A qualified and characterized standard reagent is a critical component in the development and standardization of serological assays and provides a benchmark for the comparative analysis of new assay technologies (1
). In this study, we report the quantification of total IgG and IgG subclasses and the assignment of mass values for total and subclass-specific anti-PA IgG in human standard anthrax reference serum AVR414. The concentration of total IgG assigned for AVR414 (8.33 mg/ml) was within the established range for human serum IgG in healthy adults (8 to 16 mg/ml) (2
). The concentrations of IgG1 (4.48 mg/ml) and IgG3 (0.37 mg/ml) in AVR414 were lower than the established range for normal healthy human adults (5.0 to 12.0 mg/ml and 0.5 to 1.0 mg/ml, respectively) (2
). The percentages of total IgG represented by IgG1 (52.7%) and IgG3 (4.4%) were also lower than the range in normal adult sera (60.3 to 71.5% and 5.0 to 8.4%, respectively) (8
). The concentration of IgG2 (3.35 mg/ml) was within the established range for normal adult sera with a dynamic range of 2.0 to 6.0 mg/ml (2
), but the IgG2 percentage of total IgG (39.4%) was higher than the established range (19.4 to 31.0%) (8
). The AVR414 standard reference serum pool contains anti-PA-specific IgG of all four subclasses, with concentrations of PA-specific IgG1, IgG2, and IgG4 being the highest (55.5, 24.6, and 17.6% of total anti-PA IgG) and the concentration of IgG3 being the lowest (2.2% of total anti-PA IgG). A possible explanation for the low concentration of anti-PA IgG3 in AVR414 is affinity maturation and isotype switching to IgG2 and IgG4 (4
) due to the plasma donors in this study receiving a minimum of four vaccinations of the licensed AVA regimen.
The anti-PA IgG concentrations assigned by the two methods were not significantly different (P = 0.42). This level of probability from two different approaches for mass values assignment is an indication of the robustness and reliability of the assigned units. The utility of AVR414 has been clearly demonstrated in its application to anti-PA IgG mass value assignments in a panel of QC and positive vaccine control sera and the assignment of PA-specific values to an additional standard reference serum pool, AVR801.
It has been our objective to create a standardized platform technology and reagents that will enable the comparative analyses of human anti-PA IgG responses to clinical anthrax and anthrax vaccines. The characterization of the anti-PA-specific total IgG and IgG subclasses of the standard AVR414 described here provides a strong basis for this standardization. AVR414 has been successfully applied in quantitative serological assays to evaluate human humoral antibody immune responses to vaccination with AVA (N. Marano, J. Lingappa, P. Pittman, V. Semenova, S. Leitman, P. Plikaytis, C. Quinn, and B. Perkins, Abstr. 5th Int. Conf. Anthrax, abstr. O609, 2003), for analysis of the immune response to PA in individuals with bioterrorism-associated cutaneous and inhalation anthrax (5
), and for evaluation of anti-PA IgG subclass distribution in anthrax vaccinees and confirmed cases of clinical anthrax (V. A. Semenova, P. M. Dull, D. S. Schmidt, T. H. Taylor, E. Steward-Clark, M. M. Ballard, and C. P. Quinn, Int. J. Infect. Dis. vol. 8, abstr. 35.010, p. S111, 2004). For each of those studies, the AVR414 standard reference serum was the pivotal unifying reagent. Qualified reagents, such as AVR414, will facilitate assay technology transfer and reduce interassay and interlaboratory variance (3
). In addition, the determination of assay end points using a 4-PL model to describe the standard curve parameters (29
) also makes a significant contribution to assay standardization by reducing the number and type of possible errors related to interpolating antibody concentrations from the standard curve (30
). Together these are valuable tools in standardizing the evaluation of the human anti-PA IgG immune response in recipients of PA-containing vaccines and in cases of human anthrax.