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Highly Sensitive EnzymeImmunoassays for theDetection of β-LactamAntibioticsAndreas Grubelnik , Celestino Padeste & LouisTiefenauerVersion of record first published: 01 Jul 2010.

To cite this article: Andreas Grubelnik , Celestino Padeste & Louis Tiefenauer(2001): Highly Sensitive Enzyme Immunoassays for the Detection of β-LactamAntibiotics, Food and Agricultural Immunology, 13:3, 161-169

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ISSN 0954-0105 (print)/ISSN 1465-3443 (online)/01/030161-09 © 2001 Taylor & Francis Ltd

DOI: 10.1080/09540100120075817

Food and Agricultural Immunology (2001) 13, 161–169

Correspondence to: A. Grubelnik. Fax: + 41 56 310 26 46; E-mail: Andreas.Grubelnik@psi.ch

Highly Sensitive Enzyme Immunoassays for the Detectionof b -Lactam Antibiotics

ANDREAS GRUBELNIK, CELESTINO PADESTE AN D LOUIS TIEFENAUER

Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut, CH-5232Villigen PSI, Switzerland

(Original manuscript received 17 January 2001; revised manuscript accepted 10 May 2001)

Specific and sensitive antibodies against b -lactam antibiotics are difficult to raise due to thechemical reactivity of the b -lactam ring. The antibiotic–protein conjugates used as theimmunogen can easily react with primary amino groups as for instance the « -amino groupof lysine side chains in proteins. This leads to the degradation of the immunogen to a complexmixture and, therefore, to an unpredictable immune response of the host animal. We producedantisera against the hydrolyzed form of b -lactam antibiotics by immunizing rabbits withstable conjugates mimicking this form. Addition of penicillinase in the immunoassay leads tohydrolysis of the b -lactam antibiotic, which is recognized by the antiserum. In a competitiveenzyme-immunoassay, benzylpenicillin could be detected at levels of 0.05 ng ml–1 andcloxacillin at levels of 0.1 ng ml–1 in pasteurized milk. The sensitivity and also the selectivityof these EIAs are remarkably high and offer a wide range of different applications. Theprocedure should be applicable for other b -lactam antibiotics, and may therefore in futureplay an important role in food quality control and assurance.

Keywords: Penicillin, cloxacillin, penicillinase, b -lactam antibiotics, food qualityassessment

INTRODUCTION

The b -lactam antibiotics, in particular the penicillins, are most frequently used for theprophylaxis and therapy of infectious diseases in veterinary medicine. Unauthorized druguse, prolonged drug excretion and neglect of the withdrawal times may result in residues ofpenicillins in milk and animal tissues. Antibiotic residues in milk can inhibit bacterial startercultures used in diary food production, increase the resistance of pathogenic bacteria towardsantibiotics and may cause allergic reactions in penicillin-sensitized persons. To protectcustomers health, the European Union (EU) fixed maximum residue limits (MRL) in milkand animal tissues for some antibiotics (EEC, 1990), e.g. ampicillin, amoxicillin and

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benzylpenicillin (4 ng ml–1 in milk, 50 ng g– 1 in animal tissues), cloxacillin and relatedisoxazolyl antibiotics (30 ng ml– 1 in milk and 300 ng g–1 in animal tissues) and nafcillin(30 ng ml– 1 (g–1 ) in both).

The specific determination of antibiotics in this concentration range is still difficult. Theanalytical methods applied are not specific and sensitive enough or they are expensive and slow.The most common screening methods are based on growth inhibition of test microorganisms.These microbiological tests are in general sufficiently sensitive for commonly used antibiotics,but they are unspecific and require a long incubation time (for a review, see Aerts et al., 1995).Physicochemical methods such as HPLC or GC-MS are laborious due to extraction procedures,they are expensive and often not sensitive enough and therefore not suitable for screening ofmilk or meat samples (for reviews, see Boison, 1992 and 1995).

