Haitang Zhang

Monoclonal Antibody-Based ELISA and Colloidal Gold Immunoassay for Detecting 19-Nortestosterone Residue in Animal Tissues

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.

Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin.

Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method was employed to synthesize the artificial antigen of enrofloxacin (ENR), and New Zealand rabbits were used to produce anti-ENR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (ELISA) standard curve was established. This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg (R2=0.9567), with the half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 9.4 μg/kg and 0.2 μg/kg, respectively. Of all the competitive analogues, the produced pAb exhibited a high cross-reactivity to ciprofloxacin (CIP) (87%), the main metabolite of ENR in tissues. After optimization, the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork. The overall recoveries and coefficients of variation (CVs) were in the ranges of 86%–109% and 6.8%–13.1%, respectively. It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.

Preparation of Anti-Nortestosterone Antibodies and Development of an Indirect Heterologous Competitive Enzyme-Linked Immunosorbent Assay to Detect Nortestosterone Residues in Animal Urine

The steroid 19-Nortestosterone (NT) has been illegally used in horse racing, dog games to boost physical performance, and also in animal husbandry to accelerate weight gain. This paper reports an indirect heterologous competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody for rapid, sensitive analysis of NT in animal urines. After derivation, NT haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method and mixed-anhydride technology respectively. Two antisera raised in rabbits and two coating antigens synthesized were screened and optimized using homologous and heterologous ELISA formats. The most sensitive heterologous icELISA (antisera generated from NT-17-BSA, with NT-3-OVA as coating antigen) gave an IC50 value of 27 ng/mL, with a dynamic range from 4 to 198 ng/mL, and the limit of detection (LOD) of 1.9 ng/mL. Except for cross-reactivity (CR) toward 17α-Nortestosterone (67%) and β-Boldenone (16.4%), no significant CR was observed for other chemical analogues tested. When applied to the authentic animal urines, the intra- and inter-assay precision values were below 8.4%, while the recoveries were in the range of 95–110%. The correlation coefficient between the established icELISA and LC-MS/MS method was greater than 0.98.

Establishment and Optimization of Monoclonal Antibody-based Heterologous dcELISA for 19-Nortestosterone Residue in Bovine Edible Tissue

This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion procedures, and the development of mAb-based heterologous direct competitive enzyme-linked immunoabsorbent assay (dcELISA) methods to detect NT residue using one of these hybridomas (clone 3B8-E6). Under optimal experimental conditions, this assay exhibited a working range of 0.004 to 19 ng/mL with IC₅₀ and limit of detection values of 0.28 and 0.002 ng/mL, respectively, when it was run in 0.01M phosphate-buffered saline (pH 7.4). Except for minor cross-reactivity with β-boldenone (6.9%) and trenbolone (1.2%), other interference to the assay was negligible (<0.05%). No significant differences (P > 0.05) were found for IC₅₀ values when the pH of the assay buffer ranged from 6 to 8 and phosphate ion concentration was less than 20 mM. The dcELISA can tolerate higher concentrations of methanol than other organic solvents tested. When applied to bovine sample, the correlation coefficients (R) of the dcELISA and GC-MS data were 0.9918 in muscle, 0.9834 in liver, and 0.9976 in kidney. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of NT residue in food.

Analysis of 19-nortestosterone residue in animal tissues by ion-trap gas chromatography-tandem mass spectrometry

