中国科技论文在线 http://www.paper.edu.cn Anal Bioanal Chem DOI 10.1007/s00216-013-7311-5

RESEARCH PAPER

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

Juan Peng & Guyue Cheng & Lingli Huang & Yulian Wang & Haihong Hao & Dapeng Peng & Zhenli Liu & Zonghui Yuan

Received: 8 June 2013 /Revised: 1 August 2013 /Accepted: 15 August 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract β-Lactam antibiotics, including and detection in milk, beef, and chicken muscles with cefquinome , are commonly used in veterinary medicine. matrix calibration were 2.10, 30.68, and 31.13 μgkg−1, respec- Illegal use and abuse of β-lactams could cause allergy and tively. This study firstly established a rapid, simple, and accu- selected bacterial resistance. BlaR-CTD, the carboxy-terminal rate method for simultaneous detection of 15 β-lactams in of penicillin-recognizing protein BlaR from Bacillus edible tissues, among which 11 β-lactams controlled by Euro- licheniformis ATCC 14580, was utilized in this study to devel- pean Union could be detected below maximum residue limits. op a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was Keywords β-lactam . Receptor . BlaR-CTD . Purification . based on directly competitive inhibition of binding of horse- ELISA radish peroxidase-labeled 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 Introduction

single type of residual β-lactam was known. The IC50 values of 15 β-lactam antibiotics, including , ampicillin, β-Lactams are one class of the most important antibiotics , , , , , commonly used in veterinary medicine to treat bacterial infec- , , , cefquinome, , tions [1, 2]. The presence of β-lactam antibiotics in the food , , and its metabolite chain can cause allergic reactions in sensitized individuals, desfuroylceftiofur were evaluated and ranged from 0.18 to affect the intestinal flora of consumers, and increase drug- 170.81 μgL−1. Simple sample extraction method was carried resistant microorganisms [3]. Moreover, these resi- out with only phosphate-buffered saline, and the recoveries of dues in milk can inhibit the growth of starter cultures, affecting selected β-lactam antibiotics in milk, beef, and chicken were in the fermentation process of cheese and yogurt [4]. In order to the range of 53.27 to 128.29 %, most ranging from 60 to prevent the negative impact of β-lactam residues on human 120 %. The inter-assay variability was below 30 %. Limits of health and the entire ecosystem, withdrawal times and maxi- mum residue limits (MRLs) of β-lactams in food-producing Juan Peng and Guyue Cheng contributed equally to this work. animals have been established by regulatory authorities in most countries of the world. For example, the MRLs set by the Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7311-5) contains supplementary material, European Union for both benzylpenicillin and ampicillin is − − which is available to authorized users. 4 μgkg 1 in milk and 50 μgkg 1 in muscle [5]. β J. Peng : G. Cheng : L. Huang : Y. Wa ng : H. Hao : D. Peng : Presently, there are various methods for the detection of - Z. Liu : Z. Yuan (*) lactam residues, which are grouped into three main categories, National Reference Laboratory of Veterinary Drug Residues (HZAU) chromatographic method, microbiological approach, and immu- and MOA Key Laboratory for the Detection of Veterinary Drug noassay. The chromatographic method, normally used to confirm Residues in Foods, Huazhong Agricultural University, Wuhan, Hubei 430070, China the results of suspected contaminated samples, exhibits high e-mail: [email protected] specificity and excellent detectability. However, the application

