken feeds. Polyclonal antibodies (PAbs) with group-specificity to quinoxaline-1,4-dioxides were raised in rabbits after immunization with quinoaxline-2-carboxylic acid-para-aminobenzoic-bovine serum albumin conjugate (QCA-PABA-BSA). The specificity of anti-sera was determined by competitive indirect ELISA (ciELISA), and the 50% inhibitions (IC50) of quinocetone (QCT), cyadox (CYA), carbadox (CBX), mequindox (MEQ) and olaquindox (OLA) were 8.6, 13.1, 17.3, 24.5 and 26.4 ng ml−1, respectively. The limit of detection (LOD) for the QCT, CYA, CBX, MEQ and OLA were as low as 0.41, 0.59, 0.67, 0.80 and 0.86 ng g−1, respectively. On the basis of PAbs strong cross-reactivity to QCT (100%), CYA (49.9%), CBX (66.2%), MEQ (35.3%) and OLA (32.8%), no cross-reactivity was found with other antibiotics, indicating that the assay displayed not only high-sensitivity but also high-specificity. When used to analyse animal feed samples, acceptable recovery rates of 71.6–92.9% and coefficients of variation of 4.1–10.8% were obtained. The ciELISA for 10 spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9959 (n=10). Therefore, the optimised ciELISA may be a potential analytical tool for monitoring quinoxaline-1,4-dioxide residues in animal feed.

Development of a quantum dot-based immunochromatography test strip for rapid screening of oxytetracycline and 4-epi-oxytetracycline in edible animal tissues.

ABSTRACT A rapid and sensitive immunochromatographic test strip (ICTS) was developed in a competitive format with the quantum dot-conjugated monoclonal antibody specifically to determine the residues of oxytetracycline (OTC) and its metabolite 4-epi-oxytetracycline (4-epi-OTC). Using an ICTS reader, the 50% inhibition concentration (IC50) was found to be 3.41 ± 0.29 ng ml–1 of OTC in phosphate-buffered saline samples and the detection limit was 0.44 ± 0.05 ng ml–1. The visual cut-off level of the ICTS is 25 ng ml–1. The recoveries from tissue samples spiked with OTC of 50–600 μg kg–1 were 74.2–107.2%, with coefficients of variation below 15%. The method was used to analyse incurred tissue samples, and there was good correlation (R2 = 0.999) between the ICTS and high-performance liquid chromatography. These results indicate that ICTS is suitable for the rapid and quantitative detection of OTC and 4-epi-OTC residues in edible animal tissues.

Detecting quinoxaline-2-carboxylic acid in animal tissues by using sensitive rapid enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluoroimmunoassay (TR-FIA) based on an anti-N-butylquinoxaline-2-carboxamide (BQCA) monoclonal antibody were standardized and validated for quinoxaline-2-carboxylic acid (QCA) screening in animal tissues and its performance were compared to HPLC. The sensitivities obtained for edible tissue extracts were 1.62 and 1.12 ng ml(-1) for ic-ELISA and TR-FIA detection, respectively. Two samples were spiked with QCA and analyzed by both methods. The recovery values ranged from 92.6% to 112.2% and the coefficients of variation were less than 15% for QCA spiking into swine tissue samples at concentrations of 2.5-50.0 μg kg(-1). Excellent correlations (r(2)=0.987-0.996) of the ic-ELISA/HPLC and TR-FIA/HPLC data were observed for processed samples. The results demonstrated that the ic-ELISA and TR-FIA methods were rapid and accurate for the residue detection of QCA in animal tissues.

