Jones M A, Kilpatrick P K, Carbonell R G
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905, USA.
Biotechnol Prog. 1996 Jul-Aug;12(4):519-26. doi: 10.1021/bp960035w.
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.
开发了两种免疫吸附测定模型，它们描述了标记抗原和未标记分析物对固定在固体表面的抗体结合位点的竞争性吸附。在第一个模型中，小的标记抗原和小的未标记分析物仅在结合位点有限而无空间位阻的情况下竞争。得到一个多组分朗缪尔等温线，它足以量化竞争性吸附。该模型无需调整参数，就能描述在广泛的抗生物素抗体（ABA）表面密度范围内，使用生物素化辣根过氧化物酶（B-HRP）进行生物素竞争性测定的数据。在第二个模型中，小的未标记分析物与一个大的胶体颗粒竞争，该胶体颗粒表面附着有许多抗原和酶标记。该模型量化了大颗粒在结合时可能经历的空间干扰（大配体效应），这是由于找到合适大小的可用区域来容纳较大吸附剂的概率较低。这个大配体模型还考虑了由于每次碰撞覆盖的抗体结合位点数量较多，大颗粒结合的概率增加。所得模型用于分析生物素与已偶联许多生物素和HRP分子的脂质体竞争的竞争性测定数据。这种分析很有意义，因为先前的工作表明，相对于使用传统小标记抗原进行的测定，这些双功能脂质体可以降低本体溶液中抗原的检测限。