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of this method is limited by complexity of sample preparation lactams demanded by EU, but this method was only applied to and expensive equipments [6]. The latter two methods are often milk sample. used in screening tests. Microbiological approach is low cost, but BlaR of Bacillus licheniformis is a trans-membrane protein, unspecific and time-consuming, and the detectability is poor [7]. playing a role in the regulation for the production of β-lactamase Immunoassay is sensitive, specific, rapid, and simple but the [21]. The carboxy-terminal of BlaR (BlaR-CTD) (residues 346– antibody cannot recognize penicillins and cephalosporins simul- 601) is located on the outer face of the membrane and serves as taneously. In addition, most MRLs are defined for the intact β- penicillin receptor [22]. BlaR-CTD from B. licheniformis 749/I lactams but not for metabolites or degradation products (except could complex most members of β-lactam antibiotics in just a penethamate, cephapirin, and ceftiofur). It is difficult to obtain short time [21, 23, 24]. A patent reported four receptor assays antibodies against intact drugs due to the instability of β-lactam based on BlaR-CTD for the detection of β-lactams in milk, ring, which is easily degraded during the process of antibody among which, the colloidal gold test strip could detect ten β- preparation [8, 9]. lactams below their respective EU MRL [23]. However, the strip The mechanism of action of β-lactams is based on the inhi- was unable to do the quantitative determination of β-lactams. bition of membrane-bound penicillin-binding proteins (PBPs) The purpose of the present study was to establish a receptor- involved in the final stages of bacterial synthesis based ELISA for the detection of beta-lactam antibiotics in [10].PBPscanbebroadlydividedintohighmolecularmass different edible tissues, including milk, beef, and chicken. (HMM) PBPs (≈50–100 kDa) and low-molecular mass (LMM) BlaR-CTD from B . licheniformis ATCC14580 was PBPs (≈30–40 kDa). HMM PBPs are essential for bacterial recombinantly overexpressed in Eschericha coli BL21 (DE3), survival and are the lethal targets for β-lactam antibiotics [11]. and the purified receptor protein was immobilized on a HMM PBPs are often used as specific receptors for β-lactam microplate. The binding of HRP-labeled ampicillin to BlaR- antibiotics in receptor assay, such as Charm ROSA, the CTD was non-competitively inhibited by β-lactam antibiotics Unisensor, etc. In enzyme assays, the inhibition of activity of in the samples, and the remaining BlaR-CTD/HRP-AMP com- one of the LMM PBPs, D-alanyl-D-alanine-carboxypeptidase, is plexes were detected by the substrate of tetramethylbenzidine measured [12]. Receptor assay, recognizing the intact β-lactam (TMB). ring, has the virtue of determining a great variety of β-lactams for both penicillins and cephalosporins, with higher sensitivity as well as less cost. Materials and methods Several receptor-based screening methods have been devel- oped for detecting β-lactam antibiotics. CHARM is a radioactive Chemicals assay which is harm to human health and requires expensive equipment and a trained operator [13]. PBP2a from Bacillus Ceftiofur, desfuroylceftiofur, , cephalexin, cefquinome, subtilis wasusedtopreparetheantibodyagainstcomplexof cefazolin, , nafcillin, dicloxacillin, benzylpenicillin, PBP2a-β-lactam, which were detected by enzyme-labeled sec- amoxicillin, oxacillin, ampicillin, and were from Dr ondary antibody [14]. This assay is complex because it requires Ehrenstorfer (Deisenhofen, Germany). Cefotaxime, ceftriaxone, preparation of antibody. Enzymatic assay, using DD -carboxypep- cefoperazone, cefapirin, 1–ethyl–3–(3–dimethylaminopropyl) tidase from Actinomadura R39 that specifically hydrolyzes the carbodiimide hydrochloride (EDC), N–hydroxysuccinimide D -alanyl-D -alanine groups of various peptides and certain (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, thioesters, is limited to the threshold of detection limits and the USA). Bovine serum albumin (BSA) was obtained from types of β-lactams [15–17]. PBP2x* (PBP2x with deletion of Bovogen (East Keilor, VIC, Australia). β-isopropyl-D-thio- amino acid residues 19–48) of pneumoniae R6 galactopyranoside (IPTG) and horseradish peroxidase (HRP) has high affinities for penicillins and cephalosporins [18]. were purchased from Solarbio (Beijing, China). TMB and Ni Cacciatore et al. [19] disclosed a biospecific interaction assay sepharose fastflow were from Thermo Fisher Scientific (Wal- based on the non-competitive inhibition between the binding of tham, MA, USA). Luria–Bertani (LB) were purchased form digoxigenin-labeled ampicillin (DIG-AMP) to PBP2x* and other Qingdao Hope Bio-Technology (Qingdao, China). Kanamycin β-lactams. It required an expensive equipment (surface plasmon was obtained from TianYuan (Wuhan, China). All other reagents resonance) and a labor-intensive sample pretreatment. Later, were purchased from Sinopharm Chemical Reagent Co., Ltd. Lamar et al. [9] developed a receptor-based microplate assay (Shanghai, China) and were all of analytical grade. with PBP2x* for detection of β-lactams in different matrices. The complexes of DIG-AMP/PBP2x* were detected by using Bacteria

