Jones M A, Kilpatrick P K, Carbonell R G
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7904.
Biotechnol Prog. 1993 May-Jun;9(3):242-58. doi: 10.1021/bp00021a003.
Small unilamellar phospholipid vesicles with covalently attached biotin and horseradish peroxidase (HRP) were prepared and characterized in terms of hydrodynamic diameter, amount and activity of immobilized enzyme, and number of biotin molecules on the outer vesicle surface. In addition, the specific adsorption of these bifunctional vesicles and commercially available biotin-labeled horseradish peroxidase (B-HRP) to antibiotin antibody (ABA) coated polystyrene microtiter plate wells was examined. At low antibody surface densities, the signal (delta A/min) generated by the vesicles adsorbed to the surface was approximately 100 times higher than the signal generated by B-HRP. It was also found that the biotin-conjugated vesicles were able to compete effectively with free biotin in solution for surface ABA sites. These results indicate that this type of vesicle may be used in competitive and sandwich-type enzyme-linked immunoassays to improve the detection limits, increase the signal, and decrease the reaction time necessary to detect a given analyte concentration in solution.
制备了共价连接生物素和辣根过氧化物酶（HRP）的小单层磷脂囊泡，并对其流体动力学直径、固定化酶的量和活性以及囊泡外表面生物素分子的数量进行了表征。此外，还研究了这些双功能囊泡和市售生物素标记的辣根过氧化物酶（B-HRP）对包被抗生物素抗体（ABA）的聚苯乙烯微量滴定板孔的特异性吸附。在低抗体表面密度下，吸附到表面的囊泡产生的信号（ΔA/分钟）比B-HRP产生的信号高约100倍。还发现生物素偶联的囊泡能够与溶液中的游离生物素有效竞争表面ABA位点。这些结果表明，这种类型的囊泡可用于竞争性和夹心型酶联免疫测定，以提高检测限、增加信号并减少检测溶液中给定分析物浓度所需的反应时间。
