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skills/adaptyv/reference/experiments.md
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skills/adaptyv/reference/experiments.md
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# Experiment Types and Workflows
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## Overview
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Adaptyv provides multiple experimental assay types for comprehensive protein characterization. Each experiment type has specific applications, workflows, and data outputs.
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## Binding Assays
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### Description
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Measure protein-target interactions using biolayer interferometry (BLI), a label-free technique that monitors biomolecular binding in real-time.
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### Use Cases
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- Antibody-antigen binding characterization
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- Receptor-ligand interaction analysis
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- Protein-protein interaction studies
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- Affinity maturation screening
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- Epitope binning experiments
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### Technology: Biolayer Interferometry (BLI)
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BLI measures the interference pattern of reflected light from two surfaces:
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- **Reference layer** - Biosensor tip surface
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- **Biological layer** - Accumulated bound molecules
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As molecules bind, the optical thickness increases, causing a wavelength shift proportional to binding.
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**Advantages:**
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- Label-free detection
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- Real-time kinetics
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- High-throughput compatible
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- Works in crude samples
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- Minimal sample consumption
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### Measured Parameters
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**Kinetic constants:**
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- **KD** - Equilibrium dissociation constant (binding affinity)
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- **kon** - Association rate constant (binding speed)
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- **koff** - Dissociation rate constant (unbinding speed)
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**Typical ranges:**
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- Strong binders: KD < 1 nM
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- Moderate binders: KD = 1-100 nM
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- Weak binders: KD > 100 nM
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### Workflow
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1. **Sequence submission** - Provide protein sequences in FASTA format
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2. **Expression** - Proteins expressed in appropriate host system
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3. **Purification** - Automated purification protocols
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4. **BLI assay** - Real-time binding measurements against specified targets
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5. **Analysis** - Kinetic curve fitting and quality assessment
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6. **Results delivery** - Binding parameters with confidence metrics
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### Sample Requirements
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- Protein sequence (standard amino acid codes)
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- Target specification (from catalog or custom request)
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- Buffer conditions (standard or custom)
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- Expected concentration range (optional, improves assay design)
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### Results Format
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```json
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{
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"sequence_id": "antibody_variant_1",
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"target": "Human PD-L1",
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"measurements": {
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"kd": 2.5e-9,
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"kd_error": 0.3e-9,
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"kon": 1.8e5,
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"kon_error": 0.2e5,
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"koff": 4.5e-4,
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"koff_error": 0.5e-4
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},
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"quality_metrics": {
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"confidence": "high|medium|low",
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"r_squared": 0.97,
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"chi_squared": 0.02,
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"flags": []
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},
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"raw_data_url": "https://..."
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}
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```
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## Expression Testing
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### Description
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Quantify protein expression levels in various host systems to assess producibility and optimize sequences for manufacturing.
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### Use Cases
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- Screening variants for high expression
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- Optimizing codon usage
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- Identifying expression bottlenecks
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- Selecting candidates for scale-up
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- Comparing expression systems
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### Host Systems
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Available expression platforms:
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- **E. coli** - Rapid, cost-effective, prokaryotic system
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- **Mammalian cells** - Native post-translational modifications
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- **Yeast** - Eukaryotic system with simpler growth requirements
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- **Insect cells** - Alternative eukaryotic platform
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### Measured Parameters
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- **Total protein yield** (mg/L culture)
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- **Soluble fraction** (percentage)
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- **Purity** (after initial purification)
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- **Expression time course** (optional)
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### Workflow
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1. **Sequence submission** - Provide protein sequences
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2. **Construct generation** - Cloning into expression vectors
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3. **Expression** - Culture in specified host system
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4. **Quantification** - Protein measurement via multiple methods
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5. **Analysis** - Expression level comparison and ranking
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6. **Results delivery** - Yield data and recommendations
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### Results Format
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```json
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{
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"sequence_id": "variant_1",
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"host_system": "E. coli",
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"measurements": {
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"total_yield_mg_per_l": 25.5,
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"soluble_fraction_percent": 78,
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"purity_percent": 92
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},
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"ranking": {
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"percentile": 85,
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"notes": "High expression, good solubility"
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}
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}
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```
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## Thermostability Testing
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### Description
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Measure protein thermal stability to assess structural integrity, predict shelf-life, and identify stabilizing mutations.
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### Use Cases
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- Selecting thermally stable variants
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- Formulation development
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- Shelf-life prediction
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- Stability-driven protein engineering
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- Quality control screening
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### Measurement Techniques
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**Differential Scanning Fluorimetry (DSF):**
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- Monitors protein unfolding via fluorescent dye binding
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- Determines melting temperature (Tm)
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- High-throughput capable
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**Circular Dichroism (CD):**
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- Secondary structure analysis
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- Thermal unfolding curves
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- Reversibility assessment
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### Measured Parameters
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- **Tm** - Melting temperature (midpoint of unfolding)
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- **ΔH** - Enthalpy of unfolding
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- **Aggregation temperature** (Tagg)
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- **Reversibility** - Refolding after heating
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### Workflow
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1. **Sequence submission** - Provide protein sequences
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2. **Expression and purification** - Standard protocols
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3. **Thermostability assay** - Temperature gradient analysis
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4. **Data analysis** - Curve fitting and parameter extraction
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5. **Results delivery** - Stability metrics with ranking
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### Results Format
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```json
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{
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"sequence_id": "variant_1",
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"measurements": {
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"tm_celsius": 68.5,
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"tm_error": 0.5,
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"tagg_celsius": 72.0,
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"reversibility_percent": 85
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},
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"quality_metrics": {
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"curve_quality": "excellent",
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"cooperativity": "two-state"
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}
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}
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```
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## Enzyme Activity Assays
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### Description
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Measure enzymatic function including substrate turnover, catalytic efficiency, and inhibitor sensitivity.