Immunochemical methods have an increasing importance for routine analysis ofantibiotics and fulfill the requirements of a sensitive, quick and cheap assay. However, forpenicillins, the production of a sensitive antiserum is difficult. Several attempts to developenzyme immunoassays (EIAs) for the detection of penicillin in the ppb range failed. Directcovalent coupling of the penicillin to the carrier protein led to antisera reactive only againstthe open ring (penicilloyl ) form and not against the original antibiotic (Singh et al., 1984;Rohner et al., 1985; Blackmore et al., 1988). Different chemical methods have been used tocouple ampicillin to the carrier protein via the amino group to synthesize immunogens, whichare able to induce antibodies against the intact penicillin structure (Kitagawa et al., 1978;Nagakura et al., 1991; Usleber et al., 1998). These antibodies, however, are not sensitiveenough to determine antibiotic concentration in the range of the mentioned MRL. Furtherattempts to produce a sensitive antiserum by coupling the b -lactam antibiotics to the carrierprotein through the carboxy-group did not succeed either (Kachab et al., 1992; Usleber et al.,1994). Even though the antibodies showed the desired group specificity for either thecephalosporins (Kachab et al., 1992) or the isoxazolyl penicillins (Usleber et al., 1994), thesensitivity was not satisfactory.

For screening purposes, it would be of great interest to develop a group specific assay inorder to determine all penicillins present in a sample (Usleber et al., 1998). For differentialanalysis, however, the development of a highly specific and sensitive EIA for individualantibiotics is required. The different antibiotics can then be determined separately, and byusing mixtures of different antibodies, assays for a wide spectrum of analytes could beestablished.

In this work, we describe the development of EIAs for the specific and sensitivedetermination of benzylpenicillin and cloxacillin using enzymatic hydrolysis of the analytesduring the assay and antibodies specific for the hydrolysed form of the antibiotics. Theapplied method can in principle be adapted to all kinds of b -lactam antibiotics and thereforecontribute to the development of new sensitive and specific EIAs in the future.

MATERIALS AND METHODS

Chemicals and EquipmentPenicillinase (EC 3.5.2.6, type I, from Bacillus cereus), goat anti-rabbit IgG peroxidaseconjugate, bovine serum albumin (BSA), avidin, complete and incomplete Freund’s adjuvant,benzylpenicillin, amoxicillin, nafcillin, phenoxymethylpenicillinic acid, cefadroxil, diclox-acillin and penicilloyl- « -aminocaproate were purchased from Sigma, 5-(biotinamido )-pentylamine from Pierce and 3,3 9 , 5,5 9 -tetramethylbenzidine (TMB) from Serva. Ovalbumin,ampicillin, cloxacillin, oxacillin, cephalosporin C, 7-aminocephalosporanic acid, 6-aminope-nicillanic acid, D2O and all other chemicals were from Fluka.

1H-NMR spectra were recorded on a 300 MHz Varian Gemini II; purification of the biotinconjugates was performed on a Shimadzu LC10 HPLC system; color development in the EIAexperiments have been measured with an Opsys MR microtiter plate reader (Dynextechnologies ).

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Synthesis of the ImmunogensThe benzylpenicillin–BSA conjugate was prepared according to Levine (1962) with slightmodifications. Briefly: BSA (10 mg) was dissolved in 2 ml of 100 mM-sodium carbonatebuffer, pH 10.5. Benzylpenicillin (5.5 mg; 100-fold molar excess) was added and the reactionmixture incubated for 16 h at 4°C. After dialysis against 20 mM -potassium phosphate buffer(KPi), pH 7.2, the protein conjugate (hBPen-BSA) was lyophilized for storage. By ananalogous reaction, cloxacillin was coupled to BSA to generate the immunogen hClox-BSA.LBPen-Ov and hClox-Ov were synthesized in the same manner by replacing BSA withovalbumin.

Synthesis of the Biotin ConjugatesBenzylpenicillin (18.5 mg, 25 m mol) and 5-(biotinamido ) pentylamine (16 mg; 25 m mol)were dissolved in 2 ml of H2O. The reaction was started by adding 200 m l of 1 M -Na2CO3

buffer, pH 10.5, and the mixture was then incubated for 4 h at room temperature. The reactioncontrol and the product purification was done by HPLC on a Brownlee RP18 (10 m m, 0.4 ´22 cm) column in 20 mM KPi, pH 7.4 with a gradient from 0 to 75% CH3CN. The product(hBPen-LC-Bio ) eluted at a CH3CN concentration of around 25%. It was characterized by1H-NMR in D2O and then lyophilized. The yield was 13.6 mg (39%). The product can bestored lyophilized at room temperature in a desiccator or as a stock solution in H2O with50 mg 1–1 NaN3 as a preservative.

