title: Development of a qualitative assay for screening of Bordetella pertussis isolates for pertussis toxin production.
abstract: Bordetella pertussis infection has been increasing in the US, with reported cases reaching over 50,000 in 2012, a number last observed in the 1950s. Concurrently, B. pertussis lacking the pertactin protein, one of the immunogens included in the acellular vaccine formulations, has rapidly emerged since 2010, and has become the predominant circulating phenotype. Monitoring the production of the remaining acellular vaccine immunogens, such as pertussis toxin (Pt), is a critical next step. To date, methods for screening Pt have been either through genomic sequencing means or by conventional ELISAs. However, sequencing limits detection to the DNA level, missing potential disruptions in transcription or translation. Conventional ELISAs are beneficial for detecting the protein; however, they can often suffer from poor sensitivity and specificity. Here we describe a rapid, highly sensitive and specific electrochemiluminescent capture ELISA that can detect Pt production in prepared inactivated bacterial suspensions. Over 340 isolates were analyzed and analytical validation parameters, such as precision, reproducibility, and stability, were rigorously tested. Intra-plate and inter-plate variability measured at 9.8% and 11.5%, respectively. Refrigerated samples remained stable for two months and variability was unaffected (coefficient of variation was 12%). Interestingly, despite the intention of being a qualitative method, the assay was sensitive enough to detect a small, but statistically significant, difference in protein production between different pertussis promoter allelic groups of strains, ptxP1 and ptxP3. This technology has the ability to perform screening of multiple antigens at one time, thus, improving testing characteristics while minimizing costs, specimen volume, and testing time.
authors.affiliation: Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Irina
authors.initials: I
authors.lastname: Gates
authors.affiliation: Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Marina
authors.initials: M
authors.lastname: DuVall
authors.affiliation: Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Ju
authors.affiliation: Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: M Lucia
authors.initials: ML
authors.lastname: Tondella
authors.affiliation: Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Lucia
authors.identifier: http://orcid.org/0000-0002-1495-0379
authors.initials: L
authors.lastname: Pawloski
mesh_terms: D000704:Analysis of Variance; D001886:Bordetella pertussis / Q000302:isolation & purification* / Q000378:metabolism*; D055664:Electrochemical Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005615:Freezing; D006801:Humans; D008163:Luminescent Measurements; D037342:Pertussis Toxin / Q000378:metabolism*; D012034:Refrigeration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D014917:Whooping Cough / Q000175:diagnosis* / Q000378:metabolism
chemical_list: D037342:Pertussis Toxin