title: Prevention of false positive binding during immunofluorescence of Staphylococcus aureus infected tissue biopsies.
abstract: Immunofluorescence is a fundamental tool used to analyse tissue and cell samples with a wide variety of available antibodies targeting specific proteins or molecules. Staphylococcal surface protein A is used both in clinical, research and industrial settings for its ability to bind mammalian immunoglobulin G. Spurious binding between protein A and IgG antibodies can lead to false-positive fluorescence and misleading results. Here we demonstrate this occurring in formalin-fixed patient samples that harbour Staphylococcus aureus infection, and characterise methods to overcome this issue. Specifically the use of F(ab') fragment antibodies or blocking with human IgG is shown to prevent antibody-protein A interaction in formalin-fixed S. aureus smears, biopsies obtained from infected patients, and experimentally infected tissue samples.
authors.affiliation: Department of Otolaryngology Head and Neck Surgery, University of Adelaide, Adelaide, Australia.
authors.forename: Neil C-W
authors.initials: NC
authors.lastname: Tan
authors.forename: Hai
authors.initials: H
authors.lastname: Tran
authors.forename: Eugene
authors.initials: E
authors.lastname: Roscioli
authors.forename: Peter John
authors.initials: PJ
authors.lastname: Wormald
authors.forename: Sarah
authors.initials: S
authors.lastname: Vreugde
mesh_terms: D001706:Biopsy; D004848:Epithelium / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D005189:False Positive Reactions; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D005557:Formaldehyde; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D015203:Reproducibility of Results; D013203:Staphylococcal Infections / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D013205:Staphylococcal Protein A / Q000276:immunology* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000276:immunology* / Q000378:metabolism; D016707:Tissue Fixation
chemical_list: D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D005557:Formaldehyde