binding-characterization

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# Binding Characterization: SPR and BLI

## SPR vs BLI Decision Matrix

| Factor | Choose SPR | Choose BLI |
|--------|------------|------------|
| **Sensitivity** | Small molecules, fragments (<500 Da) | Large complexes, antibodies |
| **Throughput** | Low-medium (serial) | High (96-well parallel) |
| **Sample purity** | Required (clogs fluidics) | Tolerates crude lysates |
| **Kinetic resolution** | Higher (better for fast kinetics) | Lower |
| **Mass transport** | More sensitive (may distort kon) | Less sensitive |
| **Maintenance** | High (fluidics system) | Low (dip-and-read) |
| **Sample consumption** | Higher (continuous flow) | Lower |
| **Cost per experiment** | Lower chip cost, higher run cost | Higher tip cost, lower run cost |

## Key differences

### SPR (Surface Plasmon Resonance)
- **Mechanism**: Detects refractive index changes at gold surface
- **Surface**: Gold chip with dextran matrix (CM5, CM7, etc.)
- **Flow**: Continuous microfluidics
- **Best for**: Small molecules, high-affinity, precise kon/koff

### BLI (Biolayer Interferometry)
- **Mechanism**: Measures optical interference pattern shift
- **Surface**: Fiber optic biosensor tips (SA, Ni-NTA, AHC)
- **Flow**: Dip-and-read (no microfluidics)
- **Best for**: High-throughput, crude samples, antibody screening

---

## Troubleshooting: Why BLI works but SPR doesn't

| Cause | Mechanism | Solution |
|-------|-----------|----------|
| **Hydrophobic CDRs** | Adsorb to SPR gold/dextran surface | Add 0.05% Tween-20, use CM7 chip with longer dextran |
| **Aggregation** | Mass transport artifacts in SPR fluidics | Filter sample (0.22μm), reduce ligand density |
| **High instability** | Degrades during continuous flow | Shorter cycle time, add stabilizers (trehalose 5%) |
| **Charge mismatch** | Nonspecific binding to charged dextran | Adjust buffer pH ±1 from pI, add BSA 1mg/mL |
| **Slow dissociation** | Long regeneration needed (damages ligand) | Use BLI (disposable tips) |

### Why SPR works but BLI doesn't

| Cause | Mechanism | Solution |
|-------|-----------|----------|
| **Small analyte** | BLI less sensitive for <10 kDa | Use SPR with appropriate chip |
| **Weak affinity (KD >10μM)** | Fast dissociation in BLI dip | Increase analyte concentration |
| **Low expression** | Not enough signal | Increase biosensor loading |

---

## Mass transport considerations

Mass transport limitation occurs when analyte cannot diffuse to the surface fast enough to maintain equilibrium. This distorts kinetic parameters.

### Symptoms
- Observed kon appears slower than true kon
- Linear association phase (instead of exponential)
- kon varies with ligand density
- Rmax varies with flow rate

### When mass transport matters
- **High-affinity interactions** (kon >10^6 M^-1s^-1)
- **High ligand density** (>500 RU)
- **Slow flow rates** (<30 μL/min in SPR)
- **Large analytes** (slow diffusion)

### Mitigation strategies

| Strategy | SPR | BLI |
|----------|-----|-----|
| Reduce ligand density | <200 RU for high-affinity | <0.5 nm shift loading |
| Increase flow rate | 50-100 μL/min | Increase shake speed (1000 rpm) |
| Use oriented immobilization | His-tag capture | Biotinylated ligand |
| Include in fitting | Mass transport model (kt) | Usually less critical |

---

## Nonspecific binding mitigation

### Buffer additives (ranked by effectiveness)

| Additive | Concentration | Mechanism | Best For |
|----------|---------------|-----------|----------|
| BSA | 0.5-1 mg/mL | Blocks hydrophobic sites | General use |
| Tween-20 | 0.02-0.05% | Prevents surface adsorption | Hydrophobic analytes |
| Trehalose | 1-5% | Stabilizes + blocks | Unstable proteins |
| Sucrose | 5% | BLI-specific blocker | BLI tips |
| Carboxymethyl dextran | 1 mg/mL | Competitive blocking | SPR with charged proteins |
| NaCl | 150-500 mM | Reduces ionic interactions | Charged proteins |

### pH optimization
- Keep buffer pH at least 1 unit away from analyte pI
- pI near 7: Use pH 6.0 or 8.0 buffer
- Acidic proteins (pI <5): Use neutral or basic buffer
- Basic proteins (pI >9): Use slightly acidic buffer

### Reference subtraction
**Always include**:
- Blank reference channel (no ligand)
- Buffer-only injections
- Non-specific binding controls

---

## Regeneration conditions

### SPR regeneration scouting (try in order)

| Condition | Targets | Caution |
|-----------|---------|---------|
| 10 mM Glycine pH 2.0-2.5 | Most protein-protein | May denature ligand |
| 10 mM Glycine pH 1.5 | Strong interactions | Harsh, limit exposure |
| 1-2 M NaCl | Ionic interactions | Mild, try first |
| 10 mM NaOH | Very stable ligands | Can hydrolyze proteins |
| 10 mM Glycine pH 9-10 | Acid-stable proteins | Can aggregate |
| 10 mM EDTA | His-tag, metal-dependent | Strips Ni-NTA |
| 4 M MgCl2 | Hydrophobic interactions | Check ligand stability |

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