Single-Cycle Kinetics SPR Measurement of Antibody-Antigen Binding Affinity on a Biacore CM5 Sensor

Capture-based assay on a CM5 chip: flow cell 1 = reference (anti-Fc immobilized, no antibody captured); flow cell 2 = active (antibody captured). Single-cycle kinetics with a 5-point analyte series in 3-fold dilutions bracketing the expected KD (e.g. 0.37, 1.1, 3.3, 10, 30 nM), injected sequentially with one dissociation phase. Run a buffer-only (0 nM) cycle for double-referencing. Triplicate cycles (n=3) on independently captured surfaces. Antibody capture level tuned to give a theoretical Rmax of 30-100 RU to minimize mass-transport limitation.

BSL-1 for recombinant proteins (raise to BSL-2 if antigen derives from a pathogen). Hazards: EDC and NHS are irritants/sensitizers; ethanolamine and glycine-HCl regeneration solutions are corrosive/acidic; concentrated MgCl2 regenerant is an irritant. PPE: lab coat, nitrile gloves, safety glasses. Handle amine-coupling reagents fresh and on ice; dispose of acidic regeneration waste as chemical waste; route biological analytes per institutional biosafety.

Reference flow cell (anti-Fc, no antibody captured): corrects bulk refractive index, nonspecific binding, and injection artifacts. Buffer-only (0 nM analyte) cycle: enables double-referencing of baseline drift. Positive control: a well-characterized antibody-antigen pair of known KD run on the same chip to validate instrument performance. Antigen-over-reference check confirms the analyte does not bind the anti-Fc surface nonspecifically. Regeneration control verifies full ligand removal between cycles.

Sensorgrams showing stepwise increasing association responses across the 5 ascending injections and a clean monophasic dissociation. A global 1:1 fit with chi² < ~10% of Rmax and randomly distributed residuals. KD in the expected nM-or-tighter range; ka ~10⁵–10⁶ M⁻¹s⁻¹; kd ~10⁻³–10⁻⁵ s⁻¹. Capture level stable (drift <5 RU/min). Reference-subtracted buffer cycle near zero.

• Series S Sensor Chip CM5
• Human antibody capture kit (anti-human IgG Fc) or Protein A/G, immobilized by amine coupling (EDC/NHS, ethanolamine block)
• Monoclonal antibody / Fab ligand, ~2-10 µg/mL in running buffer for capture
• Purified protein antigen analyte, prepared as a 3-fold dilution series in running buffer
• Running buffer: HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20)
• Amine coupling kit (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, 1 M ethanolamine-HCl pH 8.5)
• Regeneration buffer: 3 M MgCl2 or 10 mM glycine-HCl pH 1.5 (for capture surface reuse between cycles)

To quantify the real-time binding kinetics of a Fab/antibody-antigen interaction by surface plasmon resonance (SPR) using a single-cycle kinetics (SCK) injection format on a Biacore T200 or 8K. The ligand (antibody) is captured on a CM5 sensor; antigen is injected as analyte over a 5-concentration ascending series without surface regeneration between concentrations. Deliverables: ka, kd, KD, and a global 1:1 Langmuir fit with quality metrics.

1. Dock the CM5 chip; prime the system in HBS-EP+ at 25 °C. Immobilize anti-Fc capture antibody on FC1 and FC2 by EDC/NHS amine coupling to ~8,000-12,000 RU; block with ethanolamine.
2. On FC2 only, capture the test antibody by injecting it to reach a stable capture level giving theoretical Rmax 30-100 RU.
3. Prepare the analyte 5-point 3-fold dilution series in running buffer; degas.
4. Program single-cycle kinetics: 5 ascending analyte injections (contact 120-180 s each, flow 30 µL/min) followed by a single dissociation phase of 600-1800 s.
5. Run a startup/buffer-blank cycle and a 0 nM buffer cycle for double-referencing.
6. Regenerate the capture surface (10 mM glycine-HCl pH 1.5, 30 s) and re-capture fresh antibody for each replicate cycle.
7. Repeat for n=3 independent capture cycles.
8. Reference-subtract (FC2−FC1) and blank-subtract (sample−buffer cycle) before fitting.

Independent: analyte (antigen) concentration across the 5-point series. Dependent: SPR response (RU) over time, from which ka, kd, KD are fit. Controlled: ligand capture level (Rmax), flow rate (30 µL/min to limit mass transport), temperature (25 °C), running buffer composition, contact/dissociation times, and sensor surface chemistry.

The antibody-antigen interaction follows a 1:1 Langmuir binding model. We hypothesize the antigen will associate with sub-nanomolar to low-nanomolar affinity (KD), exhibiting a measurable fast on-rate (ka ~10⁵–10⁶ M⁻¹s⁻¹) and a slow off-rate (kd ~10⁻³–10⁻⁴ s⁻¹) characteristic of a matured antibody, with response increasing monotonically across the analyte series.

Double-reference the data (subtract reference flow cell and buffer-blank cycle) in Biacore Insight/BIAevaluation. Globally fit the single-cycle kinetics dataset to a 1:1 Langmuir binding model with mass-transport term, floating ka, kd, and Rmax. Compute KD = kd/ka. Assess fit quality by chi² (relative to Rmax), residual plots, and U-value. Report kinetic constants with the standard error of the fit.

1. No or weak binding response: low capture, denatured ligand, or wrong analyte orientation — increase capture, verify ligand activity, swap ligand/analyte roles. 2. Poor 1:1 fit (curved residuals): avidity/heterogeneity — use monovalent Fab, lower capture density, or fit heterogeneous-ligand model. 3. High reference/nonspecific binding: increase P20 surfactant, add carrier/BSA, raise NaCl. 4. Mass-transport-limited kinetics: lower capture (Rmax <50 RU), raise flow rate to 50-100 µL/min. 5. Drifting/incomplete regeneration: optimize regeneration pH/time and confirm baseline return between cycles.

Kinetic parameters (ka, kd, KD) reported as mean ± SD across n=3 independent capture cycles. Fit-quality reported per cycle (chi², U-value). Where two antibodies/conditions are compared, KD values are log-transformed and compared by unpaired two-tailed t-test (α=0.05); for >2, one-way ANOVA with Tukey correction. n=3 with typical inter-cycle CV <20% gives adequate power to resolve ~2-fold KD differences.