Cell and virus propagation
Vero E6 cells (American Type Culture Collection, ATCC #CRL-1586) were grown in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Euroclone) supplemented with 10% v/v fetal bovine serum (FBS) (Euroclone), 2mM L-glutamine, 100 units ml−1 penicillin, and 100 μg ml−1 streptomycin (P/S) (Gibco). Cells were maintained at 37 °C in a humified 5% CO2 environment and passed every 3–4 days. hMPV-A1 (NL/1/00) and hMPV-B1 (NL/1/99) were purchased from the European Virus Archive—Global portal (EVAg) and provided by the Erasmus University Medical Centre Rotterdam (EVAg Ref-SKU: 011V-00930 and 011V-01003, respectively).
Viral propagation was performed in 175 cm2 tissue-culture flasks pre-seeded with 50 ml of Vero E6 cells (1.5 × 105 cells ml−1) diluted in DMEM 10% FBS. After 18–24 h at 37 °C in 5% CO2, flasks were washed with 1X sterile Dulbecco’s phosphate buffered saline (DPBS1X) (Gibco) and then inoculated with the virus at a multiplicity of infection (MOI) of 0.001. For the inoculum, stock virus was diluted in serum-free DMEM supplemented with 5µg ml−1 trypsin (TPCK-treated) (Sigma-Aldrich). Flasks were incubated with the virus for 1 h at 35 °C in 5% CO2, then filled with DMEM 2% FBS and 5µg ml−1 of trypsin and incubated at 35 °C in 5% CO2. Cells were monitored daily until 70–80% of cytopathic effect (CPE) was observed. Harvest was performed by collecting the supernatant and scraping the remaining cell layer from the flasks. The viral solution was centrifuged at 296g for 5 min at 4 °C; 5 ml of supernatant and the cell pellet underwent three freeze–thaw cycles, performed by placing the vial on dry-ice (30 s), then at 37 °C (30 s) and subsequently vortexing (30 s). Upon completion, the vial was re-centrifuged to isolate cells, and the supernatant obtained was added to the previously collected viral solution, mixed gently, aliquoted and stored at -80 °C in the presence of 20% sucrose.
Serum samples
To evaluate the sensitivity of the EMN assay, a total of 16 commercially available human serum samples (Panel F and Panel D, Clinisciences) were screened for the hMPV-A1 strain only.
The experimental protocol for the validation of the EMN assay used a human serum sample positive for hMPV by PCR as a positive homologous control, and a depleted human serum lacking IgA/IgG/IgM (Sigma‐Aldrich) as a negative control. To assess specificity, the positive sample was tested in parallel with the commercially available sheep hyperimmune sera used as heterologous samples: Influenza anti-A/Victoria/2570/2019-like (H1N1) HA Serum 21/120 (NIBSC); Influenza anti-A/Cambodia/e0826360/2020-Like (H3N2) HA Serum 21/118 (NIBSC); Influenza anti-B/Washington/02/2019-like (B-Victoria lineage) HA Serum 19/318 (NIBSC); Influenza anti-B/Phuket/3073/2013-like HA serum (B-Yamagata lineage) 19/322 (NIBSC). Also, an haemolyzed sample (INT-01H, Sun Diagnostic) was used for specificity experiments.
In order to test the reliability of the assay, a total of 105 human serum samples were randomly selected from the samples available. The samples had been collected in the Apulia region (Southern Italy). The research protocol was approved by the Ethics Committee of the University Hospital of Bari (n. 7622, prot. N. 0023599|09|03|2023). The serum survey was conducted in accordance with ethical principles (Declaration of Helsinki), and written informed consent was obtained from all the participants. Samples have been fully anonymized before testing. Serum samples were heat-inactivated by incubation at 56 °C for 30 min before testing.
ELISA-based microneutralization assay
The EMN assays were performed in 96-well, flat-bottomed, tissue-culture, microtiter plates. DMEM 1% FBS supplemented with 3μg ml−1 of trypsin was the complete medium used for the test. Regarding the virus concentration, 25 or 100 or 300 TCID50 (50% tissue culture infectious dose) per well (corresponding to 500, 2000 and 6000 TCID50 ml−1, respectively) of virus was used in the set-up experiments to determine the most appropriate infective dose; 100 TCID50 per well was the viral dose used in all the validation tests.
Serum samples were seeded at a final concentration of 1:10 and serial two-fold dilutions were performed. The appropriate dose of virus was then added to each well and the serum-virus mixture was incubated for 1 h at 35 °C, 5% CO2. At the end of the incubation time, 100 μl per well were transferred from dilution plate to cell plate pre-seeded at 24 h with 2.0 × 105 cells ml−1 in DMEM 2% FBS and incubated at 35 °C, 5% CO2.