A rapid sample treatment procedure for the gas chromatography-tandem mass spectrometry (GC-MS) determination of 19-nortestosterone (19-NT) in animal tissues has been developed. In our optimized procedures, enzymatic hydrolysis with β-glucuronidase from Escherichia coli was performed in an acetate buffer (pH 5.2, 0.2 mol/L). Next, the homogenate was mixed with methanol and heated at 60 °C for 15 min, then placed in an ice-bath at −18 °C for 2 h. After liquid-liquid extraction with n-hexane, the analytes were subjected to a normal-phase solid phase extraction (SPE) C18 cartridge for clean-up. The dried organic extracts were derivatized with heptafluorobutyric anhydride (HFBA), and then the products were injected into GC-MS. Using electron impact mass spectrometry (EI-MS) with positive chemical ionization (PCI), four diagnostic ions (m/z 666, 453, 318, and 306) were determined. A standard calibration curve over the concentration range of 1–20 ng/g was reached, with Y=467 084X-68 354 (R2=0.999 7) for 19-NT, and the detection limit was 0.3 ng. When applied to spiked samples collected from bovine and ovine, the recoveries ranged from 63% to 101% with relative standard deviation (RSD) between 2.7% and 8.9%. The procedure is a highly efficient, sensitive, and more economical method which offers considerable potential to resolve cases of suspected nandrolone doping in husbandry animals.

Preparation and validation of monoclonal antibody-based indirect competitive ELISA for detecting testosterone levels

This paper presents the generation of monoclonal antibodies (mAbs) against testosterone (TES) through cell fusion technology, and the development of a mAb-based indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect TES residue in bovine edible tissues. Under optimal conditions, this assay exhibited a working range of 0.04–19 ng/mL, with half-maximum inhibition (IC50) and limit of detection values of 0.27 and 0.02 ng/mL, respectively. After three sample pretreatment procedures were checked, a simple dilution method was adopted for further use. The stabilisation studies demonstrated that the icELISA kits can be stored for at least 180 to 240 days, at 4°C and −20°C, respectively. When applied to bovine samples, the data from icELISA and gas chromatography coupled to mass spectrometry showed excellent correlation (r2=0.9923 in muscle, 0.9884 in liver and 0.9957 in kidney). Therefore, this assay has the potential to be incorporated into a quantitative monitoring programme for the rapid screening of TES residue in foods.

Production and Characterisation of Monoclonal Antibodies against 19-Nortestosterone

OBJECTIVE To produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. METHODS Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff). RESULTS Five hybridoma cell lines, named NT-1, NT-2, NT-3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG(1) isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7 × 10(9) L/mol. The titers and IC(50) values of purified ascite fluids were in the range of (0.64-2.56) × 10(5) and (0.55-1.0) ng/mL, respectively. Of all the cross-reacting steroids, (-NT was the most reactive with the mAbs at 62% with NT-1 mAb and 64% with NT-2 mAb. Negligible cross-reactivity (<0.01%) with other steroids was observed. CONCLUSION The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.

Development of an enzyme‐linked immunosorbent assay for detection of clopidol residues in chicken tissues

BACKGROUND Clopidol is mainly used for the prevention and treatment of coccidiosis, which poses a serious potential hazard to public health, in veterinary medicine. The aim of this study was to prepare monoclonal antibodies (mAbs) against clopidol (CLOP) and develop an immunoassay for detecting CLOP residues in chicken tissues. After derivation, CLOP hapten was conjugated to carrier proteins to synthesize the artificial antigen, and immunized Balb/C mice were employed to screen mAbs. RESULTS A sensitive hybridoma named C1G3 was screened out and two indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curves were established. For the traditional two-step assay the linear range was from 0.06 to 98 ng mL(-1) , with half-maximal inhibitory concentration (IC50 ) and limit of detection (LOD) values of 2.76 ng mL(-1) and 0.03 ng mL(-1) respectively, while the rapid one-step icELISA had a working range from 0.08 to 102 ng mL(-1) , with IC50 and LOD values of 3.52 ng mL(-1) and 0.03 ng mL(-1) respectively. It was also indicated that a 10-fold dilution in chicken muscles gave an inhibition curve almost the same as that obtained in phosphate-buffered saline. When applied to spiking tests in chicken samples, the correlation coefficient (R(2) ) between concentrations added and measured was 0.9534. CONCLUSION The results of this study suggest that the immunoassay described is a promising alternative for screening CLOP residues in biological matrices and is suitable for routine diagnostics.