Serum samples
Human sera provided by Intergen Bio-Diagnostics (Purchase, NY) and Bioreclamation Inc. (Hicksville, NY), were tested for anti-RSV antibody titers. Sera were selected and pooled into 3 groups according to titer. The three groups consisted of a lyophilized reference serum, prepared under the auspices of NIAID and two control sera of high and low titer. An historical in-house control standard, C587645, was also tested. The reference serum made up the majority of tests (136 tests), whereas the control sera were each tested approximately 14 times. These sources of human sera comprised the specimens evaluated in this study and were collectively tested 180 times by both methods, in the presence and absence of complement.
Virus and cells
The A2 strain of RSV was used as the challenge virus in all tests. Vero cells (ATCC Cat #CCL 34, ATCC, Rockville, MD) were cultured in EMEM with L-glutamine, 10% FBS, 1% of antibiotic/antimycotic, and non-essential amino acids. Cells were cultured on 96-well white opaque tissue culture plates (BD Falcon, Bedford, MA) for automated counting and on transparent 96-well plates (Corning-Costar, Corning, NY) for manual plaque counting 1–3 days prior to infection.
Determination of RSV antibody titers
Microneutralization
Serum samples were heat-inactivated at 56°C for 30 minutes. Four-fold serial dilutions from 1:10 to 1:10,240 were prepared in virus diluent (EMEM with L-glutamine containing 2% FBS, 2.5% HEPES (1 M) and 1% antibiotic/antimycotic, 100×). All sera were tested in the presence and absence of 10% guinea pig complement (Cambrex/BioWhittaker, Walkersville, MD) which was added to the virus diluent prior to the addition of challenge virus. Serially diluted serum was challenged with an equal volume of the RSV-A2 strain, previously titered to give 50–100 pfu per 50 μl of inoculum. The serum/virus mixtures were incubated at 37°C, 5% CO2 for 1 h.
Vero cell monolayers, prepared in 96 well plates, were infected with 50 μl/well (in duplicate) of the serum/virus mixture. Plates were centrifuged at 1 h at 2000 rpm (700 g), followed by 30 min of rocking at room temperature. Supernatants were decanted. Plates were blotted and overlaid with 0.75% methyl cellulose (4,000 cP at 2% aqueous), prepared in MEM with 2% FBS, warmed to 37°C and inoculated at 100 μl/well. Plates were incubated at 37°C, 5% CO2 for 3 days to allow for plaque formation.
Conventional staining and plaque determination for RSV neutralization
Cells infected on transparent plates were fixed with a 50%:50% methanol:ethanol mixture at room temperature for 10 min. Plates were washed with DPBS after fixing and between staining steps. Plates were incubated for 1 h at 37°C, 5% CO2 with 50 μl/well of monoclonal antibody specific for RSV-A2 F-protein (Wyeth K6-5-1) diluted to 1:1,000 in Blotto (5% non-fat milk in PBS). Peroxidase labeled secondary goat anti-mouse IgG antibody (KPL, Gaithersburg, MD) diluted 1:100 in Blotto, was incubated at 50 μl/well for 1 h at room temperature. Plaques were developed using 100 μl/well 3,3'diaminobenzidine HRP substrate (0.5 mg/ml DAB, 0.01% H2O2) prepared in DPBS and incubated at room temperature for 5 – 10 minutes. Plates were washed with tap water to stop the reaction.
Plaques were counted manually by inverting the transparent plate under a dissecting microscope. The field of the well was separated into quadrants for ease of counting. Overlapping plaques were deemed individual when lobes were apparent.
TMB staining and plaque determination for RSV neutralization
Cells infected on opaque white tissue culture plates were fixed and washed as described above. Plates were incubated for 1 h at 37°C 5% CO2 with 50 μl/well of monoclonal antibody specific for RSV-A2 F-protein (Wyeth, K6-5-1) diluted to 1:10,000 in blotto. Peroxidase labeled secondary goat anti-mouse IgG antibody (KPL, Gaithersburg, MD) diluted 1:3,000 in Blotto was added at 50 μl/well and incubated for 1 hour at room temperature. Plaques were developed using 50 μl/well of a ready to use TMB precipitate HRP substrate, True Blue™ (KPL, Gaithersburg, MD). Higher dilutions of primary and secondary antibody were used due to the increased sensitivity of the peroxidase TMB substrate. Plates were washed thoroughly with tap water to stop the reaction and dried inverted in order to minimize bleaching.
Plates were scanned and counted by the ImmunoSpot® Image analyzer from Cellular Technology Ltd. (Cleveland, OH). The software, initially designed for use in ELISPOT analysis, has been successfully employed here for plaque detection and counting. Overlapping plaques were separated using a separation tolerance parameter set by the experimenter (Fig. 4). Minimum plaque size and sensitivity to stain comprised the major parameters that could be adjusted for counting. Parameters were adjusted on each experimental day, if necessary.
Calculation of antibody titer
Titer was calculated from the average of duplicate sample wells by extrapolating the inverse dilution of serum that produced a 60% reduction of virus according to the following formula:
X = (a-b)(e-c)/(c-d) + a
where, a = log10 of dilution above the 60% reduction point, b = log10 of dilution below the 60% reduction point, c = average plaque count above the 60% reduction point (corresponds with a), d = average plaque count below the 60% reduction point (corresponds with b) and e = value of 60% reduction of average virus control count.
Statistical analysis
All titers were reported in logarithm base 4 scale in order to visually represent a difference of one dilution (of a 4-fold dilution series) as 1 log unit. Different statistical analyses were performed to assess the agreement of titers generated by two methods. In one analysis we graphically inspected the spread of the paired titers about the 45° line and computed Pearson's correlation coefficient. In another analysis, we determined the level of equivalence between the two assays, by constructing a difference-means plot [7].