Cephalexin Residue Detection in Milk and Beef by ELISA and Colloidal Gold Based One-Step Strip Assay

Abstract

An evaluation of a rapid enzyme-linked immunosorbent assay (ELISA) and colloidal gold based one-step strip assay for cephalexin (CEX) residue detection in milk and beef is described. A monoclonal antibody (mAb) against CEX was produced using cephalexin-bovine serum albumin (CEX-BSA) conjugate as the immunogen, which exhibited no cross-reactivity with applied chemicals in the studied concentration range. The detection limit of rapid ELISA was calculated as 0.39 microg/kg in PBS and 19.5 microg/kg in beef and milk, which was quite lower than the European Union Maximum Residue Limit (MRL) of 100 microg/kg in milk and 200 microg/kg in muscle. Spiked samples were detected with a mean recovery of 82.8-124% and coefficient of variation of 4.88-25%, which indicated a good agreement with the spiked concentration. Accuracy and reproducibility were determined using spiked samples with four different final concentrations of 1, 2, 5, and 10 microg/kg of CEX (n = 7). Mean intra-assay variation of 6.67% and inter-assay variation of 10.66% were obtained. In contrast, the strip test for CEX had a visual detection limit of 0.5 microg/kg, which could be evaluated within 3-10 min. However, positive samples should be further quantified by more sensitive and accurate competitive indirect ELISA method. In conclusion, the described strip test is rapid, simple, and cost-effective as well as sensitive and specific enough for reliable and accurate on-site screening.

Identification of Ancient Silk Using an Enzyme-linked Immunosorbent Assay and Immuno-fluorescence Microscopy

“…[10][11][12][13][14][15][16][17] In recent years, immunological techniques have attracted increasing attention by those working in cultural heritage. For example, milk residues, 18,19 protein-bound materials and organic bones have been detected, [20][21][22][23][24][25] and all of these studies have demonstrated that immunoassays have the potential to identify and localize the proteins in archaeological materials both rapidly and effectively. However, most of the antibodies used in these immunoassays are readily commercially available "off-theshelf", and the need for tailored antibodies for targeted cultural heritages with high sensitivity and specificity is increasingly desirable.…”

A broadly applicable approach to prepare monoclonal anti-cephalosporin antibodies for immunochemical residue determination in milk

“…Antibody production Different approaches have been described to produce immunogenic cephalosporin conjugates, including the use of bifunctional coupling reagents such as glutaraldehyde [24,26,31], o-phthaldialdehyde [35] and carbodiimide [23,25,27,29] or heterobifunctional NHS-ester cross-linking reagents [36]. In most cases the cephalosporin-protein conjugation was performed at physiological or slightly acidic pH conditions to avoid cleavage of the beta-lactam ring.…”

“…3 A comparison of our results with previously published methods for cefquinome, ceftiofur/desfuroylceftiofur and cephalexin reveals that our mAb-based EIAs are at least similar or even better in sensitivity. Most data are available for cephalexin, and reported IC 50 values range from 0.6 to 6 ng mL −1 [24,26,29,37]. Based on mAbs against desfuroylceftiofur, Rose et al [30] established an EIA for the detection of ceftiofur, the IC 50 value was at 0.33 ng mL −1 .…”

“…However, only a few studies so far dealt with the production of specific antibodies against cephalosporins. Most authors focussed on polyclonal [23][24][25][26][27] or monoclonal [28,29] antibodies against cephalexin, while only each one study reported the production of polyclonal [26] or monoclonal [30] antibodies against ceftiofur. Recently, a rabbit antiserum enabling the specific detection of cefquinome has been described [31].…”

Surface Plasmon Resonance Biosensors for Highly Sensitive Detection of Small Biomolecules

“…To help address these issues, there has been much research devoted to the development of immunochromatographic test strip technology (Weller, 2000). This technology has been utilised for detection of drug residues (Chen et al, 2009) and drugs of abuse by transferring the immunoassay process onto a single-use test strip that generates a colour response after the sample has flowed over the immobilised protein conjugate. Such tests however, are largely semi-quantitative and so can be of limited use when a concentration result is required rather than a simple yes/no answer.…”