horseradish peroxidase (HRP)-anti-digoxigenin Fab fragments. Labeling ampicillin with digoxigenin increased separation diffi- B. licheniformis ATCC14580 was obtained from the CICIM- culties and costs. Recently, Zeng et al. [20] reported a receptor- CU (Wuxi, China). E. coli BL21(DE3) and E. coli DH5α were based microplate assay with PBP2x* which could detect 15 β- purchased from TransGen Biotech (Wuhan, China). 中国科技论文在线 http://www.paper.edu.cn Development of a direct ELISA based on BlaR-CTD

Preparation of BlaR-CTD receptor protein possess similar procedure like ELISA. In detail, a fixed amount of BlaR-CTD protein was immobilized on the Chromosomal DNA was prepared from B. licheniformis surface of the 96-well Maxisorp microtiter plates (Nunc, ATCC14580, and the gene encoding BlaR-CTD was amplified Roskilde, Denmark) overnight at 4 °C (covered with a lid), by PCR, using chromosomal DNA as template and primers of 5'- 100 μL per well. After washing with PBST (PBS contain- cgcGGATCCATGCAAAGAGATACGCACTT-3' and 5'- ing 0.5 % Tween-20), the microplate was blocked with ccgCTCGAGTTATCGGGAAGCGGATGG-3' (underlined are 250 μL per well 1 % BSA in PBS for 12 h at 4 °C to restriction enzyme cutting sites for BamHIandXho I, respec- avoid unspecific binding. After washing three times with tively). An obtained 0.7-kb DNA fragment was digested with PBST, the microplates were dried and stored away from restriction enzymes of BamHIandXho Ifollowedbyinserting moisture at 4 °C or −20 °C (for longer time). To each well, into BamHI–Xho I site of pET-28a(+) to form the recombinant 50 μLofstandardsolutionorsampleand50μLofHRP- plasmid pET-28a(+)-BlaR-CTD. After the recombinant plasmid AMP solution was added in each well and incubated for was confirmed by sequencing analysis, it was transformed into 45 min at 37 °C. The plate was washed three times with E. coli BL21(DE3). PBST followed by addition of the enzyme substrate TMB. E. coli BL21(DE3) harboring pET-28a(+)-BlaR-CTD was After incubation for 15 min at 37 °C, the enzyme reaction −1 grown overnight in LB broth containing 50 μgmL kana- was stopped by addition of 2 M H2SO4, and the absor- mycin at 37 °C with vigorous shaking. Then the culture was bance at 450 nm was measured on Sunrise 2.5 microplate diluted 100-fold into fresh LB broth containing 50 μgmL−1 reader (Tecan, Seestrasse, Schweiz). kanamycin and incubated with vigorous shaking at 37 °C until The assay sensitivity was evaluated based on the concen-

the OD600 reached 0.6. The culture was induced with 1 mM tration of β-lactam antibiotic necessary to displace 50 % IPTG and cultured for 12 h at 18 °C. Bacteria were harvested inhibition of HRP-AMP from binding to the receptor (IC50). by centrifugation at 6,000×g for 10 min, washed twice with In order to determine the specificity of the BlaR-CTD, stan- ice cold phosphate-buffered saline (PBS) (8 g NaCl, 2.9 g dard curves for available β-lactam antibiotics, including