By an analogous reaction, cloxacillin was coupled to biotin to generate hClox-LC-Bio:25 m mol (22.9 mg) cloxacillin was used and the yield was 19.1 mg (49%).

Immunization and Antibody Titer DeterminationThe immunogens hBPen-BSA and hClox-BSA (each 500 m g), were first dissolved in 1 mlH2O and then emulsified with 1.5 ml of complete Freund’s adjuvant. White New Zealandrabbits were immunized with 2 ml portions of the emulsion by subcutaneous injections.Booster injections with the same amount of antigen emulsified in incomplete Freund’sadjuvant were given 14 and 26 weeks after the primary injection. Blood was collected 3and 4 weeks after the second booster injection, combined and used for all the subsequentexperiments described in this paper. The relative antibody titer was determined in EIAsusing ovalbumin conjugates coated on activated microtiter plates (Nunc-Immuno™ PlateMaxiSorp™ Surface), whereas the sensitivity and specificity of the antiserum wasdetermined in competitive EIAs on avidin coated microtiter plates with immobilizedbiotin-antibiotic conjugate as described below.

Enzyme ImmunoassaysThe antisera were diluted 1:10 in PBS with 40% ethylene glycol and 0.05% NaN3 andstored at –20°C. All incubations were performed at room temperature in a total volumeof 200 m l/well. A washing procedure, consisting of three cycles with washing buffer(50 mM -KPi , pH 7.4, 500 mM NaCl, 0.1% Tween 20) and two cycles with assay buffer(50 mM -KPi , pH 7.4, 0.05% fat free dry milk), was carried out after each incubation step.The coating of the microtiter plates with avidin has been described in details (Bodmer etal., 1989). The antigens, hBPen-LC-Bio or hClox-LC-Bio (10 ng ml–1 in assay buffer)were bound to the avidin coated microtiter plates in a 30 min incubation step. Serialdilutions of antibiotics in assay buffer were incubated for 1 h at room temperaturetogether with diluted antiserum and 0.2 U ml–1 to 1 U ml– 1 penicillinase. Finally, thebound antibodies were visualized using a secondary goat anti-rabbit peroxidase conjugateand TMB as substrate. Cross-reactivity was calculated as the ratio of the 50% inhibition

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of each antibiotic and the 50% inhibition value for benzylpenicillin or cloxacillin,respectively.

For a rough estimation of the antibody titer, hBPen-Ov and hClox-Ov, were immobilizedon activated microtiter plates: a solution of 1 m g ml–1 hBPen-Ov (hClox-Ov) in 50 mM -KPi ,pH 7.4, was incubated for 2 h at room temperature and the surface subsequently blocked withblocking buffer (1% fat free dry milk in 50 mM -KPi , pH 7.4) at 4°C overnight. The furthersteps were done according to the description above.

Preparation of milk samples for the recovery experiments were done as follows;pasteurized milk samples were spiked with different concentrations of antibiotic. These milksamples (100 m l of each) were added to the same volume of diluted antiserum, containing0.4 U ml– 1 penicillinase in the benzylpenicillin EIA or 2 U ml–1 penicillinase in the cloxacillinEIA. Six independent recovery experiments were carried out with four-fold determination ofeach standard and sample.

FIG. 1. Structures of benzylpenicillin and cloxacillin, hydrolysis with penicillinase and synthesis of theirBSA conjugates.

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EIA FOR DETECTION OF b -LACTAM ANTIBIOTICS 165

RESULTS AND DISCUSSION

Assay DevelopmentThe aim of this study was the production of sensitive and specific polyclonal antisera againstbenzylpenicillin and cloxacillin for EIAs, immunosensors and related techniques. Thechemical reactivity of the b -lactam ring of penicillin antibiotics towards nucleophiles makesit difficult to produce sensitive antisera against the active form of the antibiotic. Theimmunogen can react with various amino or thiol groups and is therefore modified duringimmunization. This implies that the antiserum is directed not only against the intactantibiotic, but also against the different degradation products and will be unspecific and lesssensitive. If the immunogen, however, is formed by reaction of the b -lactam ring with anamine of the carrier protein, stable amides are formed which are not further degraded. Suchamides are structurally similar to the hydrolyzed form of the b -lactam and induce, if used asthe immunogen, the formation of antisera specific for the hydrolyzed b -lactams (Figure 1).Thus, if the analyte is transformed into the hydrolyzed form in the EIA by addition ofpenicillinase, it is detected by the antibody.

