Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x
Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x
复制标题
DOI：
10.1016/j.aca.2004.06.060
复制
发表时间：
2004-08-23
影响因子：
6.2
通讯作者：
Bergwerff, AA
中科院分区：
文献类型：
--
作者：
Cacciatore, G;Petz, M;Bergwerff, AA
建立了一种基于表面等离子体共振（SPR）生物传感器的牛奶中青霉素类和头孢菌素类抗生素残留检测方法。该试验基于地高辛标记的氨苄青霉素（DIG-AMPI）与肺炎链球菌可溶性青霉素结合蛋白2x衍生物（PBP 2x*）结合的抑制作用。在第一步中，将样品与PBP 2x* 孵育，由此阳性样品中的β-内酰胺将结合PBP 2x*。然后在第二温育步骤中使未复合的PBP 2x* 与DIG-AMPI形成复合物。在地高辛的基于SPR的生物特异性相互作用测定（BIA）中检测所形成的DIG-AMPI/PBP 2x*-复合物，所述生物特异性相互作用测定具有固定在传感器芯片上的针对地高辛的抗体。虽然发生基质成分与传感器芯片的结合（非特异性结合），但在脱脂散装原料奶样品中可检测到苄青霉素、氨苄青霉素、阿莫西林、氯唑西林、头孢氨苄和头孢哌酮，其浓度对应于欧盟规定的最大残留限量（MRL）。通过分析来自19头奶牛的个体原料奶样品，更详细地检查了基质组分对测定性能的影响。与散装原料奶样品相比，单个样品显示出更高的基质干扰水平和变化。非特异性结合可以通过热处理步骤、离心步骤和向样品中加入羧甲基化葡聚糖降低至更低和更恒定的水平。用这种样品制备液，在单个原料奶样品中可以检测到最大残留限量（4 μ g/kg）的苄青霉素。因此，该测定可作为常规使用的筛选试验的基础。(C)2004 Elsevier B. V.保留所有权利。
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 beta-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 mug kg(-1)) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use. (C) 2004 Elsevier B.V. All rights reserved.