Na2HPO4,0.2gKCl,0.2gKH2PO4 per liter, pH 7.4). ceftiofur, desfuroylceftiofur, cefadroxil, cephalexin, Approximately 1 g cell pellet was suspended in 20 mL bind- cefquinome, cefazolin, carbenicillin, nafcillin, dicloxacillin,

ing buffer (7.6 g Na3PO4, 29.22 g NaCl, 0.68 g imidazole per benzylpenicillin, amoxicillin, oxacillin, ampicillin, cloxacil- liter, pH 7.4), and was disrupted by 1,000 bar high pressure lin, cefotaxime, ceftriaxone, cefoperazone, and cefapirin, were homogenizer. The suspension was centrifuged at 10,000×g established. The relative cross-reactivity (CR) of each antibi-

for 10 min, and the soluble fraction was loaded onto a 5 mL otic was calculated based on the IC50 values, following equa- 2+ Ni -charged chelating sepharose resin column (Novagen). tion CR=IC50 (cefquinome)/IC50 (β-lactam)×100 %. The column was washed and eluted following the manufac- turer’s instructions. The purified BlaR-CTD was dialysed Preparation of spiked samples against PBS, and judged by SDS-PAGE with Coomassie Brilliant Blue staining. The protein concentration was deter- All samples, including cow milk and the muscles of cattle and mined by the method of Bradford [25], and the protein was broiler chicken were collected from a local supermarket in stored at concentration of 500 μgmL−1 at −70 °C. Wuhan (Hubei, China). Typically, 500 g chicken or beef muscles were minced by a kitchen homogenizer and stored Horseradish peroxidase labeling of ampicillin at −20 °C until experiment. Milk was stored at 4 °C. For recovery studies, 1 g blank milk or 2 g blank homog- EDC (2.4 mg) and NHS (1.8 mg) were dissolved in 1 mL enized meat (beef and chicken muscles) were accurately PBS, and 4.4 mg HRP dissolved in 1 mL PBS was added. weighed in a centrifuge tube. β-lactam antibiotics diluted in After stirring at 4 °C for 2 h, 6.2 mg ampicillin dissolved in PBS to obtain suitable concentration were spiked to the tissue 1 mL PBS was added to the reaction mixture. The reaction samples. The spiked materials were placed on an automated mixture was stirred for 5 h at 4 °C and then was dialyzed shaking device for 15 min to ensure complete mixing of the β- against PBS. Verification of conjugated synthesis was lactam antibiotics and sample matrices. performed in an 8454 UV–visible spectrophotometer Blank and spiked milk samples were properly diluted with (Agilent, Santa Clara, USA). The conjugate solution was PBS and the mixtures were directly subjected to analysis stored at −20 °C in the presence of 50 % glycerol. without any pretreatment. For meat extraction, blank and spiked meat samples were added with certain volume of Development of a receptor-based ELISA PBS and were thoroughly mixed with a vortex mixer for 5 min. The mixed samples were centrifuged at 4,000×g for The principle of the receptor-based ELISA is illustrated in 10 min, and the supernatants were properly diluted with PBS Figure 1. Actually, this method is not a real ELISA, it for analysis. 中国科技论文在线 http://www.paper.edu.cn J. Peng et al.

Fig. 1 Schematic illustration of the principle of the receptor-based ELISA for blank sample (negative samples, left)and sample containing β-lactam antibiotics (positive samples, right)

Validation Results and discussion

Tissue samples (milk, beef, and chicken muscles) which Expression and purification of BlaR-CTD were proven by LC-MS/MS (according to the Entry-Exit Inspection and Quarantine Industry Standard of China SN/ BlaR-CTD, carboxy-terminal penicillin sensor of BlaR, was T2050–2008) to be free of β-lactams were assayed using heterogeneously expressed in E. coli BL21 (DE3) as a His- receptor assay to demonstrate the range of blank matrix tag-fusion protein (Fig. 2). We chose pET-28a as the expres- effects and to determine the limit of detection (LOD). The sion vector instead of pGEX-6P-1, which was previously used determination of LOD was based on 20 blank samples accepting no false positive rates, with an average+3× stan- dard deviation (SD). The accuracy and precision of the method were repre- sented by recovery and coefficients of variation (CVs), respectively. The recovery (%) of the each spiked β- lactam was established using five spiked duplicate blanks at indicated levels for three different analyses, and was calculated using the following equation: (concentration measured/concentration spiked)×100. CVs were determined by the analysis of the above samples spiked with each β- lactams at indicated levels for five different analyses. Each concentration level was tested three times in a time span of 2months.

