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Determination of Subset-Restricted Anti-neutrophil Cytoplasmic Antibodies (ANCA) by Immunofluorescence Cytochemistry.

Chapter in book
Authors Firoozeh Amirbeagi
Amanda Welin
Pontus Thulin
Johan Bylund
Published in Methods in molecular biology (Clifton, N.J.)
Pages 63-77
ISSN 1940-6029
Publication year 2019
Published at Institute of Odontology
Institute of Medicine, Department of Rheumatology and Inflammation Research
Pages 63-77
Language en
Subject categories Basic Medicine


Neutrophils have long been considered a homogeneous cell type where all circulating cells of a particular individual express the same proteins. Lately, however, this view is changing and distinct neutrophil subsets, defined by the presence or absence of different proteins, are being increasingly recognized. At least two separate protein markers, CD177 and Olfactomedin-4 (OLFM4) are known to be expressed by some, but not all, circulating neutrophils of a given individual. We recently described the existence of subset-restricted serum autoantibodies targeting OLFM4; these were discovered during clinical testing for anti-neutrophil cytoplasmic antibodies (ANCAs). ANCA testing is part of the clinical examinations routinely carried out to support diagnosis of suspected autoimmune conditions, especially vasculitis. Positive sera typically react with all neutrophils from a single donor, whereas subset-restricted ANCA sera (such as those containing anti-OLFM4 antibodies) only react with a fraction of neutrophils. Described in this chapter is an indirect immunofluorescence (IIF) approach to test human sera for the presence of subset-restricted ANCA as well as instructions for costaining experiments using sera and purified antibodies directed against established subset markers.

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