Quantitation of sputum NSP activity
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SECONDARY
To measure the activity of NE, PR3, and CatG in sputum, the ProAxsis ProteaseTag® Active NSP Immunoassays (ProAxsis, Belfast, UK) were used as a sandwich ELISA assay. Immunoassay plates were coated with a capture tag which allowed selective bindings to the active form of the specific NSP (NE, PR3 or CatG) on the plates. Phosphate buffered saline was added to the sputum at a 1:4 V:W ratio (volume added (mL) = mass of sample (g) × 4 mL) to homogenize the sputum samples. Sputum aliquots were then diluted with assay diluent (200-fold, 50-fold, tenfold, undiluted) and incubated in the ProAxsis plates. Then a primary antibody against each NSP was added to the plate and incubated to allow binding to the captured NSP. If the primary antibody was not conjugated, then a secondary horse radish peroxidase (HRP) conjugated antibody was added to the plate and incubated to allow binding to the primary antibody. A color forming substrate containing tetramethylbenzidine was also added to generate a colored product via an enzymatic reaction with the HRP, which was followed by an immediate absorbance reading using a Molecular Devices plate reader. Increases in NSP activity yielded increased amounts of absorbance in each of these assays.