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Factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus X

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Abstract

Key factors influencing the analyte detection limit of the sandwich immunochromatographic assay (ICA), namely, the size of gold nanoparticles, the antibody concentration, the conjugation pH, and characteristics of membranes, are discussed. The impacts of these factors were quantitatively characterized and compared for the first time using the same antigen (potato virus X). The antibody–colloidal gold conjugates synthesized at pH 9.0–9.5 (the pH was examined in the range from 7.5 to 10.0) and at an antibody concentration of 15 μg/mL (the concentration was tested from 10 to 100 μg/mL) demonstrated maximum binding with the analyte. The relationship between the size of gold nanoparticles and the ICA detection limit was determined. The detection limit decreases from 80 to 3 ng/mL (for antibodies with K D = 1.0 × 10-9 M, data were obtained using a BIAcore X instrument) for a series of particles with a diameter from 6.4 to 33.4 nm (electron microscopy and dynamic light scattering data). In the case of larger particles (52 nm in diameter), the detection limit increases and reaches 9 ng/mL. A 10 mM phosphate buffer, pH 8, and a 50 mM phosphate buffer, pH 7, were the conditions of choice for the deposition of reactants. Taking into account these facts, we developed a lateral-flow test system for the rapid (10 min) detection of potato virus X in plant leaves. The ICA provided a visual detection limit of 3 ng/mL. In the case of the instrumental processing, potato virus X can be determined in the concentration range from 3 to 300 ng/mL with a detection limit 2 ng/mL.

The detection limit of immunochromatographic test systems, corresponding average diameters of the gold nanoparticles in the conjugates and color intensities in the test zones

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Acknowledgments

We are grateful to V.G. Avdienko and A.N. Guseva (Central Scientific Research Institute of Tuberculosis, Moscow) for the production of monoclonal antibodies, and to S.N. Chirkov (M.V. Lomonosov Moscow State University, Russia) for viral preparations. This work was supported by the Federal Targeted Program “Scientific and Scientific-Pedagogical Personnel of the Innovative Russia in 2009-2013” (government contracts no. 14.740.11.1065, May 24, 2011, and no. 02.740.11.0868, June 28, 2010), and the International Targeted Program of the Eurasian Economic Community “Innovative Biotechnologies for 2011-2015” (government contract no. 16.M04.11.0022, April 29, 2011).

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  1. A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, 119071, Moscow, Russia

    Irina Safenkova, Anatoly Zherdev & Boris Dzantiev

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  1. Irina Safenkova
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  2. Anatoly Zherdev
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  3. Boris Dzantiev
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Correspondence to Irina Safenkova.

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Safenkova, I., Zherdev, A. & Dzantiev, B. Factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus X. Anal Bioanal Chem 403, 1595–1605 (2012). https://doi.org/10.1007/s00216-012-5985-8

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