Detection Of Β-lactam Antibiotics Research Articles

Block copolymer-derived recessed nanodisk-array electrodes for electrochemical detection of β-lactam antibiotics

Antimicrobial resistance (AMR) is one of the major socio-economic factors contributing to public health. β-lactams are most commonly prescribed drugs for variety of bacterial infections. Frequent use of antibiotics leads to AMR in humans and animals. The present work is focused on developing an electro-immunosensor to control and regulate the excessive use of antibiotics in animal-based food products. An amphiphilic block co-polymer poly(ethylene oxide-block-methyl methacrylate)(PEO-b-PMMA) was used to fabricate recessed nano-disk array electrode (RNE) and immobilized with Pen-Ab and Cef-Ab antibodies. The Limit of detection (LOD) of RNE working electrode was found to be 14.8 pM for penicillin and 13.8 pM for cefalexin with good selectivity in presence of non-specific antibiotics. Fabricated RNE electrode could detect trace amounts of spiked antigen in real samples of milk, egg and meat extract. Further, mesoporous thin film and microarrays can eventually be used to develop point-of-care diagnosis of antibiotics in animal-based food products.

Development and Validation for Quantification of Cephapirin and Ceftiofur by Ultraperformance Liquid Chromatography with Triple Quadrupole Mass Spectrometry.

Cross contamination of β-lactams is one of the highest risks for patients using pharmaceutical products. Penicillin and some non-penicillin β-lactams may cause potentially life-threatening allergic reactions. The trace detection of β-lactam antibiotics in cleaning rinse solutions of common reactors and manufacturing aids in pharmaceutical facilities is very crucial. Therefore, the common facilities adopt sophisticated cleaning procedures and develop analytical methods to assess traces of these compounds in rinsed solutions. For this, a highly sensitive and reproducible ultra-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the analysis of Cephapirin and Ceftiofur. As per the FDA guidelines described in FDA-2011-D-0104, the contamination of these β-lactam antibiotics must be regulated. The analysis was performed on an XBridge C18 column with 100 mm length, 4.6 mm diameter, and 3.5 µm particle size at an oven temperature of about 40 °C. The mobile phase was composed of 0.15% formic acid in water and acetonitrile as mobile phases A and B, and a flow rate was set to 0.6 mL/min. The method was validated for Cephapirin and Ceftiofur. The quantification precision and accuracy were determined to be the lowest limit of detection 0.15 parts per billion (ppb) and the lowest limit of quantification 0.4 ppb. This method was linear in the range of 0.4 to 1.5 ppb with the determination of coefficient (R2 > 0.99). This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amounts of β-lactam contamination, monitoring cross contamination in facility surface cleaning, and determining the acceptable level of limits for regulatory purposes.

Detection of β-Lactam Antibiotics Based on Conjugated Antibody with Gold Nanorods by Localized Surface Plasmon Resonance Spectrometer

Detection of β-Lactam Antibiotics Based on Conjugated Antibody with Gold Nanorods by Localized Surface Plasmon Resonance Spectrometer

Plasmonic Sensor for Detection of β-Lactam Antibiotics based on the Conjugated Antibody with Gold Nanoparticles

This study aims to detect β-lactam antibiotics using a conjugated antibody with gold nanoparticles (GNPs). For this purpose, the gold nanoparticles synthesized from Chinese lettuce leaf extract (as reductant) were used for the colorimetric detection of β-lactam antibiotics (such as ampicillin, amoxicillin, penicillin G, oxacillin, and carbenicillin). XRD, FTIR spectroscopy, TEM, and dynamic light scattering were utilized to detect the crystallinity, to identify functional groups involved in the synthesis of GNPs, and to measure the size of the GNPs; pH 8 and a concentration of 8.4 μg of antibody at 1 mL GNPs solution were selected as the best pH and concentration of antibody for the conjugation of antibody with GNPs. The maximum wavelengths of the colloidal GNPs, conjugation of antibody with GNPs, and detection of antibiotics (from 1 nM to 1 mM) with GNPs–PAb were recorded using a micro-volume spectrophotometer system. The results indicated that the localized surface plasmon resonance spectrometer absorption wavelength of GNPs red-shifted with increasing concentration of β-lactam antibiotics. With increasing concentration of ampicillin, penicillin G, and carbenicillin, the wavelength of maximum changed, and after saturation of antibiotics concentration, the curve reaches a plateau. This indicated that the antibody showed similar behavior in the detection of these antibiotics. But regarding amoxicillin, the saturation concentration is much higher, indicating that the antibody was more specific for its detection. In contrast, for oxacillin, saturation occurred very soon, which demonstrated that the antibody had an extremely low detection capability for this antibiotic. Finally, the results showed that the antibody was sensitive to 1 nM of the five β-lactam antibiotics studied.

