The assay was optimized for consistency and sensitivity. Parameters assessed included cell culture, viral titer and infection duration. Sub confluent HEp-2 cells with a passage number between 1 and 20 were determined to be the optimal cell type. The optimized condition included a cell count of 5×104
per well in a 96 well culture plate, which was freshly seeded, with a control well viral infection rate of 6–12%. As the virus replication cycle and cell growth cycle affect virus infection rates, the virus infection duration is important for accuracy of results. The assay should be completed after one round of virus replication but before a secondary round of infection could potentially begin. Based on a series of time points assessed, the ideal duration of infection is 16–18 hours and this is consistent with previous studies using this construct which indicate easily detectable infectivity within 20 hours (Hallak et al., 2000
The “percentage law ” states that the amount of virus neutralized by a given concentration of antibody is constant irrespective of the total quantity of virus present so long as antibody is present in excess above the amount of virus in the assay (Andrewes, 1933
; Klasse and Sattentau, 2001
) (Pierson et al., 2006
). Assay compliance with the percentage law provides further reassurance that the assay result reflects antibody affinity and is not related to quantity of virus present. To ascertain whether the RSV flow cytometry neutralization assay follows the “percentage law”, three dilutions of recombinant GFP-RSV at a multiplicity of infection (MOI) ranging from 0.03 to 1.5 were evaluated with serial dilutions of palivizumab with reproducible results in multiple experiments and data from a representative experiment is further described in detail (). The EC50
at an MOI of 1.5, 0.15 and 0.03 was 2660, 2369 and 2372, respectively (). The neutralization titer of palivizumab was not altered by different MOIs in the assessment of variables. Even at low virus dilutions, the EC50
of palivizumab is consistent when other variables like cell viability and age are fully controlled. While the assay follows the percentage law, it is noted that a set of conditions related to incubation time, cell number per well, and quality or infectivity of virus stock should be well controlled in this assay. For example, at low viral infectivity rates down to 6%, the EC50
of the positive control monoclonal antibody remains constant, but at viral infection rates <5% the measurement becomes unreliable. Low infection rates when using a relatively high MOI are typically caused by unhealthy cells that are at high passage number or overly confluent, or virus stock that is degraded. Therefore, to ensure consistency, we recommend control well infection rates in this assay to range from 6–12%.
Figure 1 Demonstration of reproducible neutralization of RSV-GFP by anti-RSV monoclonal antibody Palivizumab (Synagis®). EC50 of Palivizumab with 95% confidence intervals under three different MOI. Sigmoidal curves of antibody dilution (x-axis) versus (more ...)
Three further experiments were preformed in duplicate to assess neutralization by mouse serum, rabbit serum and human plasma against RSV, at a range of MOI (). EC50 of mouse serum, rabbit serum and human plasma from a representative experiment are shown to be consistent despite altered experimental conditions ().
Figure 2 Consistent neutralization (EC50) of human plasma, mouse serum, and rabbit serum under 4 different MOI (with 95% confidence intervals). Sigmoidal curves of antibody dilution (x-axis) versus GFP-RSV infected cells shown as neutralization curves at MOI of (more ...)
This flow cytometry neutralization assay has been used to assess RSV neutralizing antibody measurement in our laboratory and experimental results are consistent. Palivizumab from the same lot is used as the positive control in all experiments. To understand the degree of consistency, the EC50 of palivizumab from 33 experiments during a one-year period () were compared. Among 33 experiments, 20 were performed by operator 1 and the mean EC50 of palivizumab at a concentration of 1000 µg/ml was 1733±515.7 (mean±SD), 13 were performed by operator 2 with a mean EC50 of 1578±330.1 (mean±SD) ().
Figure 3 Reproducibility of RSV flow cytometry neutralization assay. Figure 3a. Correlation of neutralization antibody titer (logEC50) by linear regression analysis of 16 mouse serum samples tested twice (on different dates) by operator 1. Figure 3b. Correlation (more ...)
The reproducibility of the neutralization assay was further evaluated. A comparison of the intraoperator reproducibility was made. The neutralization titers (logEC50) of 16 murine serum samples were assessed by one operator, twice, in two separate experiments and the linear regression analysis shows significant correlation, R2 = 0.9898, P < 0.0001 (95% CI of slope is 0.9479–1.065) (). Next, the interoperator reproducibility was tested. Of the 16 samples tested by operator 1, 10 samples (those with sufficient volume) were available for repeat assessment by operator 2. Additionally, another 6 samples that were previously tested by operator 1 in a separate experiment were added in the interoperator comparison experiment. The linear regression analysis demonstrates that the neutralization titer (logEC50) between two operators correlates significantly, R2 =0.9789, P< 0.0001 (95% CI of slope is 0.9934–1.176) (). A Spearman’s correlation assay also demonstrates correlation of the neutralization titer (logEC50) with an intraoperator correlation of r=0.9690, p<0.0001 (95% CI is 0.9080–0.9898) and an interoperator correlation r=0.9669, p<0.0001 (95% CI is 0.9019–0.9891).
A comparison of traditional PRNT to the flow-cytometry based neutralizing antibody assay was made by assessing serum from 68 cotton rats (). The titers ranged from EC50 10- 3000 and a Spearman’s correlation analysis shows significant correlation between the PRNT and flow cytometry based neutralization assays (r = 0.9117, 95% CI is 0.8584–0.9456 and p < 0.0001).
Figure 4 Comparison of plaque reduction neutralization test and flow cytometry neutralization assay. 68 cotton rat serum samples were tested by plaque reduction neutralization test and flow cytometry neutralization. The linear regression analysis demonstrate significant (more ...)