Abstract
BackgroundRecognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system.ResultsWe show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin.ConclusionsThe application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0456-y) contains supplementary material, which is available to authorized users.
Highlights
Recognition of microorganisms by antibodies is a vital component of the human immune response
Flow cytometry allows the separation of bacterial cells according to their population structure [5, 6] and to the fluorescence emitted by antibodies bound to different human Igs [7, 8]
Flow cytometry Samples were suspended in sterile saline solution with 5 % albumin to prevent non-specific antibody binding, stained with (i) anti-human IgA or IgG labelled with FITC (Invitrogen catalog # A18782 and A18806); and (ii) the DNA-binding fluorophor SYTO62 (Invitrogen catalog # S11344) according to the manufacturer instructions
Summary
Recognition of microorganisms by antibodies is a vital component of the human immune response. Flow cytometry allows the separation of bacterial cells according to their population structure [5, 6] and to the fluorescence emitted by antibodies bound to different human Igs [7, 8]. These labeled populations of bacteria can be characterized by second-generation sequencing of PCR-amplified microbial rDNA genes, to provide a description of the bacterial and fungal diversity and taxonomic composition [9]
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