Improved Specificity and False Discovery Rates for Multiplex Analysis of Changes in Strain-Specific Anti-Influenza IgG

We describe a statistical approach to compare absolute antibody concentrations, both within and across subjects, derived from a multidimensional measurement of IgG binding to the influenza surface receptor hemagglutinin (HA). This approach addresses a fundamental problem in the field of vaccine immunology: how to accurately compare the levels of antibodies against multiple influenza strains. The mPlex-Flu assay can simultaneously measure the concentration of IgG antibodies against up to 50 influenza strains with only ≤10 μl of serum. It yields mean fluorescence intensity (MFI) over a 4-log range with low inter- and intrasample variability. While comparison of IgG binding to a single HA between subjects is straightforward, variations in binding behavior across influenza strains, coupled with reagent variations, make quantifying and comparing binding between multiple HA subtypes within subjects challenging. In this paper, we first treat such HA variations as an independent antigen and calculate each subtype antibody concentration using its own standard curve, normalizing variations in HA binding. We applied this method to the analyses of data from an H5 influenza clinical vaccine study. The results demonstrated that there are differences in coefficient estimates and in results of “comparing groups” between those with versus those without consideration of subtype antibody variations. Then, we used simulation studies to show the importance of taking the subtype antibody variations into account in HA strain antibody data analysis. Using a common standard curve for all subtype antibodies resulted in both inflated type I error and lowered specificity when comparing different treatment groups. Our results suggest that using individual standard curves for each influenza HA strain, and independently calculating anti-HA IgG concentrations, allows for adjustment of influenza HA subtype variations in treatment group comparisons in clinical vaccine studies. This method facilitates the direct comparison of serum anti-HA IgG concentrations against different influenza HA subtypes for multiplex assays