B-087 The Antifouling Properties of BSA on Magnetic Beads are Affected by BSA Grade, Bead Type, and pH

Abstract

Abstract Background BSA (bovine serum albumin) is a protein that is widely used for its antifouling properties in a variety of immunoassays. One such immunoassay is a bead-based ELISA (enzyme-linked immunosorbent assay), which can provide quantitative detection of many diverse analytes. BSA increases the sensitivity of a bead-based ELISA by coating the beads and blocking nonspecific interactions, thereby lowering background noise. However, the BSA grade chosen for the assay is frequently based on experience, as there is a lack of understanding of how BSA grades interact with different bead surfaces under various assay conditions. The objective of this study is to examine if different BSA grades work better with particular bead chemistries and how pH influences those interactions. Specifically, the aim is to quantify the amount of BSA on ELISA beads and assess the blocking of nonspecific antibody signal with different bead types, BSA grades, and pH. Methods The mass of BSA binding to beads was measured by heating BSA-coated beads in 1x SDS loading dye, then running on a denaturing PAGE gel. Total protein was measured by densitometry against a standard curve using ImageJ. The amount of blocking was measured using a modified bead-based ELISA, comparing the amount of antibody-HRP binding to the beads in the presence and absence of BSA. The amount of antibody-HRP was measured using a SpectraMax Plus 384 plate reader against a standard curve. All data were analyzed using GraphPad Prism and presented as the average of at least three independent replicates with error bars representing standard deviation. Conditions tested include different Dynabeads (hydrophilic vs. hydrophobic - Dynabeads M-270 Carboxylic Acid and M-280 Tosylactivated), BSA grade (fatty acid (FA) vs. fatty acid-free (FAF) - Proliant SKUs 68100 and 68700), and pH (7 vs. 5.2). Results The choice of pH is more influential than the BSA grade on bead surface coating and blocking, regardless of bead type. Lowering the pH from 7 to 5.2 increases the mass of BSA on the beads ∼1.7-fold for hydrophobic beads and ∼10-fold for hydrophilic beads. However, the mass of BSA on the beads is reduced after the pH returns to 7, indicating the increased BSA binding at pH 5.2 is reversible. We also observed BSA decreases nonspecific antibody-bead interactions by up to 90%, depending on assay conditions. Though the amount of BSA coating the beads between FA and FAF grade BSA is not significant, FAF grade BSA blocks the antibody-bead interactions ∼10% more than FA grade BSA, suggesting FAF grade BSA has better antifouling properties. Conclusion Herein, we show the presence of BSA reduces the noise within bead-based ELISAs by binding to the bead and blocking nonspecific antibody-bead interactions, regardless of BSA grade, bead type, and pH. FAF grade BSA demonstrates slightly better blocking than FA grade BSA, but both BSA grades decreased nonspecific antibody-bead interactions. Further, BSA shows antifouling properties at the typical assay pH of 7, but lowering the pH to 5.2 can significantly increase BSA bead coating.