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  • Screening auf Rückstände von β-Lactam-Antibiotika in Milch:

    Entwicklung eines optischen Biosensor-Assays mit Penicillin-bindenden Proteinen

    Dem Fachbereich Mathematik und Naturwissenschaften der Bergischen Universität Wuppertal

    zur Erlangung des akademischen Grades

    Doctor rerum naturalium (Dr. rer. nat.)

    vorgelegte Dissertation

    von

    Giuseppe Cacciatore

    Wuppertal

    2005

  • Diese Dissertation kann wie folgt zitiert werden:

    urn:nbn:de:hbz:468-20050138

    [http://nbn-resolving.de/urn/resolver.pl?urn=urn%3Anbn%3Ade%3Ahbz%3A468-20050138]

  • i

    ABSTRACT

    A biospecific interaction assay (BIA) for the detection of residues of β-lactam antibiotics in milk was developed using a surface plasmon resonance (SPR) biosensor. Penicillin-binding proteins (PBP), which are enzymes involved in the final stages of murein biosynthesis and represent the lethal target of β-lactam antibiotics, were used as biomolecular recognition partners for penicillins and cepha- losporins.

    PBP were mixed with samples, incubated and then labelled with an antigen-tagged ampicil- lin-derivative prior to SPR analysis with antibodies against the antigen-tag immobilised on the sensor chip. PBP complexed exclusively antigen-tagged ampicillin in samples without residues (negative samples). In samples containing residues (positive samples), PBP complexed β-lactam antibiotics before they complexed antigen-tagged ampicillin. A certain ratio of antigen-tagged ampicillin com- plexed by PBP and as free compound was formed in intermediate situations reflecting the presence or absence of residues of β-lactam antibiotics in positive or negative samples, respectively. The immobi- lised antibodies captured antigen-tagged ampicillin complexed by the PBP or as free compound. The large difference in molecular mass at > 40 kDa for antigen-tagged ampicillin complexed by PBP and at < 1 kDa for antigen-tagged ampicillin as free compound led to a ratio dependent biosensor response since SPR signal is affected by the molecular masses of binding molecules. Consequently, the re- sponse was inversely related to the concentration of β-lactam antibiotics in the sample.

    In order to detect residues of β-lactam antibiotics at concentrations corresponding to maximum resi- due limits (MRL) set by the European Union, a suitable PBP, an antigen-tagged ampicillin-derivative and an antibody against the antigen had to be found. PBP of Streptococcus thermophilus and Bacillus stearothermophilus were partly purified by ampicillin affinity chromatography and a soluble derivative of PBP 2x of Streptococcus pneumoniae was expressed as a recombinant protein in Escherichia coli. This soluble PBP 2x-derivative, called PBP 2x*, had higher affinities towards different β-lactam antibi- otics than the other studied PBP and was chosen for construction of the β-lactam-specific BIA.

    Ampicillin (AMPI) was labelled with derivatives of digoxigenin (DIG), fluorescein (FLU) or 2,4-dinitrophenol (DNP). DIG, FLU and DNP were coupled through their activated N-hydroxysuccinimide esters to the amino group of AMPI, yielding DIG-labelled AMPI (DIG-AMPI), FLU-labelled AMPI (FLU-AMPI) and DNP-labelled AMPI (DNP-AMPI), respectively. DIG-AMPI, FLU- AMPI and DNP-AMPI were examined as antigen-tagged ampicillin-derivatives.

    The formed complexes of these ampicillin-derivatives with PBP 2x* were analysed by SPR analysis using their corresponding counter antibodies. Despite the binding of DNP-labelled bovine serum al- bumin, SPR analysis revealed that DNP-AMPI being complexed by PBP 2x* was not bound neither by a polyclonal anti-DNP antibody nor by a monoclonal anti-DNP antibody in a reproducible manner. On the other hand, anti-FLU antibodies possessed insufficient affinity for FLU-AMPI. Of the monoclonal and polyclonal antibodies against DIG tested, the monoclonal antibody was chosen, since regenera- tion of the sensor-chip surface containing immobilised polyclonal antibody was not complete.

    Benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in commingled raw milk at concentrations below their respective MRL using PBP 2x*, DIG-AMPI and the monoclonal antibody against DIG for SPR analysis of milk samples. As part of the sample preparation, commingled raw milk samples were defatted prior to addition of PBP 2x* and DIG-AMPI. The SPR analysis of 20 raw milk samples from individual cows confirmed that matrix interferences are a critical point in BIAs as non-specific binding of matrix components to the sensor chip elevated the detection limit dramatically. Matrix interferences could be reduced to a lower and a more constant level by a heat treatment step and the addition of carboxymethylated dextran to the samples. With this additional sample preparation, individual samples containing benzylpenicillin at the MRL were identified as posi- tive. The limit of detection of benzylpenicillin in individual raw milk samples was 2 ng/ml. Thus, the assay could be the basis for a screening test for routine use.

