title: A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.
abstract: Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.
authors: Department of Biochemistry, University of Illinois, Urbana, Illinois, United States of America.
authors: Preeti
authors: P
authors: Sharma
authors: ImmuVen, Inc., University of Illinois Research Park, Champaign, Illinois, United States of America.
authors: Ningyan
authors: N
authors: Wang
authors: ImmuVen, Inc., University of Illinois Research Park, Champaign, Illinois, United States of America.
authors: Adam S
authors: AS
authors: Chervin
authors: ImmuVen, Inc., University of Illinois Research Park, Champaign, Illinois, United States of America.
authors: Cheryl L
authors: CL
authors: Quinn
authors: Department of Biochemistry, University of Illinois, Urbana, Illinois, United States of America.
authors: Jennifer D
authors: JD
authors: Stone
authors: Department of Biochemistry, University of Illinois, Urbana, Illinois, United States of America.
authors: David M
authors: DM
authors: Kranz
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis*; D001681:Biological Assay / Q000379:methods*; D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay; D005098:Exotoxins / Q000032:analysis*; D005434:Flow Cytometry; D008863:Microspheres; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry; D013211:Staphylococcus aureus / Q000737:chemistry*; D013297:Streptococcus pyogenes / Q000737:chemistry*
chemical_list: D001427:Bacterial Toxins; D005098:Exotoxins; D016693:Receptors, Antigen, T-Cell, alpha-beta