Wenxiao Jiang

Simultaneous Determination of 13 Fluoroquinolone and 22 Sulfonamide Residues in Milk by a Dual-Colorimetric Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor two classes of antimicrobial residues in different food matrixes. In this paper, we describe a dual-colorimetric ELISA for the simultaneous detection of 13 fluoroquinolone and 22 sulfonamide residues. The limit of detection for fluoroquinolones and sulfonamides was 2.4 and 5.8 ng/mL, respectively. The developed immunoassay is suitable for high-throughput screening of these low-molecular weight contaminants. This is the first report where two different enzymes (alkaline phosphatase and horseradish peroxidase) were used in one immunoassay and together in a single well for simultaneous detection of multiple low-molecular weight chemical residues.

Development of a sensitive enzyme-linked immunosorbent assay for the detection of fumonisin B1 in maize

Fumonisin B₁ (FB₁) is a mycotoxin, mainly produced by Fusarium fungi and present in food and feed. It causes harmful effects on human and animal health. Therefore, it is necessary to develop sensitive and reliable screening methods. In this study, a highly sensitive monoclonal antibody (MAb) against FB₁, clone 2D7, was produced, and the 50% inhibition concentration (IC₅₀) of the MAb was 2.2 ng/mL in buffer. The MAb showed high cross-reactivity with fumonisin B₂ (FB₂), and negligible cross-reactivity with other mycotoxins. A sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this MAb was developed for the determination of FB₁ in maize. In spiked samples (100, 200 and 500 μg/kg), the average recoveries ranged from 78 ± 11 to 107 ± 4%, and the coefficient of variation ranged from 3 to 15%. The limit of detection of the icELISA was 5.4 μg/kg. This method was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) using naturally contaminated samples, and the correlation coefficient was above 0.82. These results show the reliability of the icELISA method for the determination of FB₁ in maize.

Development of a multiplex flow-through immunoaffinity chromatography test for the on-site screening of 14 sulfonamide and 13 quinolone residues in milk

In this paper, a rapid and sensitive multiplex flow-through immunoaffinity chromatography test (FTIACT) was developed for the on-site screening of 14 sulfonamide and 13 quinolone residues in milk. The developed FTIACT method combines the purification, preconcentration and immunochemical detection of multiple antibiotics on the sepharose gel test layers. The use of liposome-encapsulated quantum dots (LQDs) with the FTIACT method exhibited the best results, with limits of detection (LODs) of 1 and 0.5ng/mL for the sulfonamides (SAs) and quinolones (QNs), respectively, through qualitative analysis (visual detection by the naked eye). In order to achieve low detection limit, the color intensity of the images were converted into relative optical density values to enable a quantitative evaluation. Quantitative analysis of the samples enabled the detection of SAs (0.13ng/mL) and QNs (0.062ng/mL) in spiked milk samples. The FTIACT described in this work shows promise as a multiplex immunoassay for the qualitative and quantitative screening of multiple chemical residues in milk.

Multi-residue analysis of veterinary drugs, pesticides and mycotoxins in dairy products by liquid chromatography–tandem mass spectrometry using low-temperature cleanup and solid phase extraction

A multi-class multi-residue analysis method for determination of veterinary drugs, pesticides and mycotoxins in dairy products by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been established. These 17 classes, a total of 40 kinds of target compounds were chosen because their administration to food-producing animals is banned or regulated in China and may be potentially abused or misused. Samples were extracted with acetonitrile-ethyl acetate-acetic acid (49.5+49.5+1, v/v/v). Most of lipids in the extract were removed by low-temperature cleanup, prior to solid phase extraction on HLB cartridges. The quantification and confirmation of the 40 analytes were performed by LC-MS/MS with electro-spray ionization (ESI) interface in multiple reaction monitoring (MRM) mode. The limits of detection (LODs) and limits of quantification (LOQs) were 0.006-0.3μg/kg and 0.02-1.0μg/kg, respectively. The spiked recoveries in milk, yogurt, milk powder and cheese samples were from 67.3% to 106.9%. The repeatability and the within-laboratory reproducibility were less than 12.7% and 13.9%. Applying this method, our results revealed the presences of chloramphenicol, cimeterol, and flunixin at the concentration of 0.027-0.452μg/kg in some samples.

Simultaneous screening analysis of 3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid residues in edible animal tissues by a competitive indirect immunoassay.

