The overall goal of this project was to develop an ELISA kit that could accurately and precisely measure the concentrations of IgG anti-PT in the range of interest for diagnostic assays. Critical reagents were prequalified so that the assay could be performed by any laboratory with immunoassay equipment and experience. Because a variety of clinical laboratories have adopted proposed threshold cutoffs of 49 to 200 EU/ml, the assay and validation protocol were designed to measure samples within this range. We sought optimal performance around 100 EU/ml. Based on these considerations, an assay kit was assembled containing six lyophilized serum standards that tested the limits of the assay from 15 to 480 EU/ml. The kit also included a commercially available, well-characterized monoclonal antibody and lyophilized positive, negative, and intermediate controls. The controls were devised to test whether the assay could be used in either a quantitative or qualitative manner to aid disease diagnosis. Because the most appropriate cutoffs have yet to be confirmed, the current kit employs assay controls targeting the concentrations of 0, 49, and 94 EU/ml as proposed by Baughman et al. (2
). When studies to confirm the cutoffs are completed, the assay controls will be revised.
Several benefits were obtained by performing an interlaboratory collaborative study prior to the analytical validation. Most importantly, we learned that the assay could be transferred and that analysts with no experience with this specific assay could perform the test successfully in both the quantitative and qualitative formats. Additionally, the results and feedback from the participants provided valuable insight for improvements in the method. Specifically, the written procedures were refined and clarified, the number of successful assays required for qualification of an analyst was increased, and reagents were adjusted to bring the OD readings into a narrower range.
The data presented here demonstrate that the kit met all prespecified criteria for precision, linearity, and accuracy for samples with IgG anti-PT antibody concentrations in the range of 50 to 150 EU/ml, the range believed to be most relevant for serodiagnosis. The assay met the precision and linearity criteria for a wider range, 50 to 200 EU/ml; however, the primary accuracy criterion was not met for the sample at 200 EU/ml. Specifically, the concentration of the 200-EU/ml sample of CBER3 was underestimated by approximately 28%. The reason for the underestimation of CBER3 at 200 EU/ml remains unknown; however, a follow-up study using NIBSC 06/140 demonstrated that the assay met the predefined criterion at all concentrations tested, including 200 IU/ml. Additionally, the linearity assessment with three positive sera (Table ) indicated excellent linearity over the range from 42 to 352 EU/ml. These data suggest that the underestimation is limited to the CBER3 samples, and an investigation has begun to determine whether the results for CBER3 were influenced by the age or storage conditions of the preparations tested. Testing to understand the discrepancy between high-titer samples of NIBSC 06/140 and CBER3 are ongoing. The failure of CBER3 to meet acceptance criteria should not compromise the applicability of this assay provided future diagnostic thresholds are limited to values lower than 200 EU/ml.
Together, the interlaboratory collaborative and analytical validation studies indicate that the kit is relatively easy to transfer and perform. Both studies suggest the importance of establishing training and technical proficiency requirements prior to test implementation. For future studies, we recommend that one or more known positive serum samples be added to each assay in addition to the lyophilized controls. The serum control would be diluted along with the unknown samples and tracked over time to assess the stability of the kit and continued qualification of the analyst. In addition, a proficiency panel of serum should be established and provided to all testing laboratories to evaluate the continued performance of each testing laboratory. Results of the proficiency panel and the positive-control serum can be used to qualify new personnel and evaluate the ongoing competency of personnel performing the assay.
Tetanus-diphtheria-acellular pertussis vaccines, which were introduced into the adult population in 2005 (5
) may induce elevated anti-PT IgG titers for a short time after vaccination, requiring reevaluation of the diagnostic threshold (18
). Finally, while the assay was designed and evaluated to quantify anti-PT titers, the preliminary results presented here demonstrate that the assay can serve in a qualitative format once optimal threshold cutoffs are defined, thus eliminating the need for a full set of standards. Such a format would reduce the number of procedural manipulations needed to physically perform the assay, and it would simplify the interpretation of the results by eliminating the need for a statistical analysis program. This would facilitate adoption and use by regional or state health departments to assess pertussis outbreaks and measure the burden of disease in adolescents and adults. In fact, the possibility of making this kit widely available has been proposed for future evaluation.
In conclusion, the anti-PT IgG ELISA met all assay validation parameters within a range of 50 to 150 EU/ml. This ELISA was developed and analytically validated to be a user-friendly kit that can be used for both qualitative and quantitative purposes. With the analytical validation of this test, the first part of a two-phase project has been completed. In the second phase, now under way at CDC, the test is being evaluated in a prospective multisite clinical trial that will evaluate the sensitivity, specificity, and predictive values of this kit and two other diagnostic tools (PCR and culture). Once subjected to appropriate clinical assessment and once diagnostic thresholds are established, this assay should be considered for wide-scale use in public health, which would add an important tool for pertussis diagnosis.