Integration of antibody-antigen and receptor-ligand reactions to establish a gold strip biosensor for detection of 33 β-lactam antibiotics

A novel lateral flow immunoassay biosensor, combining the receptor-ligand reaction and antigen-antibody reaction was developed for the detection of β-lactam antibiotics in milk. The receptor protein, the carboxy-terminal domain of β-lactam sensor-transducer mutant from Bacillus. licheniformis, served as the recognition element. Colloidal gold (CG)-labelled receptor antibody-receptor ternary complex was applied as a signal transducer probe. The presence of β-lactams was identified through a color change in the test zone, where the reaction between the complex of CG/anti-receptor antibody/receptor and β-lactam antigen on the test line could be inhibited by β-lactam residues in the sample, thus leading to a reduction in color signal. Based on the indirect labeling of the receptor with an antibody as a linker, the ability to detect 33 β-lactams was below or near the corresponding maximum residue limit. The proposed lateral flow immunoassay biosensor appears to be an excellent field-based screening tool for the qualitative screening of β-lactams in milk.

Evanescent Wave Optical Fiber Sensors Using Enzymatic Hydrolysis on Nanostructured Polyaniline for Detection of β-Lactam Antibiotics in Food and Environment.

β-Lactam antibiotics such as penicillins and cephalosporins are extensively used for human infection therapy. Consistent unintended exposure to these antibiotics via food and water is known to promote antibiotic-resistant bacterial pathogenesis with high morbidity and mortality in humans. An optical enzymatic biosensor for rapid and point-of-use detection of these antibiotics in food and water has been developed and tested. Enzymatic hydrolysis of β-lactams, on the electroactive polyaniline nanofibers, altered the polymeric backbone of the nanofibers, from emeraldine base form to emeraldine salt, which was measured as an increase in evanescent wave absorbance at 435 nm. The sensors were calibrated by spiking antibiotic-free milk with ceftazidime (as a model β-lactam analyte) in a linear range of 0.36-3600 nM (R2 = 0.98). The calibration was further validated for packaged milk, local cow milk, and buffalo milk. A similar calibration was devised for chicken meat samples in a linear range of 9-1800 nM (R2 = 0.982) and tap water in a linear range of 0.18-180 nM (R2 = 0.99). Interestingly, it was possible to use the same calibration for the determination of other β-lactam antibiotics (ampicillin, amoxicillin, and cefotaxime), which reflects the usefulness of the sensor for wide-scale deployment. The sensor performance was validated with a wastewater sample, from a wastewater treatment plant (WWTP), qualitatively analyzed by high-resolution liquid chromatography coupled with mass spectroscopy for detection of β-lactams. The sensor scheme developed and tested is of grassroot relevance as a quick solution for measurement of β-lactam residues in food and environment.

Crystal structure of the periplasmic sensor domain of histidine kinase VbrK suggests indirect sensing of β-lactam antibiotics

Bacterial two-component regulatory systems (TCS) play important roles in sensing environmental stimuli and responding to them by regulating gene expression. VbrK/VbrR, a TCS in Vibrio parahaemolyticus, confers resistance to β-lactam antibiotics through activating a β-lactamase gene. Its periplasmic sensor domain was previously suggested to detect β-lactam antibiotics by direct binding. Here, we report a crystal structure of the periplasmic sensing domain of VbrK (VbrKSD) using sulfur-based single-wavelength anomalous diffraction (S-SAD) phasing. Contrary to most bacterial sensor domains which form dimers, we show that VbrKSD is a monomer using size exclusion chromatography coupled with multi-angle light scattering. This observation is also supported by molecular dynamics simulations. To quantify the binding affinity of β-lactam antibiotics to VbrKSD, we performed isothermal titration calorimetry and other biophysical analyses. Unexpectedly, VbrKSD did not show any significant binding to β-lactam antibiotics. Therefore, we propose that the detection of β-lactam antibiotics by VbrK is likely to be indirect via an as yet unidentified mechanism.

