Table 1.
RSV ECL assay conditions.
Fig 1.
Representative example of an antigen titration experiment to determine optimal coating density.
For each antigen, a stock solution was serially diluted and coated onto 96-well MSD plates. In this example, three dilutions of the assay control (MN log2 IC50 titer = 9.9) were tested at a fixed concentration of goat anti-human IgG SULFO-TAG antibody (0.5 ug/ml). Symbols represent mean values from eight replicates.
Fig 2.
Linear range comparison between RSV ECL assays and a diagnostic RSV F/G ELISA.
The assay control was used to illustrate the linear range of the four ECL assays and the diagnostic F/G ELISA. Linearity samples were prepared from the assay control and serially diluted before addition to the MSD or ELISA plates. MSD RLU or ELISA OD (optical density) values were log10-transformed and plotted versus the log10-transformed sample dilution. The upper and lower limits of the linear range were calculated using a 4-parameter logistic model and marked by dashed red lines. The fold difference was calculated for each assay as the ratio between limits of the linear range.
Table 2.
Analytical sensitivity using an anti-RSV F neutralizing monoclonal antibody (motavizumab).
Fig 3.
Comparison of infant and elderly serum samples in four ECL assays.
The mean log2 titer is represented for each group. The dashed line represents the lower limit of quantitation (log2 20 = 4.32). Significance between age groups was determined using a two sample t-test.
Fig 4.
Seroresponse of naturally infected elderly subjects.
(A) The fold change of serum titers between acute and convalescent serum samples from elderly subjects (59 RSV-positive, 30 RSV-negative) was measured by four ECL assays, a microneutralization assay and a diagnostic ELISA. A four-fold rise in serum titer, marked by a red dashed line, was used to indicate seroresponse following natural infection. Due to the limited dynamic range of the diagnostic F/G ELISA, acute and convalescent serum samples were diluted until the acute sample fell within the assay range and at least 4-fold below the ULOQ. To simplify the testing and analysis, all sample pairs showing at least a four-fold rise in ELISA titer were assigned a fold change value of four. (B) A Venn diagram illustrates the seroresponse profiles to RSV F, N, Ga and Gb from RSV-positive elderly subjects (n = 59), as measured by ECL assays. A ≥4-fold rise in serum titer was used to indicate seroresponse following natural infection. Three subjects did not show a ≥4-fold rise in any ECL assay. The Venn diagram was prepared by Venny software http://bioinfogp.cnb.csic.es/tools/venny/index.htm (C) Scatterplot showing the fold change between acute and convalescent serum pairs of RSV A (n = 29) and RSV B (n = 30) positive elderly subjects, as measured by RSV Ga and Gb IgG ECL assays.
Table 3.
Comparison of diagnostic sensitivity of RSV serology assays a,b.