AVS 50th International Symposium
    Biomaterial Interfaces Monday Sessions
       Session BI-MoA

Paper BI-MoA6
Comparison of Immunoassay Blocking Strategies on Metal Oxide Substrates

Monday, November 3, 2003, 3:40 pm, Room 307

Session: Non-fouling Surfaces and Biolubrication
Presenter: A.N. Scribner, Naval Research Laboratory
Authors: A.N. Scribner, Naval Research Laboratory
C.L. Cole, Naval Research Laboratory
R.J. Colton, Naval Research Laboratory
L.J. Whitman, Naval Research Laboratory
Correspondent: Click to Email
We have developed an alumina filter-based immunosensor that is 10 times faster and ~3 orders of magnitude more sensitive than an analogous microtiter well-based format. The assay is based on a standard sandwich immunoassay but uses magnetic microbeads and magnetic forces to differentiate between specific and nonspecific interactions. The combined use of magnetic force discrimination with PEG-based surface chemistries that minimize nonspecific binding forces result in a demonstrated specificity of >98%. Additionally, a more traditional blocking agent can also be added to compensate for lot-to-lot variability in the surface chemistry of commercially available alumina membranes. However, immunoassays on metal oxide supports not based on electrochemical detection are uncommon, so comparatively little is known about the effectiveness of different blocking agents for such surfaces. We examine agents typically used to block polystyrene plates for their relative effectiveness at blocking PEGylated and non-PEGylated alumina membranes, including detergents, proteins, hydrophobic, and hydrophilic molecules. The effectiveness of each substance as a blocker is determined quantitatively by measuring the amount of IgG-HRP remaining after incubation on a pre-blocked surface. Our results suggest that traditional reagents such as gelatin or BSA do not have the same ability to block nonspecific binding on PEGylated alumina as on polystyrene, and that casein and charged reagents such as SDS may be more appropriate choices for the blocking of modified metal oxide surfaces. Supported by ONR and the DoD JSTPCBD. ANS and CLC are employees of Nova Research, Inc., Alexandria, VA.