High-performance chromatographic immunoassays with a biotin-streptavidin platform for the selective analysis of vaccine antigens

There is an ongoing need for rapid, accurate, and reliable methods for analyzing antigens in commercial or developmental vaccines, such as for quality control. This report examines and compares various approaches for the selective measurement of antigens in vaccine products by using high-performance immunoaffinity chromatography. A general platform for these chromatographic immunoassays was developed using microcolumns that contained immobilized streptavidin and adsorbed biotin-labeled antibodies against the target antigen. The amount of streptavidin and adsorbed antibodies in the microcolumns was first determined, along with their application/elution properties and stability. These microcolumns could be used with a cycle time as low as ∼10 min per sample and were stable over several hundred application/elution cycles. This platform was then evaluated for use in both the direct capture and detection of the target antigen and in sandwich immunoassays that used a pre-incubation step (30-60 min) of samples with indicator and/or labeled secondary antibodies. The detection limit, assay range, and precision of each approach were determined, along with the feasibility of using these methods at antigen levels expected in some developmental vaccines for bacterial respiratory infections. A format using this platform with direct fluorescence detection was suitable for an antigen present in a vaccine in the low-to-mid μg/mL range (detection limit, ∼ 4 μg/mL). However, a sandwich format with combined indicator/labeled antibodies was required for selective measurement of a vaccine antigen at low μg/mL levels (detection limit, ∼ 0.15 μg/mL). The same platform can be modified for use in analyzing other vaccines or biopharmaceutical products.