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Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane

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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.

A Schematic 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, B CL-imaging detection of Dex-Bio-ALP probes and ALP adsorbed on a nylon membrane, and C CL-imaging detection of b-Ab on a nylon membrane using Dex-Bio120-ALP28 probe. Amounts (femtomole) of b-Ab per spot: a 1 = 0.32, a 2 = 0.63, a 3 = 1.25, a 4 = 2.5, a 5 = 5, and a 6 = 10

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Acknowledgements

This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and partly by the Global Center of Excellence Program of Nagasaki University.

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki, 852-8521, Japan

    Md. Golam Azam, Takayuki Shibata, Tsutomu Kabashima & Masaaki Kai

  2. Global Center of Excellence Program, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki, 852-8521, Japan

    Masaaki Kai

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  1. Md. Golam Azam
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  2. Takayuki Shibata
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  3. Tsutomu Kabashima
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  4. Masaaki Kai
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Correspondence to Masaaki Kai.

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Azam, M.G., Shibata, T., Kabashima, T. et al. Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane. Anal Bioanal Chem 401, 1211–1217 (2011). https://doi.org/10.1007/s00216-011-5196-8

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  • DOI: https://doi.org/10.1007/s00216-011-5196-8

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