Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by ADAMTS13 autoantibodies. The diagnosis and follow-up of iTTP patients is challenging, and therefore novel biomarkers are highly desired. Recently, open ADAMTS13 was identified as a novel biomarker for iTTP, as it allows to diagnose acute and subclinical iTTP even with undetectable ADAMTS13 autoantibodies. The ADAMTS13 conformation ELISA requires well-trained staff, a long-time-to-result and the need to collect multiple patient samples. Accordingly, an easy-to-use technology, allowing single sample ADAMTS13 conformation determination is needed for implementation in clinical laboratories.

Aims: To develop the ADAMTS13 conformation assay on the easy-to-use fiber optic surface plasmon resonance (FO-SPR) platform.

Methods: The antibody 1C4 (recognizing a cryptic ADAMTS13 epitope) was coupled to gold-coated optical fibers using EDC/NHS chemistry. After blocking, open ADAMTS13 in iTTP plasma samples was captured. Next, ADAMTS13 was detected with biotinylated 3H9, followed by signal amplification with anti-biotin IgG labeled gold nanoparticles (AuNPs). To validate this assay, ADAMTS13 conformation was determined in 20 healthy donor (HD) and 18 acute iTTP plasma samples by ELISA and FO-SPR. In ELISA, optical density values were corrected for ADAMTS13 antigen to determine the ADAMTS13 conformation index (CI). In FO-SPR, signal amplification SPR shifts were corrected for ADAMTS13 antigen to determine the CI.

Results: (A) Set-up FO-SPR ADAMTS13 conformation assay. The anti-ADAMTS13 antibody 1C4 is covalently coupled to the FO-SPR fiber using EDC/NHS chemistry, followed by a blocking step with superblock. Plasma ADAMTS13 (open conformation) is captured by 1C4 and detected with biotinylated 3H9, followed by signal amplification via goat anti-biotin coupled gold nanoparticles. (B) Read-out FO-SPR ADAMTS13 conformation assay. The SPR shift in function of time of the complete FO-SPR ADAMTS13 conformation assay. (C) The ADAMTS13 conformation indices determined by FO-SPR in HD and iTTP plasma samples.
Effective immobilization of 1C4 and subsequent capturing of open ADAMTS13 from iTTP plasma, followed by specific signal amplification with biotinylated 3H9 and anti-biotin IgG labeled AuNPs was obtained. ADAMTS13 conformation was closed in all 20 HD samples and open in 17 of the 18 iTTP samples (Figure), which was in accordance with the ELISA results.

Conclusions: A novel, easy-to-use FO-SPR ADAMTS13 conformation assay has been established, with a total assay time of 2.5 hours. Currently, the ADAMTS13 activity, antigen and autoantibody FO-SPR assay are also under development, to allow simultaneous determination of all ADAMTS13 parameters.  

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/development-of-an-easy-to-use-adamts13-conformation-assay-using-fiber-optic-surface-plasmon-resonance-technology/