A fluorescent immunochromatographic strip test using a quantum dot-antibody probe for rapid and quantitative detection of 1-aminohydantoin in edible animal tissues

AbstractA rapid, simple, and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) has been developed to detect 1-aminohydantoin (AHD), a major metabolite of nitrofurantoin in animal tissues. To achieve this, QD-labeled antibody conjugates, which consist of CdSe/ZnS QDs and monoclonal antibodies, were prepared by an activated ester method. Under optimal conditions, with the nitrophenyl derivative of AHD as the target, the ICST had a linear range from 0.1 to 100 ng/mL, with a correlation coefficient of 0.9656 and a 50% inhibitory concentration of 4.51 ng/mL. The limit of detection was 0.14 ng/g, which was below the minimum required performance limit of 1 μg/kg for AHD established by the European Commission. The recoveries for AHD ranged from 81.5% to 108.2%, with coefficients of variation below 13%, based on intraday and interday analysis. Furthermore, for AHD in real samples, the ICST showed high reliability and high correlation with liquid chromatography–tandem mass spectrometry (correlation coefficient of 0.9916). To the best of our knowledge, this is the first report of a novel and sensitive method based on a fluorescent ICST to detect AHD below the minimum required performance limit. The ICST demonstrated high reliability, and could be ideally suited for rapid, simple, and on-site screening of AHD contamination in animal tissues. Graphical abstractA rapid, simple, and sensitive fluorescent immunochromatographic strip test that is based on quantum dots was developed to detect 1-aminohydantoin (AHD), a major metabolite of nitrofurantoin in animal tissues. 2-NBA 2-nitrobenzaldehyde, NP nitrophenyl

An immunochromatography test strip for rapid, quantitative and sensitive detection of furazolidone metabolite, 3-amino-2-oxazolidinone, in animal tissues

ABSTRACT 3-amino-2-oxazolidinone (AOZ) is the metabolite of furazolidone, which has been banned as a veterinary drug due to the potential harmful effects on human health. In this study, a novel immunochromatography test strip (ICTS) based on colloidal gold–McAb probe for detection of AOZ within tissue samples was developed. With this method, the visual cut-off levels for the test strip was achieved at 10 μg/L. Using a test strip reader, the 50% inhibition was 1.3 μg/L. The limit of detection and limit of quantification in four kinds of animal tissues were 0.15 μg/kg and 0.31 μg/kg, respectively. When used to analyze fortified samples of animal tissue, acceptable recovery rates of 76.3–98.4% were obtained. The fortified samples were detected by ICTS and confirmed by LC-MS/MS. The results of the two methods were in good agreement. The proposed ICTS was demonstrated to be a rapid and simple analytical method for detecting AOZ in animal tissue.

Development of monoclonal antibody-based ultrasensitive enzyme-linked immunosorbent assay and fluorescence-linked immunosorbent assay for 1-aminohydantoin detection in aquatic animals

HighlightsThis is the first report of QDs‐FLISA to detect AHD developed based on a specific monoclonal antibody.The FLISA offers higher sensitivity in comparison with ic‐ELISA.Excellent correlations of the ic‐ELISA/LC–MS/MS and FLISA/LC–MS/MS data were observed for processed samples. ABSTRACT Monitoring and rapid evaluation of nitrofurantoin metabolite, 1‐aminohydantoin (AHD), are important for food safety and human health. Herein, we established the monoclonal antibody‐based indirect competitive enzyme‐linked immunosorbent assay (ic‐ELISA) and quantum dots (QDs)—fabricated fluorescence‐linked immunosorbent assay (FLISA). Monoclonal antibody specific to nitrophenyl derivative of AHD was derived from hybridoma cell lines 3.2.4/5A8. For another, CdTe core QDs with emission wavelength of 605 nm were also synthesized. The performances of the proposed ic‐ELISA and FLISA were further examined and the corresponding results were also validated by standard LC–MS/MS analysis. The obtained results indicated that both ic‐ELISA and FLISA exhibited good dynamic linear detection for NPAHD over the range from 0.1 to 3.0 ng mL−1. Meanwhile, proposed immunosorbent assays are characterized by satisfactory recovery rates of 81.5–113.7%. The experimental data suggested these two immunoassays could be facile, cost‐effective and rapid tools for the prospective quantitative method for AHD analysis in food matrix.