Detection of CRP binding to FcγRIIa depends on Fc region of anti-CRP antibodies. Polymorphonuclear leukocytes (PMNs) were isolated from donors genotyped for FcγRIIa-R131 or H131 polymorphic forms. CRP was isolated from peritoneal fluid (kindly provided by C.E. Hack, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), and 100 μg/ml CRP was incubated with 3 × 10⁵ PMNs (a and b) or FcγRIIa-transfected IIA1.6 cells (c and d) in PBS with 10% BSA (Roche Nederland BV, Mijdrecht, The Netherlands) and 0.05% sodium azide for 1 hour at 4°C. Cells were washed and incubated with 50 μg/ml of a whole mouse IgG1 anti-CRP antibody (Clone CRP 8; Sigma Chemical Co., St. Louis, Missouri, USA) (a and c) or F(ab′)₂ fragments of anti-CRP (b and d) for 30 minutes, washed again, and further incubated with an FITC-labeled goat F(ab′)₂ anti-mouse κ light chain antiserum (Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA) (a, b, and d) or FITC-labeled goat F(ab′)₂ fragments of anti-mIgG1 (Southern Biotechnology Associates, Birmingham, Alabama, USA) (c) for 30 minutes. An FITC-labeled mIgG1 isotype control (DAKO A/S, Glostrup, Denmark) was included in all experiments, and cells were analyzed by flow cytometry. Data are representative of more than five individual experiments yielding almost identical results.