Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2' of methicillin-resistant Staphylococcus aureus |
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| Authors: | Charles R Harrington Denise M O'Hara Peter E Reynolds |
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| Institution: | Coralab Research, Cambridge, U.K. |
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| Abstract: | The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains. |
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| Keywords: | Staphylococcus aureus (MRSA) Penicillin-binding protein Immunoaffinity purification Monoclonal antibody ELISA |
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