adaptyvbio/protein-design-skills/binding-characterization

Binding Characterization: SPR and BLI

SPR vs BLI Decision Matrix

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

Why SPR works but BLI doesn't

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

Nonspecific binding mitigation

Buffer additives (ranked by effectiveness)

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)

Regeneration protocol

1. Start with mildest condition (high salt)
2. Test 30s contact time
3. Verify complete dissociation (return to baseline)
4. Verify retained ligand activity (repeat binding)
5. Use shortest effective contact time

BLI tips

• Tips are often disposable (no regeneration needed)
• For reuse: Same conditions as SPR, but shorter exposure
• Anti-His tips: 10 mM Glycine pH 1.5, 30s
• Streptavidin tips: Generally not regenerable

Common artifacts and solutions

Biphasic binding

Symptoms: Two-rate association or dissociation Causes:

• Sample heterogeneity (aggregates)
• Ligand heterogeneity (multiple conformations)
• Avidity effects (bivalent analyte)

Solutions:

• Filter/centrifuge sample
• Use monovalent Fab fragments
• Reduce ligand density
• Fit to heterogeneous model

Negative dissociation

Symptoms: Signal increases during dissociation phase Causes:

• Ligand leaching from surface
• Analyte aggregation on surface
• Reference channel drift

Solutions:

• Use capture antibody instead of direct immobilization
• Increase buffer stringency
• Better reference subtraction

Hook effect

Symptoms: Signal decreases at high analyte concentrations Causes:

• Surface saturation + rebinding suppression
• Crowding effects

Solutions:

• Reduce analyte concentration range
• Reduce ligand density
• Use smaller analyte fragments

Kinetic data quality checklist

Before analysis

• [ ] Reference-subtracted properly
• [ ] Buffer injection shows flat baseline
• [ ] Rmax consistent across concentrations
• [ ] No systematic drift during association
• [ ] Complete regeneration (return to baseline)
• [ ] Duplicate/triplicate injections consistent

Fitting quality

• [ ] Residuals randomly distributed (no systematic deviation)
• [ ] Chi² < 10% of Rmax (or < 1 RU² for low signals)
• [ ] kon and koff errors < 20% of values
• [ ] KD from kinetics matches equilibrium KD (within 3-fold)
• [ ] Fitted Rmax reasonable (close to theoretical)

Red flags

• kon approaching the mass transport limit (>10^7 M^-1s^-1), where rates are unreliable
• koff too fast to sample (> 0.1 s^-1) or too slow to measure in the dissociation window (< 10^-5 s^-1)
• Rmax >> theoretical maximum (aggregation or avidity)
• Large difference between kinetic and equilibrium KD

References

Platform comparisons

• BLI vs SPR Comparison - Sartorius
• BLI vs SPR - Nicoya

SPR protocols

• SPR Guidelines - van der Merwe, Oxford
• SPR Experiment Guide - Duke DHVI

Troubleshooting

• 4 Ways to Reduce NSB in SPR - Nicoya
• 3 Ways to Limit Mass Transfer Effects - Nicoya
• Suppressing NSB in BLI - ACS Omega

Regeneration

• SPR Regeneration - SPRpages
• Mastering Regeneration - Nicoya

Mass transport

• Mass Transport Limitation in SPR - PMC
• Mass-Transfer Kinetics - SPRpages