College of Veterinary Medicine, China Agricultural University, Beijing 100094, People's Republic of China; National Reference Laboratories for Veterinary Drug Residue, District Haidian, Beijing 100193, People's Republic of China.
Anal Biochem. 2013 Nov 15;442(2):158-65. doi: 10.1016/j.ab.2013.07.042. Epub 2013 Aug 6.
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.
肺炎链球菌可溶性青霉素结合蛋白 3（sPBP3（∗））以六组氨酸融合蛋白的形式在大肠杆菌中表达。该蛋白经过纯化后，被用于开发一种基于直接竞争的微孔板检测方法，用于检测牛奶中的青霉素和头孢菌素类抗生素。该检测方法基于游离β-内酰胺抗生素在牛奶中竞争性抑制辣根过氧化物酶标记的氨苄青霉素（HRP-Amp）与 sPBP3（∗）结合。在优化条件下，大多数β-内酰胺抗生素（11 种青霉素和 16 种头孢菌素）可以在欧盟规定的最大残留限量（MRL）对应的浓度下被检测到。对添加牛奶样品的分析表明，在脱脂牛奶中的回收率为 74.06%至 106.31%，在全脂牛奶中的回收率为 63.97%至 107.26%，变异系数（CV）小于 16%。基于 sPBP3（∗）的开发方法具有高灵敏度和广泛的青霉素和头孢菌素亲和力，有望成为一种快速检测牛奶中β-内酰胺类抗生素的筛选检测方法。