Statistical analysis Fig. 2 SDS-PAGE analysis of purification of BlaR-CTD. Proteins were separated on a 12 % separation gel and stained with Coomassie Brilliant Blue. Lane 1 total cellular protein of E. coli BL21(DE3) harboring pET- Descriptive statistical parameters such as mean value, SD, and 28a(+) induced with IPTG; Lane 2 total cellular protein of E. coli CV were calculated. Statistical analysis of the data was BL21(DE3) harboring pET-28a(+)-BlaR-CTD induced with IPTG; Lane performed using Microsoft Excel 2003. 3 purified BlaR-CTD. M molecular mass standard 中国科技论文在线 http://www.paper.edu.cn Development of a direct ELISA based on BlaR-CTD

was expressed in large amount but as a form of inclusion body (data not shown). When the temperature was reduced to 18 °C, 60∼70 % of the target protein was expressed in soluble form. It is suggested that manipulation of protein translation rate using low temperature could reduced the production of inactive and misfolded protein in the inclusion body [27], but too low temperature would retard the growth of bacteria. With increased concentration of IPTG ranging from 0.1∼1.0 mmol/ L, the yield of target protein increased. Therefore, the optimal condition for the expression of recombinant BlaR-CTD was set at 18 °C with 0.1 mmol/L IPTG for induction, and the inducing time was for 12 h to obtain more target proteins. Fig. 3 The standard curve of the receptor-based ELISAwith cefquinome. As shown in Fig. 2, the band of BlaR-CTD was obvious The test was repeated five times with five replicates per concentration. after induction with IPTG compared to E. coli harboring Data were shown as means±standard deviations (error bars) (n =25) empty vector (lane 1), and was in a good agreement with the expected size of 26 kDa (lane 2). BlaR-CTD was purified in the preparation of PBP2x* [19], because pET-28a has a His- from the cytosol by immobilized metal ion affinity chroma- tag for purification which is much shorter than GST-tag in tography. The protein was eluted at 100 mM imidazole, and pGEX-6P-1. It was reported that His-tag would not influence revealed as one single band in SDS-PAGE (Fig. 2,lane3). the transpeptidase activity of PBP2x [26], and this tag did not Approximately 20 mg of 95 % pure BlaR-CTD was obtained need to be removed after purification because of its short size. from 1 L of cells. The purified BlaR-CTD was stored at Moreover, the vector of pET-28a employs kanamycin resis- −70 °C at a concentration of 500 μgmL−1. tance gene for screening, avoiding the use of β-lactams in PBP production, while ampicillin resistance gene in pGEX- Establishment of the receptor-based ELISA using BlaR-CTD 6P-1 should be changed into another non-β-lactam resistance gene in the preparation of PBPs. EDC and EDC/NHS methods have been successfully used in In the overexpression procedure, when the inducing tem- antigen production of β-lactams [20]. EDC links the carboxyl perature were 37 or 28 °C, the target proteins of BlaR-CTD group and amine group of targets with zero-length. The

Table 1 Linearity and cross-reactivity of BlaR-CTD with various β-lactams

−1 −1 Analytes Calibration curve Correlation coefficient (r)IC50 (μgL ) CR (%) EU MRL (μgkg )

Milk Muscle

Cefquinome y =−0.5462x +0.6546 0.9950 2.04 100 20 50 Benzylpenicillin y =−0.6482x +0.0529 0.9907 0.18 1150.85 4 50 Dicloxacillin y =−0.5765x +0.3068 0.9954 0.46 439.96 30 300 Amocillin y =−0.5516x +0.3340 0.9957 0.49 413.18 4 50 Ampicillin y =−0.5438x +0.3370 0.9957 0.50 405.78 4 50 Oxacillin y =−0.5039x +0.4383 0.9959 0.75 270.88 30 300 Cefoperazone y =−0.6406x +0.4436 0.9898 0.82 248.41 50 – Cefapirin y =−0.4696x +0.5391 0.9967 1.30 156.57 60 50 Cefalotin y =−0.5684x +0.5700 0.9962 1.33 153.74 –– Nafcillin y =−0.5362x +0.6391 0.9976 1.51 135.09 30 300 Cefazolin y =−0.4825x +1.0313 0.9976 12.79 15.93 50 – Ceftiofur y =−0.4988x +1.0328 0.9960 13.11 15.54 100 1000 Ceftriaxone y =−0.4624x +1.1000 0.9990 19.83 10.27 –– Cefotaxime y =−0.5297x +1.3251 0.9980 40.51 5.03 –– Desfuroylceftiofur y =−0.4868x +1.3267 0.9946 45.36 4.49 100 1000 Cefalexin y =−0.4945x +1.5882 0.9972 170.81 1.19 100 200 中国科技论文在线 http://www.paper.edu.cn J. Peng et al.

