HLA-B27 PHENOTYPING WITH FLOW-CYTOMETRY - FURTHER IMPROVEMENT BY MULTIPLE MONOCLONAL-ANTIBODIES

To establish the optimal flow cytometric method for HLA-B27 phenotypin g, we compared several strategies, using three monoclonal anti-B27 ant ibodies (from the HLA-ABC-m3, GS145.2, and FD705 clones). We used a tr iple-color direct immunofluorescence assay, including a T-lymphocyte-s pecific 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 sing le typing reagent. The efficiency of the test was affected by cross-re actions with other HLA antigens, notably the HLA-B7 antigen. Preincuba tion 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 typi ng reagent. Additional typing of Bw4 is not valuable, whereas inhibiti on of cross-reactions due to the B7 antigen will considerably improve the performance of the test. We recommend that two different monoclona l anti-B27 antibodies be used for accurate and reliable HLA-B27 phenot yping with flow cytometry.