Generation and validation of small ADAMTS13 fragments for epitope mapping of anti-ADAMTS13 autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

Research and Practice in Thrombosis and Haemostasis
Kadri KangroKaren Vanhoorelbeke

Abstract

In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments. We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti-ADAMTS13 autoantibodies in iTTP patients. A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2-T5, T6-T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2-T8, CUB1-2, MDTCS, T2-C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti-ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples. Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1-2 fragments were selected instead o...Continue Reading

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Citations

Apr 20, 2021·Research and Practice in Thrombosis and Haemostasis
Mar 18, 2021·Journal of Thrombosis and Haemostasis : JTH·Charlotte DekimpeKaren Vanhoorelbeke
Sep 25, 2021·Blood Advances·Laure De WaeleKaren Vanhoorelbeke

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Methods Mentioned

BETA
enzyme‐linked immunosorbent assay
ELISA
PCR
transfection
gel filtration
electrophoresis

Software Mentioned

Prism
GraphPad

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