Ma. Jones et al., COMPETITIVE IMMUNOSORBENT ASSAYS FOR BIOTIN USING BIFUNCTIONAL UNILAMELLAR VESICLES, Biotechnology progress, 10(2), 1994, pp. 174-186
Competitive immunosorbent assays for the model antigen biotin were per
formed using both unilamellar vesicles with covalently attached biotin
and horseradish peroxidase (HBVs) and commercially available biotin-l
abeled horseradish peroxidase (B-HRP) as the enzyme-labeled antigen. T
he assays were performed using anti-biotin antibody (ABA) surface dens
ities ranging from one-tenth to full monolayer coverage. It was found
that assays using HBVs strongly depended on the antibody surface densi
ty, while assays using B-HRP were relatively insensitive to the antibo
dy surface density. The HBV assay dependence on the ABA surface densit
y was most likely due to multiple point attachment of vesicles to the
surface. The lowest detectable antigen concentration (least detectable
dose) for vesicles (similar to 10(-9) M) was an order of magnitude lo
wer than the value found for B-HRP (similar to 10(-8) M). The sensitiv
ity (slope of the response vs biotin concentration curve) of assays wi
th B-HRP was comparable to the sensitivity of assays with HBVs at low
antibody surface density, probably due to less extensive multipoint at
tachment. It was also found that assays could be performed with vesicl
es at antibody surface densities that were at least 5 times lower, in
terms of the bulk antibody concentrations used to coat the wells, than
antibody surface densities at which B-HRP gave comparable signals (si
milar to 1 Delta A/min).