Development of an Immunochromatographic Strip for Simple Detection of Penicillin-Binding Protein 2′

Abstract

ABSTRACT 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.