Table 2 Recoveries and coefficients of variation (CVs) of β-lactams require NHS for stabilization. Here, we chose ampicillin as spiked in milk (n =15) the conjugate in our assay for two reasons. Firstly, it has an Analytes Spiked level Mean recovery ± SDa CV amino group of side chain. Secondly, it is relatively stable (μgkg−1) (%) (%) under aqueous conditions at neutral pH, thus avoiding the cleavage of the β-lactam ring during the conjugation proce- Amocillin 2 75.97±12.58 16.55 dure [28]. In order to avoid the reaction of carboxyl group of 3 80.71±11.47 14.21 the β-lactam ring and amino group of HRP, the carboxyl of 4 89.83±11.65 12.98 HRP was activated first, and then the β-lactam was added, Ampicillin 2 98.31±29.52 30.03 offering amino group of side chain. Recently, it was demon- 3 92.82±24.27 26.15 strated that ampicillin and amoxicillin conjugates showed 4 104.69±28.12 26.85 stronger binding to PBP2x* than benzylpenicillin, cloxacillin, Benzylpenicillin 2 128.29±9.30 7.25 and cefquinome [20]. In our study, the OD450 value for BlaR- 3 117.18±8.86 7.76 CTD (>3) was much higher than that for PBP2x* (<1) in the 4 97.74±8.60 8.79 reaction with HRP-AMP (data not shown), indicating BlaR- Dicloxacillin 2 82.67±13.26 16.04 CTD has much stronger affinity towards ampicillin than 3 85.30±15.23 17.86 PBP2x*. 4 123.29±9.95 8.07 BlaR-CTD was screened against HRP-AMP for the best Oxacillin 2 72.94±11.34 15.55 dilutions of the coating proteins and conjugates at which the 3 69.54±10.99 15.81 OD450 value was near 2.0. The coating and blocking buffers 4 83.22±6.83 8.20 were also optimized according to OD450 values. As shown in Nafcillin 10 100.18±17.82 17.78 Electronic Supplementary Material Table S1, the optimal re- 20 92.99±12.38 13.31 ceptor assay conditions were set as 1.25 μgmL−1 of BlaR- 40 63.33±6.61 10.44 CTD concentration and 1:300 dilution of HRP-AMP deter-

Cefquinome 10 96.18±10.99 11.43 mined by square matrix titration. The OD450 value of the assay 20 94.24 ±7.07 7.50 was clearly better when the coating buffer was PBS than 40 74.20 ±4.49 6.06 carbonate buffer solution (see Electronic Supplementary Ma- Cefoperazone 10 119.57±9.14 7.65 terial Table S2), and BSA was better as blocking reagent than 20 102.24±6.65 6.51 ovalbumin (see Electronic Supplementary Material Table S3). 40 58.28±3.80 6.51 Moreover, when coating and blocking process were done at Cefapirin 10 78.76±10.73 13.63 low temperature of 4 °C, better results are gained. The reaction 20 80.18±5.42 6.76 condition for binding of β-lactam standards or samples was 40 63.96±2.53 3.96 set as at 37 °C for 45 min since the temperature was optimal Cefalotin 10 111.32±14.51 13.04 for the binding activity of BlaR-CTD and the OD450 value 20 96.73±10.09 10.43 reach the highest level for a time period of 45 min (data not 40 64.10±1.74 7.40 shown). Ceftiofur 50 82.44±10.90 13.22 The above conditions were the best to obtain the linear 100 91.43±13.12 14.35 portion of the response curve for cefquinome matrix calibra- − 200 83.29±10.63 12.76 tion (Fig. 3). The standard curve was y = 0.5462x +0.6546 Cefazolin 50 98.34±22.81 23.19 with good linearity of R =0.9950 at concentration ranged from μ −1 100 107.89±13.54 12.55 0.5 to 8 gL .TheIC50 value of cefquinome was 2.04± μ −1 200 96.69±7.4 7.72 0.40 gL (n =5). Ceftriaxone 50 117.74±14.05 11.93 β 100 121.21±11.22 9.25 Cross-reactivity of -lactam antibiotics 200 114.86±6.22 5.41 β Cefotaxime 50 104.15±15.02 14.42 For quantitative determination, a set of -lactam standards were used in the optimized receptor assay to establish the 100 92.87±14.34 15.44 calibration curves, and IC value was determined for each 200 95.06±14.61 15.37 50 β-lactam compared to cefquinome. As shown in Table 1,the

