Development and validation of an Australian in-house anti-pertussis toxin IgG and IgA enzyme immunoassay

Summary Aims Although anti-pertussis toxin (PT) immunoglobulin G (IgG) is considered one of the most specific serological markers for Bordetella pertussis infection, there are few commercial kits available in Australia. We aimed to present the process of development, quality control and on-going clinical validation of an anti-PT IgG and IgA enzyme immuno-assay (EIA) in use since 1999, and discuss the application of such tests in the diagnosis of B. pertussis infections. Methods A total of 1311 serum samples were used during multiple clinical validations from 1998 to 2010. The samples were drawn from healthy adults, children, patients with other respiratory infections, and patients with confirmed pertussis. Assay reproducibility, accuracy and precision criteria conformed to National Pathology Accreditation Advisory Council (NPAAC) guidelines. Results Using the World Health Organization clinical and/or laboratory definition of a definite case as the comparative standard, sensitivity was 84% [95% confidence interval (CI) 75–93] and specificity was 98% (95%CI: 90–100) for anti-PT IgG. Sensitivity was 72% (95%CI 64–80) and specificity was 98% (95%CI 90–100) for anti-PT IgA. There was minimal background positivity in either healthy adults or children using the established cut-offs. There was no appreciable effect of immunisation or cross reactions with other respiratory pathogens. Conclusion Serological evaluation of various populations enabled the development of an anti-PT IgG and IgA EIA assay which was suitable for the diagnosis of acute infection in convalescent samples from clinically confirmed cases. Repeated evaluations of population-based cut-offs are required for in-house assays to ensure they remain clinically relevant. The subsequent validation of the cut-offs with WHO international standards has been published in a recent prospective study. 6