One-tube HLA-B27/B2708 typing by flow cytometry using two "Anti-HLA-B27" monoclonal antibody reagents.

Background: Flow cytometry-based methods are widely used to detect the ankylosing spondylitis-associated HLA-B27/B2708 antigens. However, the generally used "HLA-B27" monoclonal antibodies (moabs) cross-react with many HLA specificities, including the common HLA-B7 antigen. Thus, using two "B27" moabs is highly recommended.

Methods: The assay used two "HLA-B27" reagents, FITC and PE conjugated, respectively and a PE-Cy5 anti-CD3 antibody. Assay verification used 51 reference subjects possessing B*2705, B*2702, and B*2708 and a range of cross-reactive HLA antigens. A total of 1,006 consecutive patients' samples, referred for "HLA-B27 typing", were assayed alongside our standard flow cytometry method. A further 12 low frequency HLA-B*27 specificities were tested. Samples reacting with one "B27" moab only were B*27 allele typed by PCR using sequence-specific primers.

Results: All patient B27/B2708 positives (28.3%) were identified by our one-tube method which detected B*2705, B*2702, B*2708, and 8/12 other B*27 specificities. It was unaffected by HLA-B7 and other cross-reactive antigens but required a minor adjustment, a reduction in the volume of one of the "B27" moabs used, to avoid detecting a minority of HLA-B57 subjects.

Conclusions: Our one-tube B27/B2708 assay is simple, robust, uses two "B27" moabs for typing precision and security, does not suffer from interference by HLA-B7 or other cross-reactive antigens and has the obvious advantage of using a single tube per typing.