Development of a Direct ELISA Based on Carboxy-Terminal of Penicillin-Binding Protein Blar for the Detection of Β-Lactam Antibiotics in Foods

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 penicillin-binding protein BlaR for the detection of β-lactam antibiotics 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 penicillins and detection in milk, beef, and chicken muscles with cefquinome cephalosporins, 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 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 Introduction single type of residual β-lactam was known. The IC50 values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, β-Lactams are one class of the most important antibiotics amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, commonly used in veterinary medicine to treat bacterial infec- cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, tions [1, 2]. The presence of β-lactam antibiotics in the food cefotaxime, cefalexin, ceftiofur 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 antibiotic 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 maximum 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 转载中国科技论文在线 http://www.paper.edu.cn J. Peng et al. 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 peptidoglycan 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 developed 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, cefadroxil, cephalexin, cefquinome, subtilis wasusedtopreparetheantibodyagainstcomplexof cefazolin, carbenicillin, nafcillin, dicloxacillin, benzylpenicillin, PBP2a-β-lactam, which were detected by enzyme-labeled sec- amoxicillin, oxacillin, ampicillin, and cloxacillin 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 Streptococcus 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

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