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

Summary 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.