Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane |
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Authors: | Md Golam Azam Takayuki Shibata Tsutomu Kabashima Masaaki Kai |
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Institution: | (1) Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan;(2) Global Center of Excellence Program, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan; |
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Abstract: | 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. |
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