The test was repeated three times with five replicates per concentration IC50 values of 15 β-lactam antibiotics and one metabolite −1 a Standard deviation ranged from 0.18 to 170.81 μgL , exhibiting >1 % cross- reactivity relative to cefquinome. When the identity of a single intermediate product in EDC reaction is unstable in aqueous type of antibiotic is known, this method could be applied to solutions; therefore, the two-step conjugation procedures semiquantitative detection. 中国科技论文在线 http://www.paper.edu.cn Development of a direct ELISA based on BlaR-CTD

BlaR-CTD can detect β-lactam antibiotics with intact β- affinity than in the case of BlaR-CTD from B. licheniformis lactams exclusively [22]. The use of the BlaR-CTD receptors 749/I [23]. It is not surprising that BlaR-CTD exhibits lower for the detection of β-lactam antibiotics shows the following affinity for some of β-lactams. Since cloxacillin has a appreciable advantages. Firstly, BlaR-CTD can recognize a nucleus, and cephalexin is a kind of devoid of great number of β-lactam antibiotics, such as benzylpenicillin, leaving group, this may result in the lower acylation rate [29]. ampicillin, amoxicillin, carbenicillin, methylcillin, cloxacillin, Compared with PBP2x* [20], BlaR-CTD exhibited differ- 6-APA, monolactam, , , cephalexine, ent substrate profile of β-lactams. BlaR-CTD has much higher cephaloglycine, , nitrocephine, cefatoxime, affinity to penicillins than PBP2x* (e.g., benzylpenicillin, cefuoroxime, ceftiofur, cephapyrine, 7-ACA [23], which is dicloxacillin, amocillin, ampicillin), but the capability of bind- more than PBP2x* can recognize [18]. Secondly, the BlaR- ing to cephalosporins are much weaker than PBP2x* (e.g., CTD is capable of complexing a very large number of β- cefazolin, ceftiofur, cefalexin), with the exception of lactam antibiotics in a short time with high affinity [23, 29]. cefoperazone, cefapirin, and cloxacillin. Thirdly, BlaR-CTD protein can be produced in commercially useful amounts due to its recombinant origin, and the small Performance of the receptor-based ELISA size of BlaR-CTD favors its solubility in the recombinant expression. Based on the results from 20 different blank samples, the It is shown that the penicillin-binding site of BlaR-CTD LODs of cefquinome matrix calibration in milk, beef, and from B. licheniformis 749/I contains four structural elements chicken were 2.10, 30.68, and 31.13 μgkg−1,respectively. [24]. The center of the catalytic pocket is occupied by the The sample was fortified with three levels of a single β- Ser402-Thr-Tyr-Lys405 tetrad, in which the active site Ser402 lactam and then analyzed by the receptor-based ELISA. With is acylated by β-lactam. One side of the cavity is defined by the a simple and rapid extract method using only phosphate- Ser450-Thr-Thr452 motif, while the opposite side is delimited buffered saline, each sample was evaluated with three repli- by the Lys539-Thr-Gly541 triad. The bottom is closed by the cates to verify repeatability. The result of recoveries of 14 β- Ω-loop containing the Trp488-Leu489 residues. Homologous lactams in milk was shown in Table 2,withrecoveriesinthe analysis indicates that the amino acid sequence of BlaR-CTD range of 58.28∼128.29 % and the inter-assay CVs less than from B. licheniformis ATCC14580 has 90.2 % consensus with 30.03 %. The result of recoveries for 4 β-lactams in meat juice that of B. licheniformis 749/I. The four elements are consistent was listed in Table 3. The recoveries of β-lactams in beef in BlaR-CTD for both strains, except Thr540 is changed into ranged from 53.47∼125.24 % and the inter-assay CVs was Ala540 in BlaR-CTD from B. licheniformis ATCC14580. This less than 26.24 %. The recoveries of β-lactams in chicken may result in the distinct affinities to β-lactams displayed by ranged from 53.27∼127.10 % and the inter-assay CVs were the two different BlaR-CTDs, as in this study, benzylpenicillin, less than 29.51 %. nafcillin, oxacillin, dicloxacillin, and cefquinome exhibited Combined with the data in Tables 1 and 2, six penicillins much higher affinities, while cloxacillin showed much lower (nafcillin, dicloxacillin, benzylpenicillin, amoxicillin, oxacillin,

