Development of a monoclonal antibody assay and a lateral flow strip test for the detection of paromomycin residues in food matrices

A monoclonal antibody (MAb) was produced against paromomycin and used in the development of an immunoassay and a lateral flow strip test for the detection of paromomycin residues in food matrices. The MAb had 25.1% cross-reactivity with neomycin, but was insensitive to other aminoglycosides. Tested under optimized conditions in 0.01 M phosphate-buffered saline, the half maximum inhibitory concentration (IC₅₀) of the MAb was 0.61?ng/ml for paromomycin and 2.43?ng/ml for neomycin, with results obtained within 90?min. The mean recoveries from spiked food matrices were within the range of 64.56–105.85% for paromomycin and 54.08–100.55% for neomycin. The strip test results for different food matrices were obtained within 5?min and showed visual detection limits of 2.5–20?ng/ml (µg/kg) for paromomycin and 10–30?ng/ml for neomycin. Therefore, the developed immunoassay and strip test can be used in food analysis for routinely monitoring not only paromomycin but also neomycin residues.     

Development of an immunochromatographic assay for hexestrol and diethylstilbestrol residues in milk

Hexestrol (HES) and Diethylstilbestrol (DES) are synthetic hormones, which have been found abuse use in animal-origin food production. We developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immune-chromatographic strip to detect these compounds based on monoclonal antibody. Under optimized conditions, the half-inhibition concentration (IC₅₀) values of ic-ELISA were 0.15 µgL^?1 and 0.23 µgL^?1 for HES and DES, respectively. Spiked milk samples were analyzed. The recovery of both synthetic hormones in the milk samples was 60.48–102.19% (HES) and 89.34–100.16% (DES). In immune-chromatographic assay, the visual cutoff values at 0.5 and 1.0?µgL^?1 respectively for HES and DES, which allow us to detect milk samples of a concentration low to 10 µgL^?1 for HES and 15 µgL^?1 for DES.     

Development of monoclonal antibody and lateral test strip for sensitive detection of clenbuterol and related β2-agonists in urine samples

Being the most used and preferred β₂-agonist, clenbuterol (CLB) has been reported to be illegally abused in animal production and breeding as it improves growth rate while inhibiting fat synthesis and deposition leading to buildup of lean muscle. Due to its potential hazard to human health, causing food poisoning that can be fatal, it has been forbidden as a growth promoter for food-producing animals in most countries including China. Therefore, frequent monitoring of CLB abuse is necessary for consumer safety against its residues. We therefore report development of a monoclonal antibody and lateral flow test strip for sensitive detection of CLB in urine samples. In this investigation, male BALB/C mice were first immunized for antibody production. Thereafter, gold nanoparticles were utilized for antigen and antibody immobilization during construction of the strip. Under optimized conditions, the cut-off limit of the test strip was found to be 2.5 and 5?ng/g in phosphate-buffered saline (PBS) and urine using the wet method. When the dry method was used for CLB detection, the cut-off limit of the test strip in PBS and urine was found to be 3?ng/g. Each test was evaluated within 6?min. The data obtained show that the strip developed is sensitive and has an advantage of fast test results and simplicity and hence can be adopted for screening of CLB residues in urine samples for mitigation of CLB residue effects to human health.     

Monoclonal antibody for the development of specific immunoassays to detect Enrofloxacin in foods of animal origin

The carbodiimide active ester method was employed to synthesise the antigen of 1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid [Enrofloxacin (ENFX)], and male Balb/c mice were used to produce anti-ENFX monoclonal antibody. Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay standard curve was established. This assay was sensitive and highly specific to ENFX with the half maximal inhibitory concentration and limit of detection values of 0.15?ng?mL^?1 and 0.028ng?mL^?1, respectively. Similarly, the antibody had a high affinity as seen in A_max values. Spiked cow milk beef liver and fish samples analysed using this kit had recoveries in the range of 90.2–110.2%, 70.7–81.5% and 89.1–101%, respectively, showing satisfactory results. The results suggest that this ELISA kit could be applied as a screening method to detect and control the illegal content of ENFX in food products.     

