Single Radial Immunodiffusion Assay

A single radial immunodiffusion (SRID) assay was used to determine the antigenic activity of the H1N1 subunit vaccine either in a liquid formulation or in a dry solid formulation immediately after the formulation preparation process and after storage under study conditions. Gels were prepared by dissolving 1% agarose in phosphate-buffered saline (PBS), adding 10–20 μL/mL of influenza Anti-A/California/7/2009 (H1N1) HA serum (National Institute for Biological Standards and Control [NIBSC], UK), and dispensing on a plastic backing. Wells were punched in a slab of the solidified agarose gel. A dilution series of the reference standard influenza antigen A/California/7/09 (H1N1) (NIBSC, UK) at four concentrations from 50 to 12.5 μg/mL, and the test sample at two concentrations in the linear range of the standard, were prepared in PBS containing 1% of Zwittergent 3–14 (EMD Millipore). After the addition of 20 μL of antigen sample to the well, the antigen diffused into the agarose, resulting in an antigen–antibody reaction causing a zone of precipitation around the well. After a 24-hour diffusion period at room temperature in a humidified chamber, the gel was washed to remove unbound antibody, dried, stained with Coomassie Blue dye (0.1% in a mixture containing 45% ethanol and 10% acetic acid), and then destained. Images of precipitation zones were recorded on an AlphaImager (ProteinSimple, San Jose, CA), and the diameters of the rings were measured using ImageJ software [15]. The ring sizes of both the reference standard and test sample were compared, and HA content in the test sample was determined against the reference standards in units of μg/mL. Triplicate and duplicate measurements were made for each test sample and reference standard analysis, respectively.