Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2' of methicillin-resistant Staphylococcus aureus.
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.
Harrington
C R
CR
Coralab Research, Cambridge, U.K.
O'Hara
D M
DM
Reynolds
P E
PE
D000217:Acyltransferases / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D006602:Hexosyltransferases / Q000276:immunology / Q000302:isolation & purification*; D015151:Immunoblotting; D008712:Methicillin / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000276:immunology / Q000302:isolation & purification*; D002267:Muramoylpentapeptide Carboxypeptidase; D010403:Penicillin Resistance; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000276:immunology / Q000302:isolation & purification*; D011233:Precipitin Tests; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D000217:Acyltransferases; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D008712:Methicillin