Development of a monoclonal antibody assay and immunochromatographic test strip for the detection of amikacin residues in milk and eggs

An amikacin-sensitive monoclonal antibody (MAb) assay and immunochromatographic test strip were developed and applied for the detection of amikacin residues in bovine milk and chicken eggs. The immunoassay was specific to amikacin and showed no cross-reactivity with other aminoglycosides. The half maximum inhibitory concentration (IC₅₀) of the assay was 0.65?ng/mL and the results were obtained within 90?min. Recoveries from spiked food matrices were within the range of 73.55–84.61% for bovine milk and 73.70–105.75% for whole egg. The strip test results were obtained within 10?min and showed a visual detection limit of 5.0?ng/mL for both food matrices. These results show that the MAb immunoassay and strip test developed in this study are very specific to amikacin and sufficiently sensitive for detection and routine monitoring of amikacin residues in food.     

Development of an indirect enzyme-linked immunosorbent assay and lateral-flow test strips for pefloxacin and its analogues in chicken muscle samples

An anti-pefloxacin (PEF) monoclonal antibody (mAb), an indirect competitive enzyme-linked immunosorbent assay and lateral-flow test strip methods were developed to detect fluoroquinolone (FQ) residues in chicken muscle samples. Under optimised conditions, the anti-PEF mAb showed reasonable cross-reactivity with nine FQs with a limit of detection of 0.082?ng/mL assayed in 0.01?M phosphate buffered saline (PBS) solution. The intra- and inter-assay recoveries from spiked samples were within the range of 62.42–111.47% and 63.5–113.79%, respectively. The visual cut-off values of the lateral flow test strip in 0.01 M PBS and in food matrices were within the range of 2.5–50?ng/mL and 5–100?µg/kg, respectively. These results show that the anti-PEF mAb immunoassay and lateral flow test strip methods are suitable for simultaneous detection and routine monitoring of FQ residues in food.     

Rapid and sensitive immunoassays for the detection of lomefloxacin and related drug residues in bovine milk samples

The misuse and illegal use of fluoroquinolones (FQs) in animal-based food products have drawn considerable attention in several countries. As a result, there has been an increased demand for efficient detection methods of FQs in food products. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic strip device based on a monoclonal antibody against lomefloxacin (LFLX), a second generation FQ. Ic-ELISA had an IC₅₀ value of 0.19?ng/mL with a limit of detection of 0.04?ng/mL in 0.01?M phosphate-buffered saline (PBS). The intra- and inter-assay recovery rates of LFLX in bovine milk samples were 98.02–107.40% and 100.65–107.82%. The immunochromatographic assay of LFLX in PBS and spiked bovine milk samples had visual cutoff values at 1 and 5.0?ng/mL, respectively, with significant cross-reactivity with norfloxacin and enoxacin. The developed ic-ELISA and strip method may assist in the detection of FQs in foods.     

Development of a specific monoclonal antibody assay and a rapid testing strip for the detection of apramycin residues in food samples

A specific monoclonal antibody (MAb) against apramycin (AP) was produced and used to develop an indirect competitive enzyme-linked immunosorbent assay (idcELISA) and a rapid testing strip for the detection of AP residues in foods. MAb exhibited negligible cross-reactivity with other aminoglycosides. Under optimized conditions in 0.01 M PBS, the half maximum inhibitory concentration (IC₅₀) of MAb was 0.41?ng/ml with a limit of detection (LOD) of 0.15?ng/ml. The ELISA results were obtained within 90?min. The mean recoveries from all the spiked food samples were within the range of 79.02–105.49%, with coefficients of variation in the range of 2.21–11.4%. The strip test results obtained within 5?min had visual LODs in the range 2.5–5?µg/kg (ng/ml) for all food samples tested. Therefore, the developed strip test represents a fast and convenient detection method of AP residues in foods.