A fiber optic nanoplasmonic biosensor for the sensitive detection of ampicillin and its analogs.

A novel optical immunosensor for the screening of ampicillin (Amp) residues has been developed. The biosensor is based on fiber optic particle plasmon resonance detection and uses an enhancement method called as fiber optic nanogold-linked immunosorbent assay (FONLISA) for the sensitive detection of antibiotics. A commercial antibody which had a higher affinity for ampicillin than for other β-lactam antibiotics was chosen. A surface competitive binding assay was used in which a fixed concentration of antibiotic-conjugated gold nanoparticles (AuNPs) competes with free unlabeled antibiotic molecules to measure the amount of binding with antibody molecules immobilized on an optical fiber. The synthesis of the 11-mercaptoundecanoic acid (MUA)-ampicillin conjugate facilitates the attachment of the Amp molecules to AuNPs via MUA which acts as a linker between them. This AuNP-Amp conjugate was then used for the detection of β-lactam antibiotics. The practical limit of detection obtained for Amp was 0.74ppb (7.4 × 10-10g/mL) which is lower than the recommended maximum residue limit (MRL) for β-lactams. The method also shows a wide linear range of 4 orders. Its applicability to the determination of ampicillin in spiked milk samples has been demonstrated with good recovery and reproducibility. Graphical abstract.

Europium chelate-labeled lateral flow assay for rapid and multiple detection of β-lactam antibiotics by the penicillin-binding protein.

For the multiple and rapid detection of β-lactams, the broadly specific penicillin-binding protein (PBP) that recognizes the β-lactam structure was prepared. A chromatographic strip based on europium chelate-loaded fluorescent microspheres was assembled with a goat anti-mouse antibody (C line) and penicillin coating (T line). The penicillin coating competes with free β-lactam antibiotics to bind PBP-labeled fluorescent microspheres. The strip can theoretically detect all kinds of β-lactams, including amoxicillin, ampicillin, oxacillin, ceftazidime, lenampicillin, cefoperazone, sultamicillin, cefotaxime, ceftriaxone, sulbenicillin, piperacillin, cephalothin, flucloxacillin, and mezlocillin, in samples within 10 min. The europium chelate-labeled lateral flow assay does not cross-react with other antibiotics, including chloramphenicol, tetracycline, sulfamethazine, enrofloxacin, gentamicin or lincomycin. In short, we developed a very useful method for preliminary screening of β-lactams.

Conductive imprinted polymers for the direct electrochemical detection of β-lactam antibiotics: The case of cefquinome

A biomimetic sensor for cefquinome (CFQ) was designed at multi-walled carbon nanotubes modified graphite screen-printed electrodes (MWCNTs-G-SPEs) as a proof-of-concept for the creation of a sensors array for β-lactam antibiotics detection in milk. The sensitive and selective detection of antibiotic residues in food and environment is a fundamental step in the elaboration of prevention strategies to fight the insurgence of antimicrobial resistance (AMR) as recommended by authorities around the world (EU, WHO, FDA). The detection strategy is based on the characteristic electrochemical fingerprint of the target antibiotic cefquinome. A conductive electropolymerized molecularly imprinted polymer (MIP) coupled with MWCNTs was found to be the optimal electrode modifier, able to provide an increased selectivity and sensitivity for CFQ detection. The design of CFQ-MIP was facilitated by the rational selection of the monomer, 4-aminobenzoic acid (4-ABA). The electropolymerization process of 4-ABA have not been fully elucidated yet; for this reason a thorough study and optimization of electropolymerization conditions was performed to obtain a conductive and stable poly(4-ABA) film. The modified electrodes were characterized by electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and cyclic voltammetry (CV). CFQ-MIP were synthesized at MWCNT-G-SPEs by electropolymerization in pH≈1 (0.1 M sulphuric acid) with a monomer:template ratio of 5:1. Two different analytical protocols were tested (single and double step detection) to minimize unspecific adsorptions and improve the sensitivity. Under optimal conditions, the lowest CFQ concentration detectable by square wave voltammetry (SWV) at the modified sensor was 50 nM in 0.1 M phosphate buffer pH 2.