The reaction of benzylpenicillin and cloxacillin with penicillinase I (from B. cereus; EC3.5.2.6) and the further degradation of the hydrolyzed form was investigated with HPLC and1H-NMR. Although cloxacillin is ‘penicillinase resistant’, it can be hydrolyzed bypenicillinase I within reasonable time (Fink et al., 1987). HPLC experiments showed that 1 Upenicillinase is sufficient to hydrolyze 1 m g of cloxacillin within 30 min in potassiumphosphate buffer as well as in 1:1 diluted milk samples. Under the same conditions,benzylpenicillin is completely hydrolyzed within seconds. These products react further to acomplex mixture of degradation products within hours. Detailed studies of these degradation

FIG. 2. Calibration curve for benzylpenicillin with and without penicillinase (n = 4). (———) withpenicillinase; 50% inhibition at 0.32 ng ml–1; (– – – –) without penicillinase; 50% inhibition at4.5 ng ml–1; MRL: maximum residue limit of the European Union in milk (4 ng ml–1 ).

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processes by HPLC and 1H-NMR revealed that the stability of the hydrolyzed antibiotics issufficient to run an EIA, if the enzymatic hydrolyzation takes place during the assay. After5 h, only 10% of the hydrolyzed benzylpenicillin and less than 5% of the hydrolyzedcloxacillin are further degraded. On the other hand, the hydrolyzed antibiotic is not too stable,so that its concentration in a milk sample will be very low. 24 h after the addition ofpenicillinase, more than 60% of the hydrolyzed benzylpenicillin and 30% of the hydrolyzedcloxacillin are degraded and after 3 days, no hydrolyzed antibiotic is detectable anymore. Thestability of the hydrolyzed antibiotics allows that in the described EIAs, the values for active,intact antibiotics will be determined, and not the value for a mixture of active antibiotics anddegradation products.

In the EIA, the biotin conjugate of the hydrolyzed antigen is first bound to avidin coatedmicrotiter plates, the samples are then incubated together with the diluted antiserum,followed by the detection of bound antibody using a secondary antibody–peroxidaseconjugate. If penicillinase is added to the sample, the 50% inhibition concentration is shiftedfrom 3.5 ng ml–1 to 0.2 ng ml–1 in the case of benzylpenicillin (Figure 2) and from 2 ng ml–1

to 0.65 ng ml–1 in the case of cloxacillin (Figure 3). These 50% inhibition concentrationvalues allow the determination of antibiotic concentrations in the range of 0.1 ng ml–1, i.e. farbelow the MRL of the EU (4 ng ml– 1 in milk, 50 ng g–1 in animal tissues for penicillin,30 ng ml– 1 in milk, 300 ng g–1 in animal tissues for cloxacillin).

Specificity of the Antisera

Benzylpenicillin. The cross-reactivity with other b -lactam antibiotics was analyzed incompetitive EIAs (Table 1). The values indicate that the assay is not only very sensitive, but

FIG. 3. Calibration curve for cloxacillin with and without penicillinase (n = 4). (———) with penicillinase;50% inhibition at 0.75 ng ml–1; (– – – –) without penicillinase; 50% inhibition at 2.3 ng ml–1 MRL:maximum residue limit of the European Union in milk (30 ng ml–1 ).

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EIA FOR DETECTION OF b -LACTAM ANTIBIOTICS 167

also very specific for benzylpenicillin. Only the structurally closely related phenoxy-methylpenicillinic acid (penicillin V) and ampicillin show a cross-reactivity exceeding 1%.For all other tested b -lactam antibiotics of the penam type (penicillins ), the cross-reactivityis between 1 and 0.01%, whereas no cross-reactivity with b -lactam antibiotics of the cephemtype (cephalosporins ) was observed. Penicilloyl- « -aminocaproate, a commercially availableamide derivative of benzylpenicillin, which is structurally very similar to the immunogen,binds even better than benzylpenicillin itself. As this substance has no antibiotic activity andis used neither in veterinary nor in human medicine, it has no relevance in practice.

