The miniaturized format of the NI assay was developed by reduction and optimization of the conventional assay described in the 2002 WHO Manual on Animal Influenza Diagnosis and Surveillance10 and is summarized below. Fetuin was diluted in phosphate-buffered saline (PBS) to 25 mg/ml, except where noted. Each virus was titrated in PBS (pH 7·4) containing 0·1% bovine serum albumin (PBS–0·1% BSA) to determine the dilution that yields an optical density at wavelength 550 nm (OD550) of 1·0 in the final extracted chromophore layer. Please note that NAs of some other H1N1 and H3N2 strains have shown a marked dependence on divalent cations in diluent (e.g., saline with titrated CaCl2 or Dulbecco’s PBS with Ca2+and Mg2+) for optimal activity. Twofold serial dilutions of serum were made in duplicate across wells of a 96-well polypropylene PCR plate (Eppendorf, Hamburg, Germany). Six sera were typically titrated across seven dilutions in one plate that included controls. In each well, 5 μl serum diluted in PBS was mixed with 5 μl virus and incubated for 30–45 minutes at room temperature. Volumes were dispensed by a low-volume multi-channel pipette. Next, 5 μl fetuin (25 mg/ml) was added per well. Four fetuin control wells for background signal contained the substrate alone with respective buffers in place of serum and virus, and four virus control wells for non-inhibited signal contained all components except serum (PBS in place of serum). The plates were sealed, mixed, and incubated at 37°C in a PCR thermocycler or cabinet incubator for 15–16 hours or for 2 hours, where noted. Detection of free sialic acid was initiated with addition of 5 μl periodate reagent per well (200 mm NaIO4, 53% H3PO4, stored in the dark) for a 15–20 minutes incubation at room temperature. Each well then received 25 μl arsenite reagent (1M AsNaO2, 700 mm Na2SO4, 0·3% concentrated H2SO4), and the plate was agitated until the yellow color disappeared. Next, 50 μl of TBA reagent (50 mm TBA, 625 mm Na2SO4,) was added per well, wells were closed with cap strips, and the plate was incubated 15 minutes at 99°C on a PCR thermocycler block with heated lid. Plates were chilled on an ice bath for 5–10 minutes. Warrenoff reagent (95% 1-butanol, 5% concentrated HCl) was dispensed at 75 μl per well, wells were sealed with cap strips or MicroAmp optical adhesive film (Applied Biosystems, Foster City, CA, USA), and each plate was vortexed vigorously until the extraction of pink chromophore to the organic layer was clearly evident. Plates were centrifuged at 250 x g for 5 minutes to separate phases, and 50 μl of the upper phase per well was transferred to Costar 96-well half area flat bottom plates (Corning Life Sciences, Corning, NY, USA). Sample absorbance was analyzed on a Victor3 V multi-label reader with a 550 nm filter (PerkinElmer, Waltham, MA, USA). BSA, fetuin, and chemical compounds, including pure N-acetyl neuraminic acid (NANA), were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The mean background absorbance obtained in fetuin control wells (no virus or serum) was subtracted from readings of all other wells. Based on the results of an empirical test to determine the ratio of sample absorbance of 50 μl in a 96-well half area plate and in a standard spectrophotometer (data not shown), the background-corrected readings were multiplied by 3·1 to derive OD values as defined in a 1-cm cuvette spectrophotometer. Assay results were accepted if the mean OD550 values of virus control samples (no serum) was 0·7–1·3 and fetuin control samples were <0·1. NI titers were defined as the inverse of the highest serum dilution at which the mean absorbance was ≤50% of the mean signal of virus controls. Human samples with titers <5 were assigned a value of 2·5 for the purpose of calculating geometric mean titers.
The conventional NI assay was performed in test tubes as described in the 2002 WHO manual,10 with the following adjustments and differences from the miniaturized assay. Each virus preparation was diluted in PBS–0·1% BSA to a concentration providing NA activity that yielded an end OD549 reading of 0·5 by a cuvette spectrophotometer in the absence of serum (using a fetuin control sample to blank the instrument). Following incubation of 50 μl virus with an equal volume of each serial dilution of serum for 30 minutes at room temperature, 100 μl fetuin solution (12·5 mg/ml) was added per sample and incubated at 37°C for 15–16 hours. Volumes of periodate, arsenite, TBA, and Warrenoff reagents added per sample were 0·1, 1·0, 2·5, and 3·0 ml, respectively. Arsenite reagent contained 770 mm AsNaO2 and 500 mm Na2SO4, with 0·3% H2SO4, while TBA reagent contained 42 mm TBA and 500 mm Na2SO4. Incubation of samples with TBA reagent was performed in a boiling water bath. Absorbance was measured in 1-cm cuvettes with a SmartSpec 3000 spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) at wavelength 549 nm.