Phenotypic Detection of Β-Lactam Antibiotics, Methicillin and Inducible Clindamycin Resistance Among Bacterial Isolates in Patients with Otitis Externa

Background: Otitis is a general terminology used for inflammation or infection of the ear; Staphylococcus aureus and Pseudomonas spp. are the most common causes of otitis externa. The resistance mechanism against the beta-lactams group is due to the production of β-lactamase enzymes by the bacteria; the enzymes in staphylococci are encoded by erm genes that confer inducible Clindamycin resistance. Objectives: This study aimed at investigating bacterial resistance by evaluating samples collected from Otitis Externa patients admitted to Ayatollah Roohani Hospital of Babol, Iran. Methods: Ear samples were collected from 72 patients with Otitis Externa referred to Ayatollah Roohani hospital during May 2012 to 2013. At first, the isolated bacteria were identified using appropriate differential and selective media, and then were tested for antimicrobial susceptibility testing following the disk diffusion method. Special diagnostic tests were also performed for the identification of ESBL, iAmpC, pAmpC, metallo beta lactamase producers and inducible resistance to clindamycin and methicillin resistant strains. Data were analyzed by the SPSS 22 statistical software. Results: Among the 65 isolated bacteria, 24 (36.9%) cases were found to be gram negative and 41 (63.1%) were gram positive; pAmpC beta-lactamase producers were found to have the highest frequency in gram negative bacteria. From 36 (87.8%) isolated CoNS, 18 (50%) bacteria were found to be resistant to the methicillin group and 4 (11.1%) cases had inducible resistance to clindamycin; All isolated S. aureus were sensitive to methicillin and clindamycin. Conclusions: Considering that some bacteria are concurrently able to produce different types of resistance enzymes, and also the fact that high prevalence rate of resistance belongs to CoNS, it is important and necessary to perform antimicrobial susceptibility testing as per clinical and laboratory standards institute (CLSI) methods in clinical laboratories.

Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of β-lactam antibiotics in foods

β-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of β-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by β-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual β-lactam was known. The IC50 values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 μg L(-1). Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected β-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 μg kg(-1), respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 β-lactams in edible tissues, among which 11 β-lactams controlled by European Union could be detected below maximum residue limits.

Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.

An amperometric affinity penicillin-binding protein magnetosensor for the detection of β-lactam antibiotics in milk

The preparation, characterization and performance evaluation of an amperometric affinity disposable magnetosensor, based on the use of a recombinant penicillin-binding protein (PBP) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of β-lactam antibiotic residues in milk are reported. The PBP was immobilized onto His-Tag-Isolation-modified magnetic beads (His-Tag-Isolation-MBs), and a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response obtained at -0.20 V vs. the Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as the transduction signal. The developed methodology showed very low detection limits (in the low ppb level) for the 6 antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. Due to the bioreceptor employed, this methodology was able to detect only the active form of β-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min.

Lysine Nζ-Decarboxylation Switch and Activation of the β-Lactam Sensor Domain of BlaR1 Protein of Methicillin-resistant Staphylococcus aureus

The integral membrane protein BlaR1 of methicillin-resistant Staphylococcus aureus senses the presence of β-lactam antibiotics in the milieu and transduces the information to the cytoplasm, where the biochemical events that unleash induction of antibiotic resistance mechanisms take place. We report herein by two-dimensional and three-dimensional NMR experiments of the sensor domain of BlaR1 in solution and by determination of an x-ray structure for the apo protein that Lys-392 of the antibiotic-binding site is posttranslationally modified by N(ζ)-carboxylation. Additional crystallographic and NMR data reveal that on acylation of Ser-389 by antibiotics, Lys-392 experiences N(ζ)-decarboxylation. This unique process, termed the lysine N(ζ)-decarboxylation switch, arrests the sensor domain in the activated ("on") state, necessary for signal transduction and all the subsequent biochemical processes. We present structural information on how this receptor activation process takes place, imparting longevity to the antibiotic-receptor complex that is needed for the induction of the antibiotic-resistant phenotype in methicillin-resistant S. aureus.

