Improved enzyme immunoassay for group-specific determination of penicillins in milk

Polyclonal antibodies against penicillin antibiotics were raised in rabbits after immunization with an ampicillin-bovine serum albumine conjugate. The specificity and sensitivity of these antibodies were tested in a competitive direct enzyme immunoassay (EIA), using an ampicillin-horseradish peroxidase conjugate as the labelled antigen, and penicillin G as the reference antibiotic. The 50% binding inhibition concentration of penicillin G standard curves was at 2-4 ng ml(-1). with a detection limit (20% binding inhibition) at 0.5-1 ng ml(-1). The antiserum showed strong relative cross-reactivities with all penicillin-type betalactam antibiotics tested, including those regulated by maximum residue limits (MRLs) within the European Union (ampicillin 64%, amoxicillin 24%, oxacillin 1000, cloxacillin 29%, dicloxacillin 27%, and nafcillin 120%). After alkaline hydrolysis of the betalactam ring, no cross-reactivity of the respective penicillins was observed anymore. Cephalosporin-type betalactam antibiotics did not react in this test system. The recovery rates for penicillin G in artificially contaminated milk samples (2-32 ng ml(-1)) were at 89-97%. Penicillin G, ampicillin, amoxicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin were all detectable in spiked milk samples at or below the respective MRL level. Comparison analysis of the EIA and a microbiological inhibition assay using violative milk samples obtained from routine milk testing scheme in Bavaria showed excellent agreement between both methods.