Entwicklung eines Biosensors zum Nachweis von Antibiotika und Sulfonamiden in Milch- Herstellung der immunchemischen Komponenten

This paper describes the development and application of enzyme
immunoassays for the detection of antimicrobials in milk, aiming at
the establishment of a biosensor system for the on-line analysis of
drug residues in milk. For the development of group-specific
antibodies against penicillins rabbits were immunized with an
ampicillin-BSA-conjugate. The resulting antiserum was employed for
the development of a direct competitive enzyme immunoassay (EIA),
giving a detection limit of 1 ng/ml for penicillin G in milk. Due
to broad cross-reactivities the sensitive detection of those
penicillins regulated by MRLs within the European Union
(ampicillin, amoxicillin, oxacillin, cloxacillin, dicloxacillin and
nafcillin) was enabled. The practical use of the enzyme immunoassay
was demonstrated by analyzing artificially contaminated and
violative incurred milk samples (n = 321). For the development of
indirect competitive enzyme immunoassays for the detection of
streptomycin, sulfonamides and penicillins, previously established
direct assays, based on monoclonal antibodies, were adapted to
indirect formats. For this purpose a wide range of coating-antigens
was prepared by linking haptens to carrier-proteins. After
optimizing test sensitivity and characterizing the test specificity
indirect EIAs could be developed for each antimicrobial compound,
fulfilling the MRL requirements due to EU regulation 2377/90. Only
for ampicillin and penicillin G colorimetric measurements resulted
in detection limits of 7 and 6 ng/ml, respectively, which were
slightly above the MRL of 4 ng/ml. By using a luminescent
substrate, however, the MRL could be reached for these antibiotics
as well. Based on the results of the individual EIAs rapid
multianalyte tests both on microtitre plates and planar microarray
chips were developed. Due to altered assay conditions again the
systems were optimized and characterized regarding sensitivity and
specificity. The sensitivities achieved on the biochip were well
comparable with those obtained in the microtitre plate, whereas
total assay time could be reduced to 15 min (microtitre plate) and
5 min (biochip), respectively.