Jones M A, Kilpatrick P K, Carbonell R G
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905.
Biotechnol Prog. 1994 Mar-Apr;10(2):174-86. doi: 10.1021/bp00026a007.
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 (approximately 10(-9) M) was an order of magnitude lower than the value found for B-HRP (approximately 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 at low antibody surface density, probably due to less extensive multipoint attachment. It was also found that assays could be performed with vesicles 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 (approximately 1 delta A/min).
使用共价连接生物素和辣根过氧化物酶的单层囊泡（HBVs）以及市售生物素标记的辣根过氧化物酶（B-HRP）作为酶标记抗原，对模型抗原生物素进行了竞争性免疫吸附测定。测定时使用的抗生物素抗体（ABA）表面密度范围从十分之一到完全单层覆盖。结果发现，使用HBVs的测定强烈依赖于抗体表面密度，而使用B-HRP的测定对抗体表面密度相对不敏感。HBV测定对ABA表面密度的依赖性很可能是由于囊泡与表面的多点附着。囊泡的最低可检测抗原浓度（最低可检测剂量）（约10^(-9) M）比B-HRP的测定值（约10^(-8) M）低一个数量级。在低抗体表面密度下，B-HRP测定的灵敏度（响应与生物素浓度曲线的斜率）与HBVs测定的灵敏度相当，这可能是由于多点附着程度较低。还发现，就用于包被孔的本体抗体浓度而言，使用囊泡进行测定时的抗体表面密度可以比B-HRP产生可比信号时的抗体表面密度（约1 ΔA/分钟）至少低5倍。
