Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of ß-lactam antibiotics in foods.

ABSTRACT

ß-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of ß-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of ß-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by ß-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual ß-lactam was known. The IC50 values of 15 ß-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 µg L(-1). Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected ß-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 µg kg(-1), respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 ß-lactams in edible tissues, among which 11 ß-lactams controlled by European Union could be detected below maximum residue limits.