title: A radioimmune microfilter plate assay for the detection of anti-granulocyte antibodies.
abstract: A radioimmune assay (RIA) for IgG anti-granulocyte antibodies (AGA) was developed which utilized 96 well microfilter plates, required less than 50 microliters plasma, and yielded reproducible autologous determinations on patients with absolute granulocyte counts as low as 300 cells/microliter. Granulocytes were isolated by a single density ficoll-hypaque gradient procedure with red blood cell (RBC) removal by lysis and washing. Anti-granulocyte IgG coating the cells was detected by the addition of 125I labeled anti-human IgG. Six patients with autoimmune granulocytopenia and three multiply transfused patients with febrile transfusion reactions believed to be secondary to allogeneic granulocyte antibodies had increased granulocyte IgG. Plasma containing anti-a and/or anti-B titers greater than or equal to 512 gave positive results when tested against ABO incompatible granulocytes. This technique was capable of detecting antibodies which were reactive in granulocyte agglutination and immunofluorescence tests as demonstrated with known anti-NA-1, anti-NB-1, and anti-Mart sera. Comparison with granulocyte agglutination test (GAT) using clinical criteria for immune granulocytopenia demonstrated concordance with 19 negative samples and 8 positive samples, 1 false negative and 3 false positives were observed using the agglutination method. The multimeric and monomeric IgG fractions of G200 purified Cohn Fraction II were nonreactive by both RIA and GAT methods. However, granulocyte activation by phorbol myristate acetate (PMA) produced false positives in 5 out of 5 cases by GAT but 0 out of 4 by RIA. This RIA assay is not effected by nonimmune granulocyte activation and is more reproducible when compared to existing techniques.
authors.affiliation: Department of Medicine, Michigan State University, East Lansing.
authors.forename: A M
authors.initials: AM
authors.lastname: Braman
authors.forename: K A
authors.initials: KA
authors.lastname: Schwartz
mesh_terms: D000017:ABO Blood-Group System / Q000276:immunology; D000328:Adult; D000372:Agglutination Tests; D000906:Antibodies / Q000032:analysis*; D002469:Cell Separation; D005260:Female; D006098:Granulocytes / Q000276:immunology* / Q000502:physiology; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D011863:Radioimmunoassay / Q000295:instrumentation / Q000379:methods*; D012016:Reference Values; D015203:Reproducibility of Results
chemical_list: D000017:ABO Blood-Group System; D000906:Antibodies; D007074:Immunoglobulin G