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.
Darke
Chris
C
Welsh Transplantation and Immunogenetics Laboratory, Welsh Blood Service, Wales, United Kingdom. chris.darke@wbs.wales.nhs.uk
Coates
Ernest
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000145:classification; D005434:Flow Cytometry / Q000379:methods*; D015796:HLA-B27 Antigen / Q000097:blood*; D006650:Histocompatibility Testing; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D010446:Peptide Fragments / Q000097:blood*
D000911:Antibodies, Monoclonal; C090660:HLA-B protein, human; C464936:HLA-B*27:05 antigen; D015796:HLA-B27 Antigen; D007202:Indicators and Reagents; D010446:Peptide Fragments; C118185:decapeptide B 2702