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### Use Cases
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- Screening enzyme variants for improved activity
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- Substrate specificity profiling
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- Inhibitor testing
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- pH and temperature optimization
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- Mechanistic studies
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### Assay Types
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**Continuous assays:**
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- Chromogenic substrates
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- Fluorogenic substrates
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- Real-time monitoring
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**Endpoint assays:**
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- HPLC quantification
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- Mass spectrometry
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- Colorimetric detection
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### Measured Parameters
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**Kinetic parameters:**
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- **kcat** - Turnover number (catalytic rate constant)
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- **KM** - Michaelis constant (substrate affinity)
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- **kcat/KM** - Catalytic efficiency
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- **IC50** - Inhibitor concentration for 50% inhibition
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**Activity metrics:**
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- Specific activity (units/mg protein)
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- Relative activity vs. reference
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- Substrate specificity profile
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### Workflow
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1. **Sequence submission** - Provide enzyme sequences
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2. **Expression and purification** - Optimized for activity retention
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3. **Activity assay** - Substrate turnover measurements
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4. **Kinetic analysis** - Michaelis-Menten fitting
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5. **Results delivery** - Kinetic parameters and rankings
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### Results Format
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```json
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{
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"sequence_id": "enzyme_variant_1",
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"substrate": "substrate_name",
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"measurements": {
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"kcat_per_second": 125,
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"km_micromolar": 45,
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"kcat_km": 2.8,
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"specific_activity": 180
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},
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"quality_metrics": {
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"confidence": "high",
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"r_squared": 0.99
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},
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"ranking": {
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"relative_activity": 1.8,
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"improvement_vs_wildtype": "80%"
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}
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}
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```
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## Experiment Design Best Practices
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### Sequence Submission
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1. **Use clear identifiers** - Name sequences descriptively
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2. **Include controls** - Submit wild-type or reference sequences
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3. **Batch similar variants** - Group related sequences in single submission
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4. **Validate sequences** - Check for errors before submission
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### Sample Size
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- **Pilot studies** - 5-10 sequences to test feasibility
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- **Library screening** - 50-500 sequences for variant exploration
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- **Focused optimization** - 10-50 sequences for fine-tuning
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- **Large-scale campaigns** - 500+ sequences for ML-driven design
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### Quality Control
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Adaptyv includes automated QC steps:
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- Expression verification before assay
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- Replicate measurements for reliability
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- Positive/negative controls in each batch
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- Statistical validation of results
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### Timeline Expectations
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**Standard turnaround:** ~21 days from submission to results
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**Timeline breakdown:**
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- Construct generation: 3-5 days
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- Expression: 5-7 days
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- Purification: 2-3 days
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- Assay execution: 3-5 days
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- Analysis and QC: 2-3 days
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**Factors affecting timeline:**
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- Custom targets (add 1-2 weeks)
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- Novel assay development (add 2-4 weeks)
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- Large batch sizes (may add 1 week)
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### Cost Optimization
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1. **Batch submissions** - Lower per-sequence cost
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2. **Standard targets** - Catalog antigens are faster/cheaper
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3. **Standard conditions** - Custom buffers add cost
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4. **Computational pre-filtering** - Submit only promising candidates
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## Combining Experiment Types
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For comprehensive protein characterization, combine multiple assays:
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**Therapeutic antibody development:**
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1. Binding assay → Identify high-affinity binders
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2. Expression testing → Select manufacturable candidates
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3. Thermostability → Ensure formulation stability
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**Enzyme engineering:**
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1. Activity assay → Screen for improved catalysis
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2. Expression testing → Ensure producibility
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3. Thermostability → Validate industrial robustness
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**Sequential vs. Parallel:**
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- **Sequential** - Use results from early assays to filter candidates
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- **Parallel** - Run all assays simultaneously for faster results
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## Data Integration
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Results integrate with computational workflows:
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1. **Download raw data** via API
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2. **Parse results** into standardized format
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3. **Feed into ML models** for next-round design
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4. **Track experiments** with metadata tags
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5. **Visualize trends** across design iterations
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## Support and Troubleshooting
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**Common issues:**
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- Low expression → Consider sequence optimization (see protein_optimization.md)
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- Poor binding → Verify target specification and expected range
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- Variable results → Check sequence quality and controls
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- Incomplete data → Contact support with experiment ID
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**Getting help:**
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- Email: support@adaptyvbio.com
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- Include experiment ID and specific question
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- Provide context (design goals, expected results)
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- Response time: <24 hours for active experiments
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