COMPETITIVE IMMUNOSORBENT ASSAYS USING LIGAND-ENZYME CONJUGATES AND BIFUNCTIONAL LIPOSOMES - THEORY AND EXPERIMENT

Two models of immunoadsorbent assays are developed that describe the c ompetitive adsorption of labeled antigen and unlabeled analyte to anti body binding sites immobilized on a solid surface. In the first model, a small labeled antigen and a small unlabeled analyte compete with on ly binding site limitations and no steric limitations. A multicomponen t Langmuir isotherm results that is sufficient to quantify competitive adsorption. This model can describe, with no adjustable parameters, t he data of competitive assays for biotin using biotinylated horseradis h peroxidase (B-HRP) over a wide range of anti-biotin antibody (ABA) s urface densities. In the second model, the small unlabeled analyte com petes with a large colloidal particle containing many antigens and enz yme labels attached to its surface. This model quantifies the steric i nterference that large particles can experience upon binding (large li gand effect) due to the lower probability of finding an available area of the right size to accommodate the larger adsorbent. This large lig and model also takes into account the increased probability of binding a large particle due to the larger number of antibody binding sites c overed per collision. The resulting model is used to analyze the compe titive assay data of biotin competing with liposomes to which many bio tin and HRP molecules have been conjugated. This analysis is of intere st because previous work has shown that these bifunctional liposomes c an reduce the detection limit for antigens in bulk solution relative t o assays performed with conventional small labeled antigens.