title: Measuring Antibody Orientation at the Bacterial Surface.
abstract: Many bacteria have the ability to interact with antibodies as a means to circumvent the immune response. This includes binding to the Fc portion of antibodies, effectively reversing the antibody orientation and thus decreasing the Fc-mediated immune signaling. Since antibody orientation at the bacterial surface has been shown to be important in human disease, it is valuable to be able to assess how antibodies are interacting with bacterial pathogens. Here, we describe a method to measure the proportion of human IgG that are bound via their Fc or Fabs to a bacterial surface. This is achieved by treating antibody-coated bacteria with the bacterial enzyme IdeS - which will cleave IgG into Fc and Fab fragments - and subsequently detect remaining fragments with fluorescent Fabs. The method is easy and fast, and the principle is most likely also applicable to other systems where distinguishing between antibody Fc and Fab binding is important.
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University, BMC B14, SE-221 84, Lund, Sweden.
authors.forename: Oonagh
authors.initials: O
authors.lastname: Shannon
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University, BMC B14, SE-221 84, Lund, Sweden. pontus.nordenfelt@med.lu.se.
authors.forename: Pontus
authors.initials: P
authors.lastname: Nordenfelt
mesh_terms: D000907:Antibodies, Bacterial / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001419:Bacteria / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D059748:Proteolysis; D013010:Sonication
chemical_list: D000907:Antibodies, Bacterial; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G
keywords: Bacteria; Flow cytometry; IdeS; IgG; IgG-binding proteins