  • ii

    DANK

    Die vorliegende Arbeit entstand in der Zeit von Juli 2000 bis September 2004 an der Bergi-

    schen Universität Wuppertal im Fachgebiet Lebensmittelchemie des Fachbereichs Mathema-

    tik und Naturwissenschaften in der Arbeitsgruppe von Herrn Prof. Dr. Michael Petz.

    Herrn Prof. Dr. Michael Petz gilt mein besonderer Dank für die interessante Aufgabenstel-

    lung, sein stetes Interesse am Fortgang meiner Arbeit, seine Diskussionsbereitschaft und die

    gewährten Freiheiten bei der Durchführung.

    Herrn Prof. Dr. Helmut Guth danke ich für die Übernahme des Korreferates.

    Ebenso nachdrücklich bedanken möchte ich mich bei Frau Prof. Dr. Regine Hakenbeck für

    die Bereitstellung des Stamms zur Expression von PBP 2x*. Mein besonderer Dank gilt

    Herrn Privatdozent Dr. Reinhold Brückner und Herrn Dipl.-Biol. Jens Rutschmann für die

    vielen praktischen Tipps bei der Isolierung des PBP 2x*.

    Danken möchte ich auch Herrn Prof. Dr. Aldert A. Bergwerff, Frau Gertie C.A.M. Bokken

    und Frau Betty G.M. Jongerius-Gortemaker für die angenehme Arbeitsatmosphäre während

    mehrerer Forschungsaufenthalte an der Universität Utrecht, viele konstruktive Diskussionen

    und hilfreiche Anregungen bei der Durchführung der Biosensor-Messungen.

    Herrn Dipl.-Chem. Marc Constapel und Herrn Dipl.-Chem. Marc Schellenträger danke ich für

    die Durchführung der massenspektrometrischen Messungen.

    Frau Lebensmittelchemikerin Ute Rettinger, Frau Lebensmittelchemikerin Katrin Keppler und

    Frau Dr. Elena Smouchkina danke ich für ihre im Rahmen von Forschungspraktika geleistete

    Mitarbeit.

    Meinen Kolleginnen und Kollegen danke ich für das freundliche Arbeitsklima, in der diese

    Arbeit entstanden ist. Frau Dr. Erika Müller-Seitz und Herrn Dr. Joachim Schlösser danke ich

    herzlich für die vielen wertvollen Diskussionen.

    Meinen Eltern danke ich dafür, dass sie mir meine Ausbildung ermöglicht haben und mich

    immer unterstützt haben, wann immer es die Umstände erforderten.

    Schließlich danke ich Frau Susanne Scherer sehr herzlich für ihre stete Geduld.

  • iii

    Für Giuseppa und Antonino

  • iv

    INHALTSVERZEICHNIS

    1 Einleitung...................................................................................................................... 1

    2 Penicilline und Cephalosporine .................................................................................. 3 2.1 Strukturen ............................................................................................................... 3 2.2 Reaktivität des β-Lactam-Ringes ............................................................................ 7

    2.2.1 Chemische Abbaureaktionen........................................................................... 8 2.3 Wirkungsmechanismus......................................................................................... 11

    2.3.1 Murein-Biosynthese....................................................................................... 13 2.3.2 DD-Transpeptidasen, Target der β-Lactam-Antibiotika .................................. 14

    2.4 Penicillin-bindende Proteine (PBP) ....................................................................... 17 2.5 Resistenz.............................................................................................................. 19

    2.5.1 β-Lactamasen................................................................................................ 19 2.5.2 PBP mit geringer Affinität gegenüber β-Lactam-Antibiotika............................ 20 2.5.3 Therapiemaßnahmen gegen β-Lactamase-produzierende Bakterien ............ 20

    2.6 Einsatz in der Veterinärmedizin............................................................................. 20 2.6.1 Pharmakokinetik und Metabolismus .............................................................. 21 2.6.2 Toxizität und allergische Reaktionen ............................................................. 21 2.6.3 Rechtliche Regelungen für Tierarzneimittelrückstände .................................. 22

    3 Analytik von β-Lactam-Antibiotika-Rückständen .................................................... 24 3.1 Screening-Verfahren..............................