B-090 Efficient Hydrophilic Surface Coating with Low Concentrations of Bovine Gamma Globulins
Authors: Nguyen V , Okerlund A , Kunkler C , Warner C 
Published: October 2024

Abstract

Background
Bovine Gamma Globulin (BGG) is a high purity immunoglobulin fraction of bovine serum. BGG is used as a blocker to prevent non-specific binding in immunoassays and, due to the mixture of immunoglobulins, its blocking capabilities differ from traditional blockers like Bovine Serum Albumin (BSA). Our objective was to evaluate the adsorption characteristics of BGG to In vitro diagnostic relevant hydrophilic surfaces compared to BSA. A bead-based immunoassay was utilized to quantify the mass of protein coating a bead surface and atomic force microscopy (AFM) to analyze physical properties of the protein-surface layer.

Methods
The mass of BGG bound to hydrophilic beads (Dynabeads M-270 Carboxylic Acid) was determined across varying concentrations ranging from 0.1% to 1.0% in pH 7.0 phosphate buffer. Coated beads were heated in 1x SDS loading dye to dissociate bound protein, then ran on a nonreducing SDS-PAGE gel. The total protein was measured using densitometry against a standard curve, using ImageJ, and analyzed with GraphPad Prism. This methodology was also used for BSA. AFM was used to image a topographical view of BGG binding to a silica surface. Samples were prepared at concentrations from 0.1% to 1.0% in phosphate buffer at pH 7.0. Followed by a series of washes to remove non-bound molecules, the surface was allowed to dry, then scratched with an AFM cantilever. The surface was then analyzed for roughness and thickness of BGG adsorption for each concentration.

Results
BGG and BSA have notable differences in their coating properties at a concentration of 1.0%. In the bead assay, the protein-surface binding is enhanced ∼4.8-fold for BGG compared to BSA. Interestingly, even at 0.1% BGG bound ∼0.8-fold more than BSA at 1.0%. Despite a 6-fold increase in mass bound to the bead surface with 1.0% BGG compared to 0.1%, AFM revealed a 1.3-fold and 1.8-fold decrease in height and roughness at the lower concentration, while maintaining effective coating. These results suggest that higher BGG concentrations result in thicker, rougher surface coating. This could lead to steric hindrance and obstruction of binding sites, affecting assay performance. Therefore, a lower concentration of BGG may lead to better surface coating and prevent reduced sensitivity and accuracy within immunoassays.

Conclusions
The bead binding assay demonstrates that BGG displays surface adsorption with comparable or improved results with lower concentrations compared to BSA. At 0.1%, BGG exhibits effective binding to a hydrophilic surface equivalent to that achieved by BSA at 1.0%. Additionally, AFM data suggest that even at 0.1%, BGG effectively coats the silica surface and potentially reduces steric hinderance compared to BGG at 1%. While BSA remains a reliable choice for many applications, our findings suggest that BGG can be an effective blocker for immunoassay applications. The choice of a suitable blocker varies depending on factors such as assay high background, sensitivity, and specificity. By tailoring the choice of blocker to the specific requirements of the assay, researchers can optimize assay performance by improving the accuracy and the reliability of the assay.