Immunoassays contribute greatly to food safety. Yet there are no reported immunoassays that simultaneously detect MQCA and QCA, the marker residues for olaquindox and carbadox, respectively. Here, a broad-specificity mAb was successfully produced, and the mAb showed good cross-reactivity with both MQCA and QCA, having IC50 values in buffer of 4.8 and 9.6 ng/mL, respectively. The calibration curves ranged from 0.3 to 81 μg/kg. The average recoveries ranged from 76% to 108% at different spiked levels (2, 4, and 8 μg/kg for MQCA; and 4, 10, and 20 μg/kg for QCA), and the intra-/interday coefficients of variation were 4.2-13.3%. The limits of detection of MQCA and QCA in chicken, fish, pork, and shrimp were 1.76, 1.32, 1.90, and 1.18 μg/kg, respectively. This method was verified by LC-MS/MS, with a correlation coefficient above 0.98. The immunoassay was rapid and reliable for simultaneous screening analysis of MQCA and QCA residues.

Technical note: Development of an enzyme-linked immunosorbent assay for the determination of florfenicol and thiamphenicol in swine feed

One polyclonal antibody against florfenicol and thiamphenicol was produced and a competitive ELISA was developed for the detection of florfenicol and thiamphenicol in swine feed. The ELISA gave a 50% inhibiting concentration of 1.02 ng/mL for florfenicol. For swine feed fortified with 0.05 to 3.0 mg/kg, the interassay recoveries of florfenicol and thiamphenicol ranged from 86.4 to 118.6%, whereas intraassay recoveries of both drug ranged from 90.1 to 126.5% with less than 15% CV. Results obtained from HPLC-tandem mass spectrometry indicated this ELISA procedure could be used as a convenient method for rapid screening of florfenicol and thiamphenicol in swine feed.

Analysis of Pirlimycin Residues in Beef Muscle, Milk, and Honey by a Biotin–Streptavidin-Amplified Enzyme-Linked Immunosorbent Assay

Food contamination by veterinary drug residues is a worldwide public health concern and requires continuous monitoring. In this study, we developed a biotin-streptavidin-amplified ELISA (BA-ELISA) using a produced monoclonal antibody for detecting pirlimycin residues in beef muscle, milk, and honey. The IC50 value of the BA-ELISA was 1.6 ng/mL for pirlimycin in buffer, and the sensitivity was improved 3 times compared to traditional ELISAs. The optimized BA-ELISA can be used to quantitate trace amounts of pirlimycin residues in beef muscle, milk, and honey. This method had limits of detection (LODs) of 4.45 μg/kg in beef muscle, 1.65 μg/L in milk, and 2.75 μg/kg in honey. The average recovery of the BA-ELISA ranged from 78 to 97%, and the coefficient of variation ranged from 5.3 to 13.5%. The developed BA-ELISA method was validated using LC-MS/MS, and the BA-ELISA can be used for routine screening analysis of pirlimycin residues.

Production of Monoclonal Antibody and Development of a New Immunoassay for Apramycin in Food

Apramycin (APR) residue in food of animal origin can cause harmful effects on human health. In this study, a monoclonal antibody (mAb) was successfully produced using APR-BSA as immunogen, which was prepared by using the glutaraldehyde method. mAb 2A2 showed low cross-reactivity (<0.1%) with other aminoglycoside antibiotics, and its IC50 value was 0.35 ng/mL. On the basis of this mAb, a novel immunoassay in the format of an immunoaffinity test column (IATC) was developed. An immunoaffinity column filled with anti-APR antibody-Sepharose 4B gel was used as solid phase. APR in sample and HRP-APR conjugate compete with each other for the limited antibody on the column. The approach was able to give a naked-eye color signal for the detection of analyte. A blue color appears for negative results and no color for positive. The method was then successfully applied to the detection of APR in animal-origin food. To further evaluate the assay, direct competitive ELISA (dcELISA) based on the same antibody was developed for comparison in different aspects. Compared to the dcELISA, the detection time of IATC is shortened to 20 min, whereas a similar sensitivity for various samples was observed. The limits of detection (LOD) for raw milk, muscles, and livers are 3 ng/mL, 3 μg/kg, and 10 μg/kg, respectively.

Monoclonal antibody production and the development of an indirect competitive enzyme-linked immunosorbent assay for screening spiramycin in milk.

To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 μg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 μg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

Development and Application of a Gel-Based Immunoassay for the Rapid Screening of Salbutamol and Ractopamine Residues in Pork.

Salbutamol (SAL) and ractopamine (RAC) have been illegally used to promote protein synthesis and to increase the feed conversion rate in livestock. However, the residues of SAL and RAC could cause potential hazards for human health. The Ministry of Agriculture of China banned the use of SAL and RAC as growth promoters. In this paper, we provide detailed information on developing a rapid and sensitive gel-based immunoassay for on-site screening of SAL and RAC residues in pork. The detection time was shortened to 20 min. The limits of detection were 0.5 μg/kg for both SAL and RAC by visual detection, whereas the quantitative gel-based immunoassay enabled the detection of SAL (0.051 μg/kg) and RAC (0.020 μg/kg) in spiked pork samples. The gel-based immunoassay showed promise as a multiplexed immunoassay for on-site surveilling of SAL and RAC residues in pork.