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Surface Plasmon Resonance (SPR) for characterizing the affinity and kinetics for monoclonal antibodies

 Surface Plasmon Resonance (SPR) is a powerful technique for characterizing the affinity and kinetics of interactions between biomolecules, such as nivolumab (a monoclonal antibody) and its target, PD-1. SPR provides real-time, label-free measurements of binding interactions, allowing determination of the binding kinetics (association and dissociation rates) and affinity constant (Kd).

Here’s a step-by-step procedure for SPR to characterize the binding affinity of nivolumab to PD-1:

1. Materials and Reagents

  • Ligand: Recombinant human PD-1 protein (ligand, which will be immobilized on the sensor chip)
  • Analyte: Nivolumab (as the binding analyte flowing over the sensor chip)
  • Running buffer: Typically PBS or HEPES-buffered saline (HBS) with added surfactant (e.g., 0.05% Tween-20) to reduce non-specific binding
  • Regeneration buffer: A solution to dissociate nivolumab from PD-1 without denaturing the immobilized PD-1 (often an acidic buffer, e.g., glycine-HCl at pH 2.5–3.5, or NaCl at high concentrations)

2. Setting Up the SPR Instrument

  • Calibrate the SPR system according to the manufacturer’s instructions.
  • Prime the fluidics system with the running buffer to ensure consistent flow and stability of baseline signals.

3. Sensor Chip Selection and Preparation

  • Sensor Chip Selection: Use a sensor chip that is compatible with protein immobilization, such as a CM5 chip (carboxymethylated dextran) commonly used for protein-protein interactions.
  • Ligand Immobilization (PD-1): Use either amine coupling, where PD-1 is covalently attached to the chip, or capture methods if the ligand needs to be regenerated frequently.
    • Amine Coupling:
      1. Activate the carboxyl groups on the chip surface by injecting a mixture of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide).
      2. Dilute PD-1 protein in an appropriate buffer (e.g., sodium acetate buffer, pH 4.5–5.5).
      3. Inject PD-1 onto the activated surface at a controlled concentration to achieve the desired immobilization level.
      4. Deactivate the surface with ethanolamine to block any remaining active sites and stabilize the ligand attachment.
  • Aim for immobilizing a low level of PD-1 on the chip to minimize mass transport effects and allow accurate kinetic measurements.

4. Running the Experiment

  • Baseline Stabilization: Allow the running buffer to flow over the sensor surface until a stable baseline is achieved.
  • Binding Assay:
    • Prepare nivolumab dilutions across a concentration range (e.g., from low nanomolar to micromolar) to characterize binding kinetics accurately.
    • Inject each concentration sequentially over the sensor surface with immobilized PD-1, allowing nivolumab to bind to PD-1.
    • Monitor real-time changes in SPR signal (in resonance units, RU) as nivolumab associates with PD-1 on the chip.
  • Dissociation Phase:
    • Stop the injection of nivolumab and allow the running buffer to flow over the chip. This phase records the dissociation kinetics as nivolumab gradually detaches from PD-1.

5. Regeneration of the Sensor Surface

  • After each cycle, inject the regeneration buffer to remove bound nivolumab from PD-1 without damaging the immobilized PD-1.
  • After regeneration, flush with running buffer to return to baseline and prepare for the next sample injection.

6. Data Analysis and Interpretation

  • Association Rate Constant (ka): Measure the rate of nivolumab binding to PD-1. The association rate is derived from the slope of the association phase in the sensorgram.

  • Dissociation Rate Constant (kd): Measure the rate at which nivolumab dissociates from PD-1. The dissociation rate is derived from the slope of the dissociation phase in the sensorgram.

  • Affinity Constant (Kd): Calculate the affinity constant using the formula:

    Kd=kdkaK_d = \frac{k_d}{k_a}

  • Fit the binding curves with a 1:1 binding model (if appropriate) or other models if the interaction displays complexity.

Key Considerations

  • Temperature Control: Conduct the experiