title: Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus.
abstract: Panton-Valentine Leukocidin (PVL) produced by community acquired methicillin Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing pneumonia comprised of two fractions, namely PVL S and PVL F. In the present study, three monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against recombinant PVL-S (35kDa) protein of S. aureus. All the three MAbs specifically reacted to confirm PVL-S positive strains of S. aureus recovered from clinical samples in Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14 different pathogenic bacteria belonging to other genera and species in Western blot analysis. Furthermore, a simple dot-ELISA method was standardized for the identification of PVL-S toxin containing S. aureus strains. Initially in dot-ELISA, Protein A (Spa) of S. aureus posed background noise problems due to the non-specific binding of antibodies resulting in false positive reactions. With the addition of 10mM diethylpyrocarbonate (DEPC) along with 5% milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL monoclonal antibodies in dot-ELISA. Once standardized, this simple dot-ELISA was evaluated with nine PVL positive strains recovered from food, environmental and clinical samples and the results were compared with PCR assay for the presence of PVL toxin genes and also with Western blot analysis. A 100% correlation was found between dot-ELISA, PCR assay and Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA system can be of immense help in providing, rapid detection of the PVL toxin containing S. aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of CA-MRSA.
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, Karnataka, India. Electronic address: nivedithaskotyan@gmail.com.
authors.forename: Niveditha Sundar
authors.initials: NS
authors.lastname: Poojary
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, Karnataka, India. Electronic address: shylram@yahoo.co.in.
authors.forename: Shylaja
authors.initials: S
authors.lastname: Ramlal
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, Karnataka, India. Electronic address: radhikamurs@yahoo.co.in.
authors.forename: Radhika Madan
authors.initials: RM
authors.lastname: Urs
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, Karnataka, India. Electronic address: drmuralihs@yahoo.co.in.
authors.forename: Murali Harishchandra
authors.initials: MH
authors.lastname: Sripathy
authors.affiliation: Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, Karnataka, India. Electronic address: drharshvardhanbatra@gmail.com.
authors.forename: Harsh Vardhan
authors.initials: HV
authors.lastname: Batra
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D015153:Blotting, Western; D017714:Community-Acquired Infections; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005098:Exotoxins / Q000276:immunology*; D007956:Leukocidins / Q000276:immunology*; D016106:Methicillin Resistance; D055624:Methicillin-Resistant Staphylococcus aureus / Q000276:immunology*; D051379:Mice; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin
keywords: Diethylpyrocarbonate; Dot-ELISA; Panton-Valentine Leukocidin (PVL); Staphylococcus aureus