An evaluation of Com-B27, a fluorescein-conjugated mouse anti-HLA-B27 reagent

We evaluated the HLA-B27 typing reagent Com-B27, which is claimed to show minimal cross-reactivity with HLA-B7, for use in flow cytometry-based typing. This combination reagent consists of the fluorescein-conjugated HLA-B27 mouse monoclonal antibody ABC-m3 and a non-conjugated HLA-B7 monoclonal antibody that is claimed to block the reactivity of ABC-m3 to B7 without affecting its reactivity to B27. It reacted well with B27 (B*2702, B*2705) and B2708 [mean median channel fluorescence intensity (MCFI) 8.40] and weakly with B7 (including cells from homozygous B*07 donors), B42/B73 and B22/B37/B44 reference cells (mean MCFI 2.05, 3.09 and 1.10, respectively). It showed a uniform discrimination between B7 and B27/B2708 with no 'overlap' in MCFI values, which was seen with the standard ABC-m3 antibody. There was complete agreement when our standard three-antibody-based B27/B2708 flow cytometry assay and the Com-B27 reagent alone were used to independently assign B27/B2708 status to 651 random patients. Thus, the Com-B27 reagent provided improved discrimination between B7 and B27/B2708 over the ABC-m3 antibody, and its B27/B2708 assignments were comparable with our standard flow cytometry assay. However, for consistently reliable B27/B2708 typing we continue to recommend the use of a minimum of two B27 reagents in a protocol that includes DNA-based testing of 'equivocal' B27/B2708 assignments.