Development and Analytical Validation of an Immunoassay for Quantifying Serum Anti-Pertussis Toxin Antibodies Resulting from Bordetella pertussis Infection

Adequately sensitive and specific methods to diagnose pertussis in adolescents and adults are not widely available. Currently, no Food and Drug Administration-approved diagnostic assays are available for the serodiagnosis of Bordetella pertussis. Since concentrations of B. pertussis-specific antibodies tend to be high during the later phases of disease, a simple, rapid, easily transferable serodiagnostic test was developed. This article describes test development, initial evaluation of a prototype kit enzyme-linked immunosorbent assay (ELISA) in an interlaboratory collaborative study, and analytical validation. The data presented here demonstrate that the kit met all prespecified criteria for precision, linearity, and accuracy for samples with anti-pertussis toxin (PT) immunoglobulin G (IgG) antibody concentrations in the range of 50 to 150 ELISA units (EU)/ml, the range believed to be most relevant for serodiagnosis. The assay met the precision and linearity criteria for a wider range, namely, from 50 to 200 EU/ml; however, the accuracy criterion was not met at 200 EU/ml. When the newly adopted World Health Organization International Standard for pertussis antiserum (human) reference reagent was used to evaluate accuracy, the accuracy criteria were met from 50 to 200 international units/ml. In conclusion, the IgG anti-PT ELISA met all assay validation parameters within the range considered most relevant for serodiagnosis. This ELISA was developed and analytically validated as a user-friendly kit that can be used in both qualitative and quantitative formats. The technology for producing the kit is transferable to public health laboratories.Despite effective immunization with pertussis vaccines, pertussis remains endemic. Recently reported disease has increased in adolescents and adults, among whom the diagnosis of pertussis is especially problematic (7, 8, 10, 16, 30). Routine surveillance, outbreak investigations, and implementation of control measures have been limited by a lack of validated and standardized tests to identify suspected cases of pertussis (4, 29, 32, 36, 38). By the time health care is sought and pertussis is suspected, isolation of Bordetella pertussis by culture or confirmation by PCR is uncommon (24, 29, 31, 36). Although detecting the physical presence of the bacterium is difficult, antibody titers directed to B. pertussis tend to be elevated during the later phase of illness and persist after the infection is resolved, so the development and validation of a simple and rapid serodiagnostic test has practical utility for diagnosing pertussis infections. The ability to confirm outbreaks quickly using a serologic test has the potential to conserve resources that have been used for outbreaks mistakenly attributed to pertussis (4). Additionally, the routine availability of a serodiagnostic test for pertussis would provide significant public health benefit by furnishing public health officials with a more accurate assessment of the burden of disease in the United States and with a better understanding of the role of adolescents and adults in the transmission of pertussis. Acellular pertussis vaccines for adults and adolescents have recently been licensed (5, 6); however, the absence of readily available, validated and standardized tests to confirm suspected cases in these older age groups has hampered the collection of the epidemiological data required to guide developers and public health officials in effective utilization of these vaccines (11, 12, 32). A serodiagnostic test could supply these data and allow the design and evaluation of control strategies.A large body of evidence is now available to demonstrate that measurement of specific antibodies could assist in the laboratory confirmation of pertussis (8, 13-15, 17, 20); however, the criteria defining the infection threshold are not well agreed upon by international and national health organizations. One proposal for threshold values was based on the measurement of antibodies against pertussis toxin (PT), filamentous hemagglutinin, and fimbria types 2 and 3 in a population of more than 6,000 U.S. residents of ages 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey (2). Based on the mixture modeling of these data to identify hypothesized exposure groups, an anti-PT immunoglobulin G (IgG) level of >94 ELISA units (EU)/ml was proposed as the diagnostic cutoff point for recent infection, with a lower value of >49 EU/ml as an intermediate cutoff that suggested possible infection (3). Alternate diagnostic thresholds have been developed and applied. Specifically, the Massachusetts State laboratory has utilized a cutoff value of 200 EU/ml for almost 20 years (23), and De Melker et al. (9) adopted a value of 125 EU/ml for routine use in The Netherlands. Thus, the above studies established a variety of threshold cutoffs for anti-PT titers that range from 49 to 200 EU/ml. Final assessment of these proposed diagnostic cutoff points requires a prospective clinical study including patients with confirmed B. pertussis infection. By establishing accurate cutoff values for anti-PT titers for patients currently or recently ill, serological detection may provide a qualitative assessment of whether a test sample has anti-PT titers that are higher or lower than appropriately defined positive and negative control values.Despite these potential benefits, no Food and Drug Administration (FDA)-approved diagnostic assays are currently available for the serodiagnosis of B. pertussis infection, and none of the published methods (1, 9, 17, 19, 23, 25-27, 33-35, 37) have been demonstrated to be readily transferable to public health laboratories. Thus, the overall goal of this project is to develop a simple and readily transferable enzyme-linked immunosorbent assay (ELISA) for the measurement of anti-PT IgG in human serum samples that subsequently could be subjected to an appropriate clinical assessment. A single-serum dilution-based ELISA procedure with ready-to-use reagents was designed and optimized to quantify the anti-PT range thought relevant for diagnosing late-stage pertussis infections. We describe the initial assay development, initial evaluation of the prototype kit by an interlaboratory collaborative study, and assay validation study.