Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane
Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane
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DOI：
10.1007/s00216-011-5196-8
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发表时间：
2011-07
影响因子：
4.3
通讯作者：
M. Azam;T. Shibata;T. Kabashima;M. Kai
中科院分区：
文献类型：
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作者：
M. Azam;T. Shibata;T. Kabashima;M. Kai
在本研究中，我们合成了基于葡聚糖(MW = ，约2000 kDa)的大分子探针，其中含有多个分子的碱性磷酸酶作为信号触发酶，生物素作为组装介体。碱性磷酸酶和生物素分子通过高碘酸氧化形成大分子后共价键合在葡聚糖骨架上。当碱性磷酸酶与葡聚糖的摩尔比为50倍时，合成的探针大分子中含有27-31个碱性磷酸酶分子。这些探针的化学发光强度是吸附在尼龙膜上的等摩尔游离碱性磷酸酶的14-20倍。AlP催化的金刚烷基-1，2-二氧杂环己烷底物的化学发光反应速度从较慢的发光(发光型)提高到较快的(闪光型)。在强碱性条件下(pH=13.0)，化学发光信号积分2分钟，约为常规条件(pH=9.5)时的10倍。因此，合成的大分子探针可以成功地用于生物素偶联的抗兔抗体的高通量化学发光检测，其检测下限为每尼龙膜上的一个斑点880微克分子。本研究为免疫分析中快速、方便、灵敏地检测目标蛋白提供了一种分析策略。图：用葡聚糖(Dex)-生物素(Bio)-ALP探针在固相膜上化学发光检测生物素标记的抗兔抗体(b-Ab)，用BCL成像检测吸附在尼龙膜上的Dex-Bio-ALP探针和ALP，用Dex-Bio120-ALP28探针CCL成像检测尼龙膜上的b-Ab。每点b-Ab量(毫微摩尔)：a1= 0.32，a2= 0.63，a3= 1.25，a4= 2.5，a5= 5，a6= 10
In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran backbone after the formation of aldehyde groups into the macromolecule by periodate oxidation. The synthesized probes contained 27–31 molecules of ALP in their macromolecules when 50-fold molar ratio of ALP to the dextran was used for the synthesis. These probes provided 14–20 times stronger chemiluminescence (CL) than that of the equimolar free ALP adsorbed on a nylon membrane. The velocity of the CL reaction of ALP-catalyzed adamantlyl-1,2-dioxetane substrate was improved from a slower emission (glow type) of CL to a faster one (flash type). The CL signal integrated for 2 min under strongly alkaline conditions (pH 13.0) was about ten times greater than that obtained by the conventional conditions (pH 9.5). Therefore, the synthesized macromolecular probe could be successfully utilized for the high-throughput CL detection of biotin-conjugated anti-rabbit IgG antibody with a lower detection limit of 880 amol per spot on the nylon membrane. This study provides analytical strategy for the rapid, convenient, and sensitive detection of target proteins in immunoassays.FigureASchematic principle for the chemiluminescence (CL) detection of biotin-conjugated anti-rabbit IgG antibody (b-Ab) on a solid-phase membrane by using a dextran (Dex)-biotin (Bio)-ALP probe,BCL-imaging detection of Dex-Bio-ALP probes and ALP adsorbed on a nylon membrane, andCCL-imaging detection of b-Ab on a nylon membrane using Dex-Bio120-ALP28probe. Amounts (femtomole) of b-Ab per spot:a1= 0.32,a2= 0.63,a3= 1.25,a4= 2.5,a5= 5, anda6= 10