Fully Automated Analysis of β-Lactams in Bovine Milk by Online Solid Phase Extraction-Liquid Chromatography-Electrospray-Tandem Mass Spectrometry

A fully automated method for the detection of beta-lactam antibiotics, including six penicillins (amoxicillin, ampicillin, cloxacillin, dicloxacillin, oxacillin, and penicillin G) and four cephalosporins (cefazolin, ceftiofur, cefoperazone, and cefalexin) in bovine milk samples has been developed. The outlined method is based on online solid-phase extraction-liquid chromatography/electrospray-tandem mass spectrometry (SPE-LC/ESI-MS-MS). Target compounds were concentrated from 500 microL of centrifuged milk samples using an online SPE procedure with C18 HD cartridges. Target analytes were eluted with a gradient mobile phase (water + 0.1% formic acid/methanol + 0.1% formic acid) at a flow rate of 0.7 mL/min. Chromatographic separation was achieved within 10 min using a C-12 reversed phase analytical column. For unequivocal identification and confirmation, two multiple reaction monitoring (MRM) transitions were acquired for each analyte in the positive electrospray ionization mode (ESI(+)). Method limits of detection (LODs) in milk were well below the maximum residue limits (MRLs) set by the European Union for all compounds. Limits of quantification in milk were between 0.09 ng/mL and 1.44 ng/mL. The developed method was validated according to EU's requirements, and accuracy results ranged from 80 to 116%. Finally, the method was applied to the analysis of twenty real samples previously screened by the inhibition of microbial growth test Eclipse 100. This new developed method offers high sensitivity and accuracy of results, minimum sample pre-treatment, and uses for the first time an automated online SPE offering a high throughput analysis. Because of all these characteristics, the proposed method is applicable and could be deemed necessary within the field of food control and safety.

Analytical methodologies for the detection of β-lactam antibiotics in milk and feed samples

Antibiotic residues in milk are of great concern to dairy farmers, milk processors, regulatory agencies and consumers. In addition, the effectiveness of antibiotics is threatened by extensive, inappropriate use. In veterinary practice, antibiotics are utilized at therapeutic levels primarily to treat diseases and to prevent infections. They are also used at sub-therapeutic levels to increase feed efficiency and to promote growth in food-producing animals. The frequent, sometimes illegal, use of antibiotics may result in residues being found at different concentration levels in products of animal origin, such as milk. β-lactams comprise some of the antibiotics most frequently used for the treatment of sick animals in Europe. This review presents analytical methods that have been developed during the past decade for penicillin and cephalosporin residues in milk and feed samples. It includes microbiological approaches, biosensors, immunochemical techniques and chromatographic methods.

Biosensor Analysis of β-Lactams in Milk Using the Carboxypeptidase Activity of a Bacterial Penicillin Binding Protein

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.

Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg −1) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.

Analysis of β-Lactam Antibiotics Using a Microbial Receptor Protein-based Biosensor Assay

An optical biosensor assay for detection of β-lactam antibiotics in milk based on a microbial receptor protein was developed. The assay uses a general sensor surface previously described with a small organic molecule (H1) immobilized. A conjugate between a β-lactam (cephalosporin C) and a monoclonal H1 antibody is injected across the sensor surface before injection of the sample mixed with receptor protein. Receptor inhibited by β-lactam residues in the milk sample will not bind to the sensor surface and the reduction in response is inversely related to the β-lactam concentration of the sample. The detection limit for a number of commonly used β-lactams was below or near the respective maximum residue limit and the relative standard deviation (CV) for penicillin G in milk was 6-12% in the interval 2.0-12.5 μ g kg-1. For application in the field further optimization is needed to solve problems related to non-specific binding to the sensor surface.