Immunoassays to detect antibody to B. pertussis
antigens, especially PT, are used by many laboratories worldwide either for diagnosis or to evaluate immune responses to vaccines (1
). Thus, accurate and precise assays are needed. Consequently, this study was designed to focus on the potential influence of the source of PT as an important parameter for obtaining comparable results in different laboratories. The results presented here indicate that the four evaluated PT antigens showed very similar behaviors in the assay systems used by the participating laboratories. This is, to our knowledge, the first interlaboratory study of pertussis immunoassays carried out thus far in which each of the participating laboratories directly compared the antigenic behavior performance of PT antigens from different manufacturers. In addition to allowing a comparison of the coating performance of the four preparations, this design allowed the comparison of the four PT preparations among four laboratories.
The optimal coating concentration varied among the laboratories. Three of the four laboratories selected the same coating concentration for the four PTs. These laboratories used concentrations from 1 to 2 μg/ml. The fourth laboratory selected 4 to 9 μg/ml for the four PTs. Thus, there was some heterogeneity in the selected coating concentrations; however, these differences appeared to be minor and method specific. Variables that may have impacted the selection of coating concentration include general difference in the assay methodologies, choice of microtiter plates, buffer compositions, coating conditions (), and the method used to determine the optimal coating concentration (29
When each of the four PTs was compared within a single laboratory, excellent agreement among the four PT preparations was observed. For the four laboratories, rc values between the pairs of antigens ranged from 0.99, 0.99 to 1.00, 1.00, and 0.97 to 1.00. Because the coefficient takes into account both variability and quantitative agreement, such high values of rc indicate very similar performances between the four PT preparations. This consistency is also illustrated in the box plot (). The largest difference observed was between PT1 and PT3 in lab D. For this comparison, the median value was 0.14 for PT1 and −0.13 for PT3.
The comparisons between pairs of laboratories also displayed a high degree of agreement for each PT preparation, with rc measurements between 0.90 and 0.98, 0.93 and 0.99, 0.92 and 0.98, and 0.93 and 0.99 for the four antigens. By using the same PTs in each laboratory, we were able to eliminate one potential source of variability (i.e., PT) and compare the precision and comparability among laboratories. The small, but consistent, systematic differences among laboratories are also illustrated in the box plot (). The basis for these systematic differences are unknown, but may be related in part to the calibration of in-house standard relative to the IS. More importantly, despite numerous differences in procedures and reagents, good agreement was obtained when laboratories used the same PT as coating antigen and CBER3 or IS for calibration. Together, these concordance analyses suggest that the PT source was not the primary contributor to interlaboratory variability and that any of these four PTs could be considered for a harmonized assay.
By including sera from a variety of sources, this study was able to obtain data relevant to an additional outstanding question, namely, whether the PT source could lead to unintentional bias. To address this, samples for this study included sera from subjects immunized with inactivated PT produced by GSK, sera from subjects immunized with inactivated PT produced by SP, and sera from individuals infected naturally by strains circulating in the population. Additionally, the panel included sera from infants, toddlers, adolescents, and adults. Analyses were done for the various subgroups; in no case was a bias observed. Specifically, no bias was observed when the coating antigen matched the immunizing antigen. Similarly, the four PTs showed very similar behavior performances when the IgG anti-PT antibody levels in recently infected individuals were measured.
Each laboratory evaluated the CBER3 and IS within the collaborative study ( and ). CBER3 was included by all laboratories as a calibrator or control sample, and all laboratories obtained values that were, in general, within 25% of the assigned value. The IS was included both as a control sample and as a blind test sample. The measured values in many cases were greater than the assigned values; however, the extent of the overestimation varied among the laboratories. The highest estimates were obtained by lab B, which was approximately 50% above the assigned value. With respect to the primary purpose of the study, the four PT preparations yielded very similar estimates within each of the laboratories; however, because the units were assigned to the IS such that the unitages of IS and CBER3 are similar (30
), the results of our study suggest that there is some heterogeneity among laboratories in the unitage of IS compared to CBER3.
The data reported here are consistent with data reported from previous collaborative studies (13
). For example, the results from the collaborative study with 32 participating laboratories (17
) suggested that, despite widely different methods and reagents, different laboratories were able to obtain reasonable agreement when a common primary reference was used for calibration. Similarly, there was generally good agreement among 22 participating laboratories in the assignment of unitage of the IS (30
). The current study expands on these previous studies in two important ways. First, this study design allowed the direct comparisons of PT antigens within the same laboratory and of laboratories with the same antigen. Second, the study evaluated defined subgroups of sera to evaluate whether the PT source could lead to unintentional bias.
Two limitations of this study should be noted. First, the study was not exhaustive in that it did not evaluate PT from all available sources. This study was designed to include commonly used PT preparations, including two commercially available sources, but was not designed to investigate the impact, if any, of the minor amino acid differences that have been observed among some recently isolated strains (22
). The PTs were from three different laboratory strains (BP165, Tohama I, and 10536), but the study did not include PTs representing all strains of B. pertussis
currently in circulation. However, recent clinical sera from individuals infected with strains currently in circulation were evaluated. Second, due to reagent limitations, there was a limit to the number of laboratories that could participate. Thus, the study was designed to include four experienced laboratories with well-established methods. All laboratories used in-house qualified reagents, but the procedures varied with respect to the number of sample dilutions, calculation methods, detection reagents, and general methodology.
The reproducibility of results obtained here suggests that future efforts to obtain comparable data may be able to focus on assay harmonization rather than the development of a fully standardized assay that uses a common protocol and shared reagents. Assay harmonization efforts are more focused on the identification of critical parameters that affect assay performance, allowing laboratories to develop or adapt procedures and reagents that conform to those critical variables (18
). Many laboratories have well-established pertussis ELISAs, and harmonization of these methods rather than formal standardization may be more feasible and practical. The data obtained here suggest that the harmonized approach requiring each laboratory to characterize and qualify reagents fully, verify methodology, and calibrate carefully to a common reference preparation may be sufficient. The recent availability of an international pertussis reference serum was a major step toward the harmonization of assays (30
In conclusion, our present findings suggest that despite variation in the PT coating concentrations, excellent agreement among the four PTs within each laboratory and among the laboratories for each PT was observed. Therefore, we conclude that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine immunogenicity evaluation. The data from this study, as well as available sera and reagents, can serve as a resource for laboratories that are trying to generate data that can be compared to data from other laboratories.