Table 3 Recoveries and coeffi- cients of variation (CVs) of β- Analytes Spiked level Beef Chicken −1 lactams spiked in beef and chicken (μgkg ) a a muscles (n =15) Mean recovery±SD CV Mean recovery±SD CV (%) (%) (%) (%)

Ampicillin 25 110.59±22.96 20.76 121.09±23.09 19.07 50 103.68±27.20 26.24 119.77±11.23 9.37 100 96.55±21.23 21.99 88.59±4.70 5.31 Benzylpenicillin 25 125.24±9.70 7.75 127.10±11.10 8.74 50 90.93±9.02 9.92 94.92±8.75 9.22 100 53.47±1.57 2.94 53.27±2.91 5.47 Oxacillin 50 107.03±25.17 23.52 114.61±23.35 20.37 100 106.52±10.21 9.58 108.53±13.19 12.15 200 74.64±10.32 13.83 74.76±5.92 7.72 The test was repeated three times Cefquinome 25 107.21±22.77 21.24 110.00±22.05 20.05 with five replicates per 50 70.90±16.08 22.68 90.51±26.71 29.51 concentration 100 80.24±14.29 17.81 99.00±22.76 22.99 a Standard deviation 中国科技论文在线 http://www.paper.edu.cn J. Peng et al.

and ampicillin) and five cephalosporins (ceftiofur, cefquinome, 9. Lamar J, Petz M (2007) Development of a receptor-based microplate cefazolin, cefoperazone, and cefapirin) could be detected at assay for the detection of beta-lactam antibiotics in different food matrices. Anal Chim Acta 586(1–2):296–303 concentrations below their respective EU-MRLs in milk by using 10. Holtje JV (1998) Growth of the stress-bearing and shape-maintaining BlaR-CTD from B. licheniformis ATCC14580. murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62(1): 181–203 11. Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P (2008) The penicillin-binding proteins: structure and role in pepti- Conclusions doglycan biosynthesis. FEMS Microbiol Rev 32(2):234–258 12. 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Cacciatore G, Petz M, Rachid S, Hakenbeck R, Bergwerff A Acknowledgments This work was supported by the National (2004) Development of an optical biosensor assay for detection Basic Research Program of China (grant No. 2013CB127201), of β-lactam antibiotics in milk using the penicillin-binding the Fundamental Research Funds for the Central Universities protein 2x*. Anal Chim Acta 520(1–2):105–115 (program No. 2013QC002 and 2011PY078) and the Program of 20. Zeng K, Zhang J, Wang Y, Wang ZH, Zhang SX, Wu CM, Shen JZ International S & T Cooperation Funded Project (program No. (2013) Development of a rapid multi-residue assay for detecting beta- 2011DFA32140). lactams using penicillin binding protein 2x*. Biomed Environ Sci 26(2):100–109 21. 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