After 24 h (for hMPV-A1) and 48 h (for hMPV-B1) of incubation, the plates were washed twice with DPBS1X. Fixation was performed by adding 100 μl per well of a cold 80% v/v solution of Acetone (Sigma-Aldrich) in DPBS1X and incubating the plates for 10 min at room temperature (RT); the plates were then emptied and air-dried before the ELISA read-out. Plates were washed three times by means of an automatic plate washer using 300 μl per well of wash buffer, a DPBS1X solution with 0.3% of Tween20 (Sigma‐Aldrich). The primary antibody, Anti-prefusion viral F protein DS7 (Absolute Antibody), was added (100 μl per well) and plates were incubated for 1 h at RT. Subsequently, the plates were washed three times, as previously, and the secondary antibody, Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Bio-Rad), was added (100 µl per well) for an incubation time of 1 h at RT in the dark. Both primary and secondary antibodies were diluted in a 1:1000 ratio by using the antibody diluent, a 5% non-fat dried milk (NFDM)(AppliChem) solution in wash buffer. Next, the plates were rinsed six times with 300 μl per well of wash buffer, and 100 μl of the substrate solution 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich) was added to each well. The plates were incubated for 20 min at RT in the dark. The reaction was stopped by adding 100 μl per well of 0,5 M Hydrochloric Acid (Fisher Scientific) and the plates were read at an optical density of 450nm by means of a SpectraMax plate-reader (Medical Device). Softmax Software (GxP Compliance) was used for data collection.
Validation of ELISA-based microneutralization assay
The EMN assay was validated by testing samples in four different analytical sessions run by two operators over 2 days and obtaining three reportable (RP) values. For each of the three replicate measurements, the GMT was calculated. Specificity and robustness were demonstrated by testing samples in two independent runs performed by two operators.
Dilutional linearity
To assessment of linearity, the PCR-positive control was tested in a two-fold dilution scheme in which at least one dilution had a titer below the lower limit of quantitation of the assay, starting from a dilution of 1:10. Thus, the range analyzed was the following: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280. The abovementioned sample dilutions were tested in one repetition per plate, in three different plates by two operators on Days 1 and 2. The parameter was evaluated by examining the relationship between the base-2 logarithm of the GMT (observed titers) and the base-2 logarithm of the serum dilutions across the factorial design. The R2, y-intercept and slope of the regression line were calculated and reported.
Relative accuracy
Data for the evaluation of relative accuracy correspond to the RP values obtained from linearity tests. According to ICH guideline Q2(R2)20, the accuracy can be tested by using either a conventional true value or an accepted reference value. The GMT of the expected values was calculated from the GMT of the results obtained from the neat sample, and by dividing this value by the corresponding factor of the two-fold serial dilution. Relative accuracy was evaluated by calculating the percentage of recovery on the GMT of the RP values and the expected (true) titer by applying the formula: 100 × (GMT observed/GMT expected).
Precision
Precision was assessed by using the results obtained in the linearity tests and considering three different aspects: repeatability, intermediate precision, and FV.
Repeatability, or intra-run variability, is the variation expected across replicates under the same operating conditions over a short period of time20.
Intermediate precision is determined from the total variance component. It represents the variations expected between different laboratories, and those due to the impact of different factors, such as days, environmental conditions, operators, and equipment.
FV represents the variation expected across GMT results yielded by multiple replicates in routine testing. Two independent runs, each of which consisting of one replicate, were considered for FV calculation.
Limit of quantitation and range
The LLOQ and ULOQ were determined as the lower and upper 95% CI of the observed GMT of the lowest and highest sample concentrations analyzed with acceptable linearity, accuracy and precision.
Specificity
Specificity was assessed by testing the hMPV PCR-positive sample in parallel with commercial anti-heterologous samples. The positive sample had to show a four-fold difference from the anti-heterologous samples tested.
Robustness
Robustness is the capacity of an analytical procedure to remain unaffected by small but deliberate variations in method parameters. Two critical conditions were evaluated: cell seeding concentration and incubation time of the virus-serum mixture.
ELISA
96-well plates (Nunc, Maxi-Sorp) were coated with either 1 µg ml−1 of hMPV strain A glycoprotein G Protein (His Tag) (Sino Biological), or 1 µg ml−1 of hMPV strain B glycoprotein G Protein (His Tag) (Sino Biological) or 1 µg ml−1 of hMPV B Fusion Glycoprotein F0, His-Tag (HEK293) (Native Antigen).
Samples were diluted 1:100 in 5% NFDM solution in TBS (Tris-buffered saline with 0.05% Tween 20, Thermo Scientific). After this step of dilution, for the sessions involving the glycoprotein G of both strains, 10 more steps of two-fold dilution, ranging from 1:100 to 1:51,200, were applied to all samples. In the case of Glycoprotein F0, instead, the range of measurement was extended to 20 dilution points from 1:100 to 1: 52,428,800. After 1 h in blocking solution (5% NFDM in TBS-Tween solution) at RT with shaking, the plates were washed; 100 µl of each serum dilution was then added to the coated plates, which were incubated for 1 h at RT with shaking. After another washing step, polyclonal goat anti-Human IgG-Fc (Bethyl Laboratories) HRP-conjugated antibody was added, and the plates were incubated for 1 h at RT with shaking. After a washing step, TMB substrate was added, and the plates were incubated in the dark at RT for 20 min. The reaction was stopped with 0,5 M Hydrochloric Acid and read at 450nm by means of SpectraMax plate-reader (Medical Device). SoftMax Pro Software – GxP edition 7.1.2 was used for data collection.
Statistics and reproducibility
All the graphs and the statistical analyses were generated and calculated by GraphPad Prism software version 10.2.3. Statistical analyses performed for validation experiments were executed on Excel 365 Apps for business and R version 4.3.1 software. Correlation between EMN assay and ELISA results was determined by Spearman’s rank correlation coefficient analysis. Further statistical details on the individual experiments are provided in the respective methods section and figure legend.
