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

Abstract

A specific monoclonal antibody (MAb) against apramycin (AP) was produced and used to develop an indirect competitive enzymelinked 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 (IC 50) 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.

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

“…The strip test procedure and principle used in this study are a slight modification of that reported in earlier studies (Isanga, Tochi, et al, 2017;Luo et al, 2014). Briefly, 50 μL of the sample was added to the conjugate layer containing colloidal gold-labeled antibodies.…”

“…Amikacin is a small molecule lacking immunogenicity so in order to make it immunogenic, it needs to be conjugated to a larger carrier protein or carbohydrate Isanga, Tochi, et al, 2017;Xu et al, 2014). This conjugation can be made possible via one of the four amino or eight hydroxyl functional groups present in the amikacin chemical structure as shown in Figure 2(a).…”

“…Preliminary checkerboard titration experiments (results not shown) were used to determine the optimum concentrations of coating antigen (AMK-EDC-OVA) and 1F11 antibody, which were 0.3 and 0.5 μg/mL, respectively. The other optimized factors were pH and salt concentration, since they significantly affect antibody performance in immunoassay; yet, they vary in different food matrices (Isanga, Tochi, et al, 2017;Tochi et al, 2016). Variation of pH also affects the isoelectric point of the antibody, as with any other protein, hence making it an indispensable factor in assay performance (Yu, Liu, Song, Kuang, & Xu, 2016).…”

“…The glutaraldehyde (GDA) and carbodiimide (EDC) coupling methods reported in previous studies (Isanga, Tochi, et al, 2017;Xu et al, 2014;Xu et al, 2011) were used to prepare the immunogens and coating antigens, as described briefly below.…”

“…An immunochromatographic strip was developed based on a method reported in previous studies (Isanga, Tochi, et al, 2017;Luo et al, 2014). Double-distilled water was used to prepare all solvents which were afterwards filtered using a 0.22-µm membrane.…”