Matsui Hidehito, Hanaki Hideaki, Inoue Megumi, Akama Hiroyuki, Nakae Taiji, Sunakawa Keisuke, Omura Satoshi
Laboratory for Antimicrobial Agents, Kitasato University, 5-9-1 Shirokane, Minato-Ku, Tokyo 108-8641, Japan.
Clin Vaccine Immunol. 2011 Feb;18(2):248-53. doi: 10.1128/CVI.00252-10. Epub 2010 Dec 22.
Infections with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MR-CNS) are a serious problem in hospitals because these bacteria produce penicillin-binding protein 2' (PBP2' or PBP2a), which shows low affinity to β-lactam antibiotics. Furthermore, the bacteria show resistance to a variety of antibiotics. Identification of these pathogens has been carried out mainly by the oxacillin susceptibility test, which takes several days to produce a reliable result. We developed a simple immunochromatographic test that enabled the detection of PBP2' within about 20 min. Anti-PBP2' monoclonal antibodies were produced by a hybridoma of recombinant PBP2' (rPBP2')-immunized mouse spleen cells and myeloma cells. The monoclonal antibodies reacted only with PBP2' of whole-cell extracts and showed no detectable cross-reactivity with extracts from other bacterial species tested so far. One of the monoclonal antibodies was conjugated with gold colloid particles, which react with PBP2', and another antibody was immobilized on a nitrocellulose membrane, which captures the PBP2'-gold colloid particle complex on a nitrocellulose strip. This strip was able to detect 1.0 ng of rPBP2' or 2.8 × 10(5) to 1.7 × 10(7) CFU of MRSA cells. The cross-reactivity test using 15 bacterial species and a Candida albicans strain showed no detectable false-positive results. The accuracy of this method in the detection of MRSA and MR-CNS appeared to be 100%, compared with the results obtained by PCR amplification of the PBP2' gene, mecA. This newly developed immunochromatographic test can be used for simple and accurate detection of PBP2'-producing cells in clinical laboratories.
耐甲氧西林金黄色葡萄球菌（MRSA）和耐甲氧西林凝固酶阴性葡萄球菌（MR - CNS）感染是医院面临的严重问题，因为这些细菌会产生青霉素结合蛋白2'（PBP2'或PBP2a），该蛋白对β - 内酰胺类抗生素亲和力较低。此外，这些细菌对多种抗生素具有耐药性。这些病原体的鉴定主要通过苯唑西林药敏试验进行，该试验需要数天才能得出可靠结果。我们开发了一种简单的免疫层析试验，能够在约20分钟内检测到PBP2'。抗PBP2'单克隆抗体由重组PBP2'（rPBP2'）免疫的小鼠脾细胞与骨髓瘤细胞杂交产生。该单克隆抗体仅与全细胞提取物中的PBP2'反应，与目前测试的其他细菌物种的提取物未显示出可检测到的交叉反应。其中一种单克隆抗体与能与PBP2'反应的金胶体颗粒偶联，另一种抗体固定在硝酸纤维素膜上，该膜可在硝酸纤维素条带上捕获PBP2'-金胶体颗粒复合物。该条带能够检测到1.0 ng的rPBP2'或2.8×10⁵至1.7×10⁷CFU的MRSA细胞。使用15种细菌物种和一株白色念珠菌进行的交叉反应试验未显示出可检测到的假阳性结果。与通过PBP2'基因mecA的PCR扩增获得的结果相比，该方法检测MRSA和MR - CNS的准确性似乎为100%。这种新开发的免疫层析试验可用于临床实验室简单、准确地检测产生PBP2'的细胞。
