Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices.
The penicillin-binding protein PBP 2x* from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the detection and determination of penicillins and cephalosporins with intact beta-lactam structure in milk, bovine and porcine muscle juice, honey and egg. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and digoxigenin as functional groups (DIG-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses anti-digoxigenin F(ab) fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more antibody fragments are bound via the digoxigenin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present. A fractional factorial design was applied to detect chemometrically effects and interactions of the assay parameters. For optimisation of the significant parameters a Box-Behnken design was used. The assay has been developed for various food matrices as screening test with the option for a quantitative assay, when the identity of the residual beta-lactam is known (e.g. elimination studies). Cefoperazon, cefquinome, cefazolin, cloxacillin, ampicillin and benzylpenicillin could be detected at levels corresponding to 1/2 EU maximum residue limit (MRL) in milk, meat juice from muscle tissue of different species, egg and honey (where applicable) without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.
Lamar
Janine
J
Department of Food Chemistry, University of Wuppertal, Gauss-Strasse 20, 42119 Wuppertal, Germany.
Petz
Michael
M
D000667:Ampicillin / Q000032:analysis; D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis*; D004076:Digoxigenin / Q000032:analysis; D004350:Drug Residues / Q000032:analysis*; D004531:Eggs; D005504:Food Analysis / Q000379:methods*; D006722:Honey; D006735:Horseradish Peroxidase / Q000378:metabolism; D008460:Meat; D008892:Milk / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D046915:Penicillin-Binding Proteins / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000378:metabolism; D047090:beta-Lactams / Q000032:analysis*
D000900:Anti-Bacterial Agents; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D000667:Ampicillin; D006735:Horseradish Peroxidase; D004076:Digoxigenin