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Multiplex Fluorescent Antibody Microarrays and Antibody Graphs for Microbial and Biomarker Detection in the Environment

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Hydrocarbon and Lipid Microbiology Protocols

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

We describe a rapid protocol for multiplex fluorescent sandwich microarray immunoassay (FSMI) for detecting microorganisms and biomarkers in environmental samples. An FSMI consists basically in two steps: (i) specific binding of antigens (or any recognizable analyte in a sample) to the immobilized antibodies (capturing antibodies) on a microarray and (ii) revealing specific antigen-antibody binding by fluorescently labelled antibodies (tracer or detector antibodies). Because multiplexing immunoassays may produce cross-reaction events, we have developed a method, based on graph theory, to disentangle such cross-reactions associated to an antibody microarray. First, we need to generate an antibody graph from the experimental cross-reactivity test of each antigen with its fluorescent antibody (tracer) by FSMI on the whole microarray. From the matrix of quantified fluorescence, it is possible to establish a weighted correlation between each cognate antigen-antibody pair and other antibodies printed on the microarray. Then, deconvolution methods allow disentangling the cross-reaction events and discriminate between true cognate antigen-antibody recognition from others due to related antigenic structures. Therefore, the multiplex FSMI implemented with antibody graphs is a powerful technique for the analysis and characterization of environmental samples, in the identification and monitoring of the microbial diversity or the detection of microbial biomarkers that might be relevant for hydrocarbon microbiology.

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Acknowledgements

This work is funded by the Secretaría de Estado de Investigación, Desarrollo e Innovación of the Spanish Ministry of Economy and Competitiveness grants No. AYA2011-24803, FIS2011-27569 and FIS2014-57686.

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Authors and Affiliations

  1. Department of Molecular Evolution, Centro de Astrobiología (INTA-CSIC), Carretera de Ajalvir km 4, Torrejón de Ardoz, 28850, Madrid, Spain

    Yolanda Blanco, Mercedes Moreno-Paz & Victor Parro

  2. Centro Nacional de Biotecnología (CSIC), c/ Darwin 3, 28049, Madrid, Spain

    Jacobo Aguirre

Authors
  1. Yolanda Blanco
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  2. Mercedes Moreno-Paz
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  3. Jacobo Aguirre
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  4. Victor Parro
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Corresponding author

Correspondence to Victor Parro .

Editor information

Editors and Affiliations

  1. University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Dept. Biologia, Univ de les Illes Balears, Palma de Mallorca, Spain

    Balbina Nogales

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Blanco, Y., Moreno-Paz, M., Aguirre, J., Parro, V. (2015). Multiplex Fluorescent Antibody Microarrays and Antibody Graphs for Microbial and Biomarker Detection in the Environment. In: McGenity, T., Timmis, K., Nogales , B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_159

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  • DOI: https://doi.org/10.1007/8623_2015_159

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-52776-4

  • Online ISBN: 978-3-662-52778-8

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