Co(III)-NTA Mediated Antigen Immobilization on a Fiber Optic-SPR Biosensor for Detection of Autoantibodies in Autoimmune Diseases: Application in Immune-Mediated Thrombotic Thrombocytopenic Purpura

Abstract

Autoantibodies are key biomarkers in clinical diagnosis of autoimmune diseases routinely detected by enzyme-linked immunosorbent as-says (ELISA). However, the complexity of these assays is limiting their use in routine diagnostics. Fiber optic-surface plasmon resonance (FO-SPR) can overcome these limitations but improved surface chemistries are still needed to guarantee detection of autoantibodies in complex matrices. In this paper we describe the development of an FO-SPR immunoassay for the detection of autoantibodies in plasma samples from immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients. Hereto hexahistidine tagged recombinant ADAMTS13 (rADAMTS13-His6) was immobilized on nitrilotriacetic acid (NTA)-coated FO probes chelated by cobalt (Co(III)) and exposed to anti-ADAMTS13 autoantibodies. Initial studies were performed to optimize rADAMTS13-His6 immobilization and to confirm the specificity of the immunoassay for detection of anti-ADAMTS13 autoantibodies with FO-SPR. The performance of the immunoassay was then evaluated by comparing Co(III)- and nickel (Ni(II))-NTA stabilized surfaces, confirming the stable immobilization of the antigen in Co(III)-NTA functionalized FO probes. A calibration curve was prepared with a dilution series of a cloned human anti-ADAMTS13 autoantibody in ADAMTS13-depleted plasma resulting in an average inter-assay coefficient of variation of 7.1 % and a limit of detection of 0.24 ng/mL. Finally, the FO-SPR immunoassay was validated using seven iTTP patient plasma samples resulting in an excellent correlation with an in-house developed ELISA (r = 0.973). In summary, the specificity and high sensitivity in combination with a short time-to-result (2.5h compared to 4-5h for a regular ELISA) makes the FO-SPR immunoassay a powerful assay for routine diagnosis of iTTP and with ex-tension for any other autoimmune disease.