Cloxacillin. Cross-reactivity of this antiserum with other antibiotics could only be detectedwith the other two isoxazolyl antibiotics oxacillin (2.2%) and dicloxacillin (0.6%). All otherb -lactam antibiotics of the penam and the cephem type showed no inhibition in the testedconcentration range. Since cloxacillin is the most frequently used isoxazolyl antibiotic andthe cross-reactivity of the two other is quite small, the antiserum is useful for cloxacillindetection.

Analysis of Milk SamplesTo demonstrate the applicability of the benzylpenicillin and cloxacillin assays for samplesrelevant in the diary industry, recovery experiments have been carried out in milk samplesspiked with antibiotics in the concentration range of 0–5 ng ml–1. The results in Table 2 showthat the detection of benzylpenicillin and cloxacillin in milk in concentrations down to0.05 ng ml–1 can easily be achieved. Non-contaminated milk samples gave reproduciblyvalues below 0.02 ng ml– 1. The excellent assay sensitivity would even allow the eliminationof matrix effects by sufficiently high dilution of the samples in more complex matrices. Insummary, benzylpenicillin and cloxacillin can be detected in concentrations more than oneorder or magnitude below the MRL of the EU. Other matrices such as honey, urine or animaltissues are under investigation and the data look very promising.

OutlookTrace levels of benzylpenicillin and cloxacillin can be detected in milk using an antiserumagainst the hydrolyzed form of the antibiotics combined with hydrolysis of the analytes in the

TABLE 1. Specificity of the polyclonal antisera against benzylpenicillin and cloxacillin.

Antibiotic

Benzylpenicillin antiserum

50% B/B0(ng ml–1 )

Cross-reactivity(%)

Cloxacillin antiserum

50% B/B0(ng ml–1 )

Cross-reactivity(%)

Benzylpenicillin 0.2 100 > 105 < 0.1Amoxicillin 2500 < 0.1 > 105 < 0.1Ampicillin 4.6 4.3 > 105 < 0.16-Aminopenicillanic acid 22 0.9 > 106 < 0.1Cloxacillin 140 0.14 0.65 100Oxacillin 3000 < 0.1 29 2.2Dicloxacillin 3000 < 0.1 105 0.6Nafcillin 145 0.14 > 104 < 0.1Penicillin V 3.2 6.2 > 105 < 0.1Penicilloyl- « -aminocaproate 0.06 330 > 105 < 0.1Cephalosporin C > 104 < 0.1 > 106 < 0.17-Aminocephalosporanic acid > 104 < 0.1 > 106 < 0.1Cefadroxil > 104 < 0.1 > 106 < 0.1

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168 A. GRUBELNIK ET AL.

assay. This strategy may also be applicable to other b -lactam antibiotics of interest, as far asthey can be hydrolyzed by b -lactamases. As shown with cloxacillin, even so-calledb -lactamase resistant antibiotics can be detected with this method. A set of specific andsensitive antisera against the most common b -lactam antibiotics could be raised byimmunization with the corresponding protein conjugates. These antisera can be utilized tospecifically determine antibiotic residues or, when combined, to determine selected b -lactamantibiotics simultaneously in a sample of interest. The approach proposed in this work maypromote the analysis of antibiotics by immunological methods and could provide a powerfultool for monitoring food quality.

ACKNOWLEDGEMENTS

This work was supported by the Swiss National Science Foundation (Swiss Priority ProgramBiotechnology ). Thanks are due to J.-J. Hefti for technical assistance.

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TABLE 2. Recovery of benzylpenicillin and cloxacillin from spiked milk samples (n = 6)

Antibiotic added (ng ml–1 )

Cloxacillin

Antibiotic found(ng ml–1 )

Recovery(%)

Benzylpenicillin

Antibiotic found(ng ml–1 )

Recovery(%)

5.0 4.99 ± 0.42 100 5.98 ± 1.09 1202.0 2.01 ± 0.26 100 2.17 ± 0.13 1091.0 0.89 ± 0.18 89 1.03 ± 0.09 1030.5 0.56 ± 0.04 112 0.43 ± 0.10 860.3 0.27 ± 0.07 90 0.25 ± 0.05 830.1 0.14 ± 0.02 140 0.100 ± 0.023 1000.05 0.06 ± 0.03 120 0.045 ± 0.017 900.0 < 0.02 < 0.02

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