Gewinnung von Antikörpern gegen ein bakterielles Zellwandpeptid als multispezifische Rezeptoren für die Rückstandsanalytik von ß-Lactam- und Glykopeptidantibiotika

Content Provider	Semantic Scholar
Author	Schlösser, Joachim
Copyright Year	2000
Abstract	A novel approach was investigated to produce multi-specific antisera equally useful for the detection of β -lactam and glycopeptide antibiotics which was based on the natural mode of action of both classes of antibiotics. In addition a novel mechanism of a specific enzyme immunoassay was established which proofed to be useful for the detection of the glycopeptide antibiotic avoparcin in animal feed. For the development of multi-specific antisera, the peptide Nα-acetyl-L-lysinyl-D-alanyl-Dalanine (NαAc-Lys-D-Ala-D-Ala) was chosen as hapten for immunization instead of using the respective antibiotics. This peptide is the natural substrate of the D,D-transpeptidase reaction which is essential for synthesis of bacterial cell walls. β.lactam antibiotics are supposed to be molecular mimicries of D-Ala-D-Ala, and binding of these antibiotics to the active site of a D,D-transpeptidase leads to an irreversible inhibition of this enzyme. In contrast, glycopeptide antibiotics form complexes with D-Ala-D-Ala-terminated peptides. In this complexes D-Ala-D-Ala is no longer available for the D,D-transpeptidase reaction. Antibodies raised against D-Ala-D-Ala can be expected to have binding characteristics similar to bacterial D,D-transpeptidases. On the one hand they are supposed to bind D-Ala-D-Alaterminated peptides as well as β -lactam antibiotics and on the other hand the binding to D-Ala-D-Ala-terminated peptides should be suppressed in the presence of glycopeptide antibiotics. Such antibodies could be useful tools in screening tests for penicillin residues in milk and meat as well as for the glycopeptide antibiotic avoparcin which was widely used as feed addi-tive until its ban in 1997. The peptide NαAc-Lys-D-Ala-D-Ala was synthesized using the NHS-ester method. In addition the preparation of further peptides e.g. Nα-acetyl-L-lysinyl-L-alanyl-L-alanine is described. All peptides and intermediates were characterized using spectroscopic (IR) and chromatographic (DC, HPLC) methods. For the development of group specific antibodies NαAc-Lys-D-Ala-D-Ala was conjugated to glucoseoxidase (GOD) using the periodate method. A GOD-ampicillin conjugate was prepared in the same manner. In order to develop several variations of enzyme immunoassays and enzyme receptor assays, peptides and antibiotics were conjugated to horseradish peroxidase (HRP) and bovine serum albumine (BSA) using the glutardialdehyde and periodate method. NαAc-Lys-D-Ala-D-Ala conjugates were characterized using a stereo-specific HPLC method to quantify the amount of bound D-Alanine. In addition the successful synthesis of all conjugates was shown in several specific enzyme immunoassays or enzyme receptor assays. The GOD-NαAc-Lys-D-Ala-D-Ala conjugate was given intradermally to three rabbits. After six weeks specific antibodies against NαAc-Lys-D-Ala-D-Ala were detected in all rabbits. Even after a re-immunisation with the GOD-ampicillin conjugate no cross-reactivity with β-lactam antibiotics was observed. This did not disapprove the concept as shown by binding studies with various peptides (epitope mapping). Although D-Ala-D-Ala was essential for binding, only NαAc-Lys-D-Ala-D-Ala gave a sufficient fit to the antibodies’ binding sites. This fact makes the binding sites of the antibodies different from that of D,D-transpeptidases with the consequence that β -lactams were not recognized. For glycopeptide antibiotics, more successful results with the produced rabbit antibodies can be reported. Glycopeptide antibiotics form a complex with the NαAc-Lys-D-Ala-D-Ala terminus of the conjugates used in several ELISA tests. Thus it prevents the NαAc-Lys-D-Ala-D-Ala terminus to be recognised by the antibodies. These results were in accordance with the concept outlined and showed that it was successful in principle. Finally, this approach was used for the development of a novel competitive enzyme immunoassay. Studies involving a wide variety of different feed samples with avoparcin concentrations commonly used before the ban of avoparcin, showed that this test system is applicable for the screening of the illegal use of avoparcin in feed for food producing animals.
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