Fluorescent silver nanoclusters as antibody label in a competitive immunoassay for the complement factor H
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Silver nanoclusters (AgNCs) were investigated as labels for the development of a fluoroimmunoassay for the complement factor H (CFH). The reductive one-pot synthesis of AgNCs using lipoic acid as a ligand was optimized by varying the concentration of NaBH4, the temperature and the reaction time. The average diameter and crystal structure of the AgNCs (which display red fluorescence) were determined by HR-TEM. The silver concentration was quantified by ICP-MS. Labelling of the antibody against CFH with AgNCs was optimized. The antibody was labeled with the AgNCs without compromising the recognition capabilities of the antibody. A competitive fluoroimmunoassay was then developed. Fluorescence is measured at excitation/emission maxima of 430/660 nm. The assay has a 0.4 ng mL−1 detection limit and a linear range that extends from 1.2 to 23 ng mL−1. The results compare favorably with those obtained by a commercial ELISA kit. The method was applied to the determination of CFH in spiked human serum.
KeywordsFluoroimmunoassay Lipoic acid One-pot synthesis Carbodiimide Labelling Human serum
This work was supported through project CTQ2016-79015-R by Agencia Estatal de Investigación (Spain) and FEDER. B. Fernandez acknowledges her contract RYC-2014-14985 to the Spanish Ministry of Economy and Competitiveness through the “Ramón y Cajal Program”. The Instituto Oftalmológico Fernández-Vega and Fundación de Investigación Oftalmológica acknowledge support from “Cátedra Rafael del Pino” and from Instituto de Desarrollo Económico del Principado de Asturias (IDEPA) and FEDER (project IDE/2016/000214).
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The author(s) declare that they have no competing interests.
- 8.Geerlings MJ, Kremlitzka M, Bakker B, Nilsson SC, Saksens NT, Lechanteur YT, Pauper M, Corominas J, Fauser S, Hoyng CB, Blom AM, de Jong EK, den Hollander AI (2017) The functional effect of rare variants in complement genes on C3b degradation in patients with age-related macular degeneration. JAMA Ophthalmol 135:39–46CrossRefGoogle Scholar
- 36.Sofat R, Mangione PP, Gallimore JR, Hakobyan S, Hughes TR, Shah T, Goodship T, D'Aiuto F, Langenberg C, Wareham N, Morgan BP, Pepys MB, Hingorani AD (2013) Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay. J Immunol Methods 390:63–73CrossRefGoogle Scholar
- 37.Hakobyan S, Harris CL, Tortajada A, Goicochea de Jorge E, García-Layana A, Fernández-Robredo P, Rodríguez de Córdoba S, Morgan BP (2008) Measurement of factor H variants in plasma using variant-specific monoclonal antibodies: application to assessing risk of age-related macular degeneration. Invest Ophthalmol Vis Sci 49:1983–1990CrossRefGoogle Scholar