Hydrocarbon and Lipid Microbiology Protocols

Part of the series Springer Protocols Handbooks pp 207-224


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

  • Yolanda BlancoAffiliated withDepartment of Molecular Evolution, Centro de Astrobiología (INTA-CSIC)
  • , Mercedes Moreno-PazAffiliated withDepartment of Molecular Evolution, Centro de Astrobiología (INTA-CSIC)
  • , Jacobo AguirreAffiliated withCentro Nacional de Biotecnología (CSIC)
  • , Victor ParroAffiliated withDepartment of Molecular Evolution, Centro de Astrobiología (INTA-CSIC) Email author 

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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.


Antibody microarray Biosensor Deconvolution method Environmental monitoring Graph theory Multiplex sandwich microarray immunoassay