Biotechnol. Prog.,
Copyright © 1996 American Chemical Society and American Institute of Chemical Engineers
Competitive Immunosorbent
Assays Using Ligand-Enzyme Conjugates and Bifunctional Liposomes: Theory
and Experiment
Accepted
Two models of immunoadsorbent assays are developed
that describe the competitive adsorption of labeled antigen and unlabeled analyte to antibody binding sites immobilized on a solid
surface. In the first model, a small labeled antigen and a small unlabeled analyte compete with only binding site limitations and no steric limitations. A multicomponent
Langmuir isotherm results that is sufficient to
quantify competitive adsorption. This model can describe, with no adjustable
parameters, the data of competitive assays for biotin using biotinylated
horseradish peroxidase (B-HRP) over a wide range of
anti-biotin antibody (ABA) surface densities. In the second model, the small unlabeled
analyte competes with a large colloidal particle
containing many antigens and enzyme labels attached to its surface. This model
quantifies the steric interference that large
particles can experience upon binding (large ligand
effect) due to the lower probability of finding an available area of the right
size to accommodate the larger adsorbent. This large ligand
model also takes into account the increased probability of binding a large
particle due to the larger number of antibody binding sites covered per
collision. The resulting model is used to analyze the competitive assay data of
biotin competing with liposomes to which many biotin
and HRP molecules have been conjugated. This analysis is of interest because
previous work has shown that these bifunctional liposomes can reduce the detection limit for antigens in
bulk solution relative to assays performed with conventional small labeled
antigens.