Wink T, van Zuilen S J, Bult A, van Bennekom W P
Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, The Netherlands.
Anal Chem. 1998 Mar 1;70(5):827-32. doi: 10.1021/ac971049z.
A recently developed liposome sandwich immunoassay for interferon-gamma (IFN-gamma), to be applied in microtiter plates, is tailored for surface plasmon resonance (SPR) spectrometry. The assay is performed on a thin (approximately 20 nm) polystyrene layer that covers a gold surface. This way, analytical data obtained from microtiter plate technology can directly be extrapolated toward SPR. For assaying the antigen IFN-gamma, a 16-kDa cytokine, a capture monoclonal antibody is physically adsorbed onto the polystyrene surface. After addition of the sample containing IFN-gamma, a biotinylated detecting antibody is added. Avidin is used as a bridging molecule between the biotinylated antibody and the biotinylated liposomes. All solutions are prepared with PBS buffer (10 mM, pH 7.4). This avoids additional changes in index of refraction caused by the use of various buffer solutions in immunoassays on microtiter plates for coating, binding, and washing procedures. It is shown that, when liposomes are used, a substantial enhancement of the detection limit is achieved. The "liposome" strategy improves the sensitivity for the IFN-gamma assay approximately 4 x 10(4) times and the detection limit to low picomolar. The method is generally applicable to other sandwich immunoassays.
一种最近开发的用于γ干扰素（IFN-γ）的脂质体夹心免疫测定法，适用于微量滴定板，是为表面等离子体共振（SPR）光谱法量身定制的。该测定在覆盖金表面的薄（约20纳米）聚苯乙烯层上进行。通过这种方式，从微量滴定板技术获得的分析数据可以直接外推到SPR。为了测定抗原IFN-γ（一种16千道尔顿的细胞因子），将捕获单克隆抗体物理吸附到聚苯乙烯表面。加入含有IFN-γ的样品后，再加入生物素化的检测抗体。抗生物素蛋白用作生物素化抗体和生物素化脂质体之间的桥接分子。所有溶液均用PBS缓冲液（10 mM，pH 7.4）配制。这避免了在微量滴定板上进行包被、结合和洗涤程序的免疫测定中因使用各种缓冲液而引起的折射率额外变化。结果表明，当使用脂质体时，检测限有显著提高。“脂质体”策略将IFN-γ测定的灵敏度提高了约4×10⁴倍，检测限降低到低皮摩尔。该方法一般适用于其他夹心免疫测定。
