[Preparation, characterization and preliminary application of monoclonal antibody against the transpeptidase domain of MRSA-PBP2a].
OBJECTIVE
To prepare monoclonal antibody (mAb) against the penicillin binding protein 2a (PBP2a) of MRSA and establish a latex agglutination assay to detect PBP2a.


METHODS
BALB/c mice were immunized with the recombinant transpeptidase domain of PBP2a expressed by gene-engineering. Mouse mAb against PBP2a was obtained with hybridoma technique. mAb's characteristics (IgG subclasses, titers, specificities, and affinities) were identified and determined by indirect ELISA and Western blot. Polystyrene beads were sensitized with mAb F2 by physical adsorption, and a latex agglutination assay was developed to detect PBP2a.


RESULTS
Two strains of hybridoma cell lines, which could stably secret anti-PBP2a mAb were obtained, named F1 and F2. The isotype of F1 and F2 was both IgG1. Ascites titers were 0.5x10(-6)-1x10(-6) and the affinity constants (Kaff) were 1.57 x 10(8) M(-1) and 5.43 x 10(9) M(-1), respectively. Western blot analysis showed that both the mAbs had specific binding abilities with both recombinant protein and clinically isolated MRSA-PBP2a. The sensitivity of the anti-PBP2a latex agglutination assay with serial dilutions of purified PBP2a was found to be 1 mg/L.


CONCLUSIONS
The obtained two strains of hybridoma cell lines can secret anti-PBP2a mAb stably, which lays the foundation for establishment of a simple and rapid anti-PBP2a latex agglutination assay kit.
Xu
Xia
X
Department of Medical Laboratory Sciences, Guangzhou Medical College, Guangzhou 510182, China. xuxia503@yahoo.com.cn
Tang
Xiao-hua
XH
Xie
Gui-e
GE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000378:metabolism; D000915:Antibody Affinity; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000276:immunology; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases