Competitive Immunosorbent Assays for Biotin Using Bifunctional Unilamellar Vesicles

Matthew A. Jones, Peter K. Kilpatrick, and Ruben G. Carbonell

Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905

Competitive immunosorbent assays for the model antigen biotin were performed using

both unilamellar vesicles with covalently attached biotin and horseradish peroxidase

(HBVs) and commercially available biotin-labeled horseradish peroxidase (B-HRP) as

the enzyme-labeled antigen. The assays were performed using anti-biotin antibody

(ABA) surface densities ranging from one-tenth to full monolayer coverage.

It was found that assays using HBVs strongly depended on the antibody surface density, while assays using B-HRP were relatively insensitive to the antibody surface density. The

HBV assay dependence on the ABA surface density was most likely due to multiple

point attachment of vesicles to the surface. The lowest detectable antigen concentration

(least detectable dose) for vesicles (~ 10 ^-9 M) was an order of magnitude lower than the

value found for B-HRP (~ 10 ^-8 M). The sensitivity (slope of the response vs biotin

concentration curve) of assays with B-HRP was comparable to the sensitivity of assays

with HBVs a t low antibody surface density, probably due to less extensive multipoint

attachment. It was also found that assays could be performed with vesicles a t antibody

surface densities that were a t least 5 times lower, in terms of the bulk antibody

concentrations used to coat the wells, than antibody surface densities a t which B-HRP

gave comparable signals (~1 DA/min).