Application of recovery tests in the validation of immunoassays for assessing the immunogenicity of B. anthracis PA vaccine.
In the quantitative assessment of polyclonal serum antibodies, the complex composition and characteristics of the analyte population (serum antibodies) restricts the capability of constructing appropriately defined calibration standards. This fact limits the application of the conservative recovery tests to the validation of immunoassays aimed at determining serum antibody levels. The present report describes a modification of recovery tests that overcomes this impediment. The modified approach is based on a dilution analysis system, where a given immune serum is serially diluted in normal serum and the antibody titers in each of the derived diluted samples are then determined. Expected sample titers (calculated on the basis of the relevant dilution factors) are plotted against the respective observed results, and the resulting recovery curve is then examined by means of a regression analysis, according to the standard rules of the conservative recovery analysis. This approach was tried with two immunoassay systems, Enzyme Linked Immunosorbent Assay (ELISA) and Neutralizing Antibodies (NtAb) immunoassays, aimed at assessing the immunogenicity in guinea pigs of B. anthracis protective antigen (PA) vaccine. In a series of feasibility studies using a recovery simulation model (dilutions made in the immunoassay diluent, rather than in normal serum) the average recovery levels in ELISA and NtAb immunoassays were 0.99 +/- 0.011 and 1.02 +/- 0.04 respectively, and the 99% confidence intervals contained the target 100% value. Regression lines were proved to be linear demonstrating R > 0.97 in all cases. The 99% confidence intervals around the observed slopes and intercepts always contained the corresponding target values 1 and 0. The relative standard deviation (RSD) in the ELISA and NtAb immunoassays was found to be 0.01 and 0.025 respectively. All of the above experimental results were not affected by the serum antibody titer, or by day-to-day variations embodied in these immunoassay systems. When true recovery tests were applied to the above immunoassays, essentially identical results were obtained. In both assays the correlation coefficients were in the range of 0.96-1, recoveries were found to be in the range of 0.90-1.06, and RSD values were in the range of 0.02-0.025. All the recovery deviations from the target value of 1 were not statistically significant. The hitherto observed experimental findings illustrate the capability of the dilution analysis system to allow the application of recovery tests to the validation of quantitative immunoassays, which are based on the procedure of serum titrations.
Halperin
G
G
Department of Biotechnology, Israel Institute for Biological Research, Israel. halperin@iibr.gov.il
Marcus
H
H
D000818:Animals; D022122:Anthrax Vaccines / Q000276:immunology*; D000907:Antibodies, Bacterial / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D006168:Guinea Pigs; D012044:Regression Analysis; D015203:Reproducibility of Results
D022122:Anthrax Vaccines; D000907:Antibodies, Bacterial