Immunoassay Systems for Detection and Quantitative Determination of Cefalexin

Beta-lactam antibiotic cephalexin is poorly recognized by bacterial beta-lactam binding proteins and therefore cannot be reliably detected by a group-specific receptor assay. For effective and rapid monitoring of cephalexin concentration in food products, the test systems based on high-affinity and selective binding of this antibiotic to polyclonal antibodies have been developed. The principle of the method for the test systems of enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) is based on the competitive interaction of (a) cephalexin, potentially contained in the samples, and (b) a cephalexin-protein conjugate immobilized in the wells of a microplate or on the membrane of a test strip, with (c) polyclonal antibodies against cephalexin conjugated with peroxidase or adsorbed on gold nanoparticles. The high specificity of the obtained polyclonal antibodies against cephalexin and the absence of cross-reactivity to a number of cephalosporins, penicillins, and antibiotics of other classes have been confirmed. In the ELISA system in a buffer solution and in a milk matrix, the detection limit, the IC₅₀ value, and the range of detected concentrations were 0.02, 1.0 and 0.025–100 ng/mL respectively. The limit of detection for visual and instrumental determination of cephalexin in the LFIA system were 1.5 and 0.1 ng/mL, and the working range of quantitatively measured concentrations was from 0.05 to 1.5 ng/mL. For both systems, the coefficient of variation of measurements was in the range of 2.5–8.0%. The test systems allow to detect cephalexin in milk without sample preparation. The recovery of cephalexin added to the milk samples was 90.0–106.7%. The presented developments can serve as the basis for kits of reagents for monitoring the content of cephalexin in food products.