Enzyme Activity and Inhibitor Screening Services
Inhibitor Screening for ADAMTS13 Modulators
Detect and quantify inhibitory autoantibodies against ADAMTS13 with mixing-test and Bethesda-type workflows, and screen candidate.
What This Service Covers
ADAMTS13 is a zinc-containing plasma metalloprotease that cleaves ultra-large von Willebrand factor (VWF) multimers into smaller, less adhesive forms. In acquired immune-mediated thrombotic thrombocytopenic purpura (iTTP), autoantibodies against ADAMTS13 either inhibit its enzymatic function or accelerate its clearance from circulation, producing severe deficiency and VWF-driven microthrombosis. An ADAMTS13 activity level below roughly 10% of normal is commonly used to distinguish TTP from other thrombotic microangiopathies, and the finding of severe deficiency is typically followed by testing for ADAMTS13 antibodies.
This service is built for drug discovery and translational teams working on iTTP and related thrombotic microangiopathies who need to know whether a sample, antibody preparation, or candidate molecule inhibits or restores ADAMTS13 function. We combine functional inhibitor detection with modulator screening so that inhibitory antibodies can be distinguished from non-inhibitory (clearing) antibodies, and so that candidate compounds can be assessed for their effect on residual enzyme activity in a controlled assay system.
Because ADAMTS13 antibodies may bind any domain of the protease, and the cysteine-rich spacer domain is consistently involved in reactivity in patients who develop acquired TTP, functional readouts remain central: only assays that measure loss of enzyme activity can confirm that an antibody or compound is truly inhibitory rather than merely binding.
Inhibitory Antibody Detection
Basic 1 + 1 mixing tests pair test plasma with normal plasma to reveal whether an inhibitory antibody is present, using residual ADAMTS13 activity as the functional readout.
- Mixtures of patient plasma and normal plasma tested for residual activity
- Loss of activity in the mixture indicates inhibitory antibodies
- Complements binding-based antibody assays that cannot separate inhibitory from clearing antibodies
Bethesda-Type Titer Measurement
Serial dilutions of test plasma against a fixed amount of normal or recombinant ADAMTS13 allow inhibitor titer to be expressed in Bethesda units.
- Dilution series measures loss of function across mixtures
- Titer reported in inhibitor units (Bethesda units)
- Useful for tracking inhibitor status over the course of disease
Modulator Screening
Candidate molecules can be screened for their ability to inhibit or restore ADAMTS13 activity in the presence of autoantibodies, using the same functional readouts.
- Residual VWF-cleaving activity as the primary endpoint
- Normal plasma or recombinant ADAMTS13 as the enzyme source
- Quality controls with normal and known inhibitor plasma
Why Functional Inhibitor Testing Matters
Antibodies against ADAMTS13 include both functionally inhibitory antibodies and antibodies that cause clearance of the enzyme. Binding assays such as ELISA detect both classes but cannot distinguish between them, and not all patients present with inhibitory antibodies — in some cases the deficiency is caused by clearing antibodies alone, which are not detectable in functional assays. Functional testing therefore answers a different and complementary question: does this sample actually reduce ADAMTS13 activity?
This distinction has direct consequences for research and development programs. Inhibitors of ADAMTS13 compromise enzyme replacement approaches and are associated with a need for alternate management strategies, so knowing whether a candidate antibody or compound is inhibitory — and at what titer — shapes assay design, mechanism studies, and translational decisions.
| Question | Functional Inhibitor Assay | Binding-Based Antibody Assay | What It Tells You |
|---|---|---|---|
| Is an antibody present? | Indirectly, via loss of activity | Yes, directly | Binding assays detect antibodies but not their functional effect |
| Is the antibody inhibitory? | Yes | No | Only activity-based readouts confirm inhibition |
| Can titer be quantified? | Yes, in Bethesda units | Binding titer only | Bethesda-type dilution series measures functional titer |
| Are clearing antibodies detected? | No | Yes | The two assay families are complementary, not interchangeable |
How the Workflow Runs
Projects follow the established functional logic of ADAMTS13 inhibitor testing: establish baseline activity, test for inhibition by mixing, quantify titer by dilution, and apply the same readouts to candidate modulator screening.
Baseline Activity Measurement
ADAMTS13 activity is measured in the test plasma to establish the starting point, since severe deficiency is the context in which inhibitor testing is typically pursued.
Mixing Test
Test plasma is mixed with normal plasma in a 1 + 1 format and incubated to allow any antibody-enzyme interaction, then residual ADAMTS13 activity is measured in the mixture.
Bethesda-Type Titration
Serial dilutions of test plasma are tested against a fixed amount of normal or recombinant ADAMTS13, and inhibitor units (Bethesda units) are calculated from the residual activity.
Modulator Screening
Candidate molecules are screened for their effect on ADAMTS13 activity in the presence of autoantibodies, using the same residual VWF-cleaving activity readouts.
Assay Options and Customization
The functional core of inhibitor testing is stable, but the enzyme source, readout, and screening format are configured per project. Scope is defined case by case in the project SOW after consultation, so the assay matches the question being asked rather than a fixed catalog configuration.
Normal Plasma or Recombinant ADAMTS13
Mixing and titration steps can use normal plasma or recombinant ADAMTS13 as the enzyme source, depending on the sensitivity and standardization needs of the project.
- Normal plasma supports classic mixing-test formats
- Recombinant ADAMTS13 supports defined enzyme input
- Source selection documented in the final report
Residual VWF-Cleaving Activity
Residual ADAMTS13 activity can be assessed by a variety of activity assays, including VWF multimer cleavage and fluorescence resonance energy transfer (FRETS-VWF73) formats.
- Activity readouts quantify loss of function
- Format chosen to match available sample volume and sensitivity needs
- Consistent readout across screening and confirmation
Inhibitor and Modulator Testing
The same functional framework supports both inhibitor detection in biological samples and screening of candidate molecules for inhibitory or restorative effects.
- Inhibitor titer in Bethesda units for antibody samples
- Candidate compounds assessed against residual activity endpoints
- Quality controls with normal and known inhibitor plasma
Typical Project Scope
Scope is agreed per project. The table below shows the parameters that are commonly defined during consultation; final values are set in the project SOW.
| Parameter | Typical project scope | How it is defined | Notes |
|---|---|---|---|
| Sample type | Plasma samples, antibody preparations, or candidate compounds | Defined at consultation | Matrix and handling requirements reviewed before assay setup |
| Enzyme source | Normal plasma or recombinant ADAMTS13 | Selected per project | Choice affects standardization and sensitivity |
| Detection format | 1 + 1 mixing test followed by Bethesda-type titration | Standard functional workflow | Mixing detects inhibition; titration quantifies it |
| Activity readout | Residual VWF-cleaving activity, format selected per project | Scoped per project | Multiple activity assay formats are compatible with the workflow |
| Titer reporting | Inhibitor units (Bethesda units) calculated from residual activity | Standard for titration projects | Reported alongside the dilution series used |
| Controls | Normal plasma and known inhibitor plasma | Included as quality controls | Supports run validity assessment |
| Screening depth | Number of candidates and concentrations scoped per project | Defined in the SOW | Screening and confirmation stages can be separated |
| Reporting | Assay format, enzyme source, readout, and results | Delivered as a project report | Documentation supports downstream mechanistic and translational use |
What You Receive
Deliverables are structured so that functional results can be interpreted in context: which assay format was used, what enzyme source supplied the activity, and how the titer or screening endpoint was calculated.
Activity and Inhibitor Data
Baseline activity, mixing-test outcomes, and, where applicable, inhibitor titer expressed in Bethesda units, with the underlying dilution series.
- Baseline ADAMTS13 activity for the test sample
- Mixing-test result indicating presence or absence of inhibition
- Inhibitor units calculated from residual activity
Modulator Screening Output
For screening projects, results are reported as the effect of each candidate on residual ADAMTS13 activity under the agreed assay conditions.
- Candidate-level activity results
- Conditions and controls documented
- Screening and confirmation stages reported separately when scoped
Assay Documentation
A project report records the assay format, enzyme source, readout, and quality-control performance so results can be reviewed and reproduced.
- Assay format and readout described
- Quality-control results included
- Interpretation notes on inhibitory versus clearing antibodies where relevant
Assay Comparison
Different assay families answer different questions about ADAMTS13 and its antibodies. The comparison below is intended to help teams decide which functional endpoint fits their program.
| Assay | What It Measures | Strength | Limitation |
|---|---|---|---|
| ADAMTS13 activity assay | Enzyme function in the test sample | Establishes the baseline deficiency context | Does not by itself detect inhibitors |
| 1 + 1 mixing test | Residual activity after mixing test plasma with normal plasma | Detects inhibitory antibodies functionally | Qualitative unless combined with titration |
| Bethesda-type assay | Loss of function across serial dilutions | Quantifies inhibitor titer in Bethesda units | Requires careful dilution series and controls |
| Binding-based antibody assay | Antibody binding, including clearing antibodies | Detects antibodies that functional assays miss | Cannot distinguish inhibitory from clearing antibodies |
Built for iTTP Research Programs
The service is designed around the questions that arise in iTTP and related thrombotic microangiopathy programs, where the functional status of ADAMTS13 is central to mechanism and translational work.
Inhibitory Versus Clearing Antibodies
Functional testing separates antibodies that inhibit ADAMTS13 activity from those that act through clearance, a distinction that binding assays alone cannot make.
- Functional readout confirms inhibition
- Clearing antibodies are not detectable in functional assays
- Complementary assay families can be combined when scoped
Modulator Development Support
For teams developing therapies that aim to inhibit or restore ADAMTS13 activity, screening against residual activity endpoints provides a functional basis for candidate triage.
- Candidate effects measured against residual activity
- Assay conditions documented for comparability
- Results framed for downstream mechanistic studies
Disease-Relevant Framework
Assays are interpreted in the context of TTP biology, where severe ADAMTS13 deficiency drives VWF multimer accumulation and microthrombosis.
- Severe deficiency is the typical testing context
- Ultra-large VWF multimers accumulate when ADAMTS13 is absent
- Inhibitor status informs management and research decisions
Scope and Limitations
Functional inhibitor testing answers whether activity is lost in a mixture; it does not by itself establish antibody binding, antigen levels, or genetic status. Where a program needs those complementary data, they are scoped separately.
Inhibitor levels may fluctuate over the course of disease and depend on free circulating antibodies, so a single negative functional result should be interpreted with the sampling context in mind.
FAQ
How does a mixing test differ from a binding-based antibody assay?
A mixing test measures function: test plasma is combined with normal plasma and residual ADAMTS13 activity is measured, so loss of activity indicates an inhibitory antibody. Binding-based assays detect antibodies against ADAMTS13, including clearing antibodies, but cannot distinguish inhibitory from non-inhibitory antibodies. The two approaches are complementary, and functional testing is the route to confirming inhibition.
What is a Bethesda-type assay and what does it report?
A Bethesda-type assay quantifies inhibitor titer by testing serial dilutions of test plasma against a fixed amount of normal or recombinant ADAMTS13 and measuring the loss of function across the dilution series. The result is expressed in inhibitor units, commonly referred to as Bethesda units, calculated from the residual activity observed.
Can you detect inhibitors in samples that show no inhibition in a functional assay?
Not all patients present with inhibitory antibodies; in some cases ADAMTS13 deficiency is caused by clearing antibodies alone, which are not detectable in functional assays. If a program needs to capture that class of antibodies, binding-based antibody testing can be scoped alongside functional inhibitor testing so the two datasets can be interpreted together.
Which enzyme source and readout will be used for my project?
Mixing and titration steps can use normal plasma or recombinant ADAMTS13 as the enzyme source, and residual activity can be assessed by a variety of activity assays, including VWF multimer cleavage and fluorescence resonance energy transfer formats. The enzyme source and readout are selected during consultation and documented in the project report so results can be compared across runs.
Can the same workflow screen candidate modulators rather than patient samples?
Yes. The functional framework used for inhibitor detection also supports screening of candidate molecules for their ability to inhibit or restore ADAMTS13 activity in the presence of autoantibodies. Screening depth, concentrations, and confirmation stages are defined per project in the SOW, and results are reported against residual activity endpoints with the relevant controls.
How should a negative functional result be interpreted?
Inhibitor levels may fluctuate over the course of disease and depend on free circulating antibodies, so a single negative functional test should be read in the context of when and how the sample was taken. Where the clinical or research question requires it, repeat sampling or complementary antibody testing can be discussed as part of project scoping.
References
- Zheng XL, Vesely SK, Cataland SR, et al. ISTH guidelines for the diagnosis of thrombotic thrombocytopenic purpura. Journal of thrombosis and haemostasis: JTH. 2020;18(10):2486-2495. View on PubMed
- Mang G, Chen J, Sun P, et al. Von Willebrand factor exacerbates heart failure through formation of neutrophil extracellular traps. European heart journal. 2024;45(37):3853-3867. View on PubMed
Discuss Your ADAMTS13 Inhibitor Screening Project
Share your sample types, candidate molecules, and the functional question you need answered. We will define the assay format, enzyme source, readout, and screening depth with you before work begins.