Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
A Novel, Simple Fluid Phase Binding Assay for the Detection of Serum Anti-Domain I Antibodies in Patients with the Antiphospholipid Syndrome.
Pericleous2, Charis, Garza-Garcia1, Acely, Driscoll1, Paul C., Latchman4, David S., Isenberg3, David A., Giles4, Ian P., Rahman4, Anisur
Domain I (DI) of beta-1359-glycoprotein I (b2GPI) has been established as a critical immunodominant region for pathogenic antiphospholipid antibodies (aPL) in patients with the antiphospholipid syndrome (APS). A direct enzyme-linked immunosorbent assay (ELISA) to detect anti-DI antibodies in APS sera could be affected by conformational changes in immobilised DI and has required considerable efforts to optimise by multiple groups including our own. Here we present an alternative concept for detecting anti-DI activity based upon inhibiting anti-b2GPI activity with recombinant DI in the fluid phase.
Sera from 15 anti-b2GPI positive patients fulfilling APS classification criteria (15/15 female; mean age 47.4±11.9; 14/15 Caucasian, 1/15 Asian; 14/15 with a thrombotic history, 7/15 with previous pregnancy morbidity), and 5 patients with circulating anti-b2GPI aPL (but without APS, aPL+/APS-) were serially diluted (from 1:50) and tested in triplicate using a standard solid phase anti-b2GPI ELISA. The appropriate dilution of serum corresponding to 50% of maximal binding (MBD) was identified for each patient. In each case this gave an OD405nm >0.4. For the new anti-DI assay, each serum sample at the 50% MBD was firstly incubated (2hr, RT) with increasing doses of in-house bacterially-expressed recombinant human DI (0, 20, 40, 60, 80 or 100 mg/ml) and then a standard anti-b2GPI assay was performed. For each patient sample the percentage binding was calculated based on a change in OD405nm in the presence of DI.
Mean percentage binding (± standard deviation, SD) to b2GPI for all 15 APS sera was calculated in the presence of increasing concentrations of DI; the same was performed for the 5 aPL+/APS- samples. The presence of DI significantly reduced binding for APS samples compared to aPL+/APS- samples, in a dose-dependent manner (Table 1).
Table 1. The inhibitory ability of recombinant human DI in the fluid phase
|Concentration of DI added (mg/ml)||Mean (SD) binding for APS samples as a percentage of binding in the absence of inhibitor (n=15)||Mean (SD) binding for aPL+/APS-samples as a percentage of binding in the absence of inhibitor (n=5)||P value|
|20||75.0 (20.3)||96.3 (12.4)||<0.01|
|40||62.0 (15.6)||99.7 (12.0)||<0.01|
|60||56.9 (18.1)||94.0 (13.2)||<0.01|
|80||44.3 (22.9)||89.1 (17.1)||<0.01|
|100||37.5 (21.7)||85.1 (16.1)||<0.01|
Using the well-established direct anti-b2GPI assay, serum inhibition of anti-b2GPI activity by fluid phase recombinant DI is both remarkably simple and selective of pathogenicity in this small sample of patients. The ability to express recombinant human DI in a bacterial system allows for the cost-effective production of large amounts of DI for use in this assay. The findings from this proof-of-concept novel and simple approach to measuring anti-DI activity in an anti-b2GPI positive patient warrant further exploration to further characterise specificity and sensitivity, and hence clinical diagnostic and prognostic utility.
To cite this abstract, please use the following information:
Pericleous, Charis, Garza-Garcia, Acely, Driscoll, Paul C., Latchman, David S., Isenberg, David A., Giles, Ian P., et al; A Novel, Simple Fluid Phase Binding Assay for the Detection of Serum Anti-Domain I Antibodies in Patients with the Antiphospholipid Syndrome. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1359