title: Development of a novel multiplex electrochemiluminescent-based immunoassay for quantification of human serum IgG against 10 Staphylococcus aureus toxins.
abstract: An electrochemiluminescent (ECL)-based multiplex immunoassay using Meso-Scale Discovery (MSD) technology was developed for detecting antibody response toward 10 Staphylococcus aureus (S. aureus) exotoxins. These 10 antigens included three different groups of toxins: 1) single component pore-forming toxins such as alpha- and delta-hemolysins, 2) the bicomponent pore-forming toxin Panton-Valentine leukocidin (PVL), comprised of LukS-PV and LukF-PV subunits, and 3) enterotoxin/superantigens - Staphylococcal enterotoxins A (SEA), B (SEB), C1 (SEC1), D (SED), K (SEK) and Toxic shock syndrome toxin-1 (TSST-1). Assay development included optimization steps with a conventional SEB ELISA-based serological assay and then optimized parameters were transferred and re-optimized in a singleplex ECL format. Finally, two pentaplex solid-phase ECL formats were developed. As proof of concept, one set of pentaplex ECL data was compared with conventional ELISA results. During the assay development controls were screened and developed for both the singleplex and multiplex assays. ECL-based multiplex assays were more sensitive with a wide dynamic range and proved more time-efficient than conventional ELISAs. Using the newly developed ECL method we showed, for the first time, that delta-hemolysin toxin can induce an immune response as antibody titers could be detected.
authors.affiliation: Integrated Biotherapeutics Inc., Gaithersburg, MD 20878, United States. Electronic address: rajan@integratedbiotherapeutics.com.
authors.forename: Rajan P
authors.initials: RP
authors.lastname: Adhikari
authors.affiliation: Integrated Biotherapeutics Inc., Gaithersburg, MD 20878, United States.
authors.forename: Christian
authors.initials: C
authors.lastname: Haudenschild
authors.affiliation: Integrated Biotherapeutics Inc., Gaithersburg, MD 20878, United States.
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Sterba
authors.affiliation: Integrated Biotherapeutics Inc., Gaithersburg, MD 20878, United States.
authors.forename: Sara
authors.initials: S
authors.lastname: Sahandi
authors.affiliation: Integrated Biotherapeutics Inc., Gaithersburg, MD 20878, United States.
authors.forename: Sven
authors.initials: S
authors.lastname: Enterlein
authors.affiliation: Integrated Biotherapeutics Inc., Gaithersburg, MD 20878, United States.
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Holtsberg
authors.affiliation: Integrated Biotherapeutics Inc., Gaithersburg, MD 20878, United States.
authors.forename: M Javad
authors.initials: MJ
authors.lastname: Aman
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood*; D001426:Bacterial Proteins / Q000276:immunology; D001427:Bacterial Toxins / Q000276:immunology*; D004768:Enterotoxins / Q000276:immunology; D005098:Exotoxins / Q000276:immunology; D006460:Hemolysin Proteins / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000097:blood*; D007956:Leukocidins / Q000276:immunology; D008163:Luminescent Measurements / Q000379:methods*; D013211:Staphylococcus aureus / Q000276:immunology*; D018089:Superantigens / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D005098:Exotoxins; D006460:Hemolysin Proteins; D007074:Immunoglobulin G; D007956:Leukocidins; C078284:Panton-Valentine leukocidin; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal; C026066:enterotoxin A, Staphylococcal; C025994:delta hemolysin protein, Staphylococcus aureus
keywords: Delta toxin; ELISA serology; Multiplex; Staphylococcal exotoxins