Anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura do not hamper ELISA‐based quantification of ADAMTS13 antigen

Dekimpe, Charlotte; Roose, Elien; Tersteeg, Claudia; Joly, Bérangère S.; Dewaele, Aurélie; Horta, Sara; Pareyn, Inge; Vandenbulcke, Aline; Deckmyn, Hans; Feys, Hendrik B.; Tellier, Edwige; Kaplanski, Gilles; Scully, Marie; Coppo, Paul; Meyer, Simon F. De; Veyradier, Agnès; Vanhoorelbeke, Karen

doi:10.1111/jth.14747

Cited by 12 publications

(9 citation statements)

References 20 publications

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“…Plasma was collected into a new Eppendorf tube and stored at −80 °C until further use. The successful depletion of ADAMTS13 was confirmed by an ELISA as previously described , with minor modifications (Figure S1). Colorimetric development was performed with TMB substrate solution.…”

Section: Experimental Sectionmentioning

confidence: 90%

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

Horta

Dekimpe

et al. 2020

Anal. Chem.

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Autoantibodies are key biomarkers in clinical diagnosis of autoimmune diseases routinely detected by enzyme-linked immunosorbent assays (ELISAs). 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 interassay 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 (2.5 h compared to 4–5 h for a regular ELISA) make the FO-SPR immunoassay a powerful assay for routine diagnosis of iTTP and with extension for any other autoimmune disease.

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Section: Experimental Sectionmentioning

confidence: 90%

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

Horta

Dekimpe

et al. 2020

Anal. Chem.

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“…In most of the patients, anti-ADAMTS13 antibodies behave as inhibitors of the ADAMTS13 protease, but in 10-20% of iTTP-patients, these antibodies demonstrate other mechanisms of interaction with ADAMTS13, such as increased clearance, 16,17 conformational changes, 14,18 or increased metalloprotease activity 19 without modifying ADAMTS13 quantification in ELISA. 20,21 Our data suggest that in addition to these various interactions, anti-ADAMTS13 antibodies may also induce endothelial activation and VWF exocytosis, the well-known second hit of the disease. 8 This observation may appear contradictory with animal models suggesting that anti-ADAMTS13 antibodies do not induce VWF release.…”

Section: Discussionmentioning

confidence: 78%

Immune-mediated thrombotic thrombocytopenic purpura plasma induces calcium- and IgG-dependent endothelial activation: correlations with disease severity

et al. 2022

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Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by a severe ADAMTS13 deficiency due to the presence of anti-ADAMTS13 autoantibodies, with subsequent accumulation of circulating ultra-large von Willebrand Factor (VWF) multimers. The role of endothelial cell activation as a trigger of the disease has been suggested in animal models but remains to be demonstrated in humans. We prospectively obtained plasma from the first plasma exchange of 25 patients during iTTP acute phase. iTTP but not control plasma, induced a rapid VWF release and P-selectin exposure on dermal human micro-vascular endothelial cell (HMVEC-d) surface, associated with angiopoietin-2 and endothelin-1 secretion, consistent with Weibel-Palade bodies exocytosis. Calcium (Ca2+) blockade significantly decreased VWF release, whereas iTTP plasma induced a rapid and sustained Ca2+ flux in HMVEC-d which correlated in retrospect, with disease severity and survival in 62 iTTP patients. F(ab)’2 fragments purified from the immunoglobulin G (IgG) fraction of iTTP plasma mainly induced endothelial cell (EC) activation with additional minor roles for circulating free heme and nucleosomes, but not for complement. Furthermore, two anti-ADAMTS13 monoclonal antibodies purified from iTTP patient B cells, but not serum from hereditary TTP, induced endothelial Ca2+ flux associated with Weibel-Palade bodies exocytosis in vitro, whereas inhibition of endothelial ADAMTS13 expression using small interference RNA, significantly decreased the stimulating effects of iTTP IgG. In conclusion, Ca2+-mediated endothelial cell activation constitutes a second “hit” of iTTP, is correlated with the severity of the disease and may constitute a possible therapeutic target.

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“…ADAMTS13 antigen levels were measured using our in-house developed ELISA as previously described. 22 Detailed information is found in the supplemental information.…”

Section: Methodsmentioning

confidence: 99%

ADAMTS13 conformation and immunoprofiles in Japanese patients with immune-mediated thrombotic thrombocytopenic purpura

et al. 2023

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Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare thrombotic disease caused by autoantibody induced ADAMTS13 deficiency. Open ADAMST13 conformation, induced by autoantibodies, was identified as a novel biomarker for iTTP. Determining immunoprofiles in iTTP patients was shown to guide the development of novel targeted therapies. However, these studies were done in mainly Caucasian iTTP cohorts. To validate those findings across other ethnic cohorts, we investigated 195 acute TTP plasma samples from the Japanese iTTP registry. Seventy-six of the 195 samples had detectable ADAMTS13 antigen levels of which 94.7% were shown to have an open ADAMTS13 conformation. A positive correlation was observed between ADAMTS13 inhibitor titers (diagnostic parameter in Japan) and anti-ADAMTS13 IgG autoantibody titers. Studying anti-M, -DT; -CS, -T2-T5, -T6-T8, -CUB1-2 autoantibodies and the corresponding immunoprofile showed that 73.0% of the patients had anti-CS autoantibodies and 25.8% anti-M autoantibodies, with the latter being higher than in Caucasians. Stratifying patients according to their immunoprofile revealed that the profile with only anti-CS autoantibodies alone was the most common immunoprofile like in Caucasians (28.9%). While this profile did not impact the one-year TTP-related mortality rate, patients with autoantibodies against all 6 ADAMTS13 fragments had a higher risk for TTP-related death than other patients (p=0.02). We here validated open ADAMTS13 as a novel biomarker for acute iTTP and determined the dominant immunoprofiling in the Japanese cohort, contributing to setting up the diagnosis and managing guidelines across different ethnic cohorts and to developing ADAMTS13 variants that do not bind to the anti-CS autoantibodies.

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Anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura do not hamper ELISA‐based quantification of ADAMTS13 antigen

Cited by 12 publications

References 20 publications

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

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

Immune-mediated thrombotic thrombocytopenic purpura plasma induces calcium- and IgG-dependent endothelial activation: correlations with disease severity

ADAMTS13 conformation and immunoprofiles in Japanese patients with immune-mediated thrombotic thrombocytopenic purpura

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