HLA-B27 phenotyping with flow cytometry: further improvement by multiple monoclonal antibodies.
To establish the optimal flow cytometric method for HLA-B27 phenotyping, we compared several strategies, using three monoclonal anti-B27 antibodies (from the HLA-ABC-m3, GS145.2, and FD705 clones). We used a triple-color direct immunofluorescence assay, including a T-lymphocyte-specific antibody as an internal control and an anti-HLA-Bw4 antibody. Blood samples from >400 subjects were tested. From ROC curve analysis none of the three antibodies appeared to be suitable for use as a single typing reagent. The efficiency of the test was affected by cross-reactions with other HLA antigens, notably the HLA-B7 antigen. Preincubation with anti-B7 serum efficiently inhibited this cross-reaction and raised the test efficiency considerably. We concluded that none of the anti-B27 antibodies investigated is suitable for use as a single typing reagent. Additional typing of Bw4 is not valuable, whereas inhibition of cross-reactions due to the B7 antigen will considerably improve the performance of the test. We recommend that two different monoclonal anti-B27 antibodies be used for accurate and reliable HLA-B27 phenotyping with flow cytometry.
Hoffmann
J J
JJ
Department of Clinical Laboratories, Catharina Hospital, Eindhoven, The Netherlands.
Janssen
W C
WC
D000911:Antibodies, Monoclonal / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D015796:HLA-B27 Antigen / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007202:Indicators and Reagents; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D015796:HLA-B27 Antigen; D007202:Indicators and Reagents