A rapid enzyme-linked assay for ADAMTS-13

Area of Science:

• Biochemistry
• Hematology
• Assay Development

Background:

• ADAMTS-13 deficiency causes thrombotic thrombocytopenic purpura (TTP) via microvascular thrombi.
• Existing ADAMTS-13 assays are complex and time-consuming.
• A need exists for a rapid and sensitive assay for ADAMTS-13 activity.

Purpose of the Study:

• To develop a rapid and sensitive assay for ADAMTS-13 activity in plasma.
• To verify the specific cleavage site of ADAMTS-13 on the designed substrate.

Main Methods:

• Constructed an enzyme-linked substrate with a core ADAMTS-13 peptide, HRP, and biotin.
• Quantified ADAMTS-13 activity by measuring unadsorbed HRP after substrate cleavage and streptavidin-agarose removal.
• Assessed inhibitory antibodies by measuring residual ADAMTS-13 activity after incubation with test plasma.

Main Results:

• The assay determined plasma ADAMTS-13 activity within 60 minutes with 5.8% CV using 1 µL of plasma.
• Amino acid sequencing confirmed cleavage at the Tyr1605-Met1606 bond.
• TTP patient plasma showed <2% ADAMTS-13 activity, with antibody titers ranging from undetectable to 81 BU mL⁻¹.

Conclusions:

• The HRP-linked substrate assay is rapid, sensitive, and reproducible.
• This assay effectively determines ADAMTS-13 activity and inhibitory antibody levels in plasma.
• The developed assay offers a significant improvement over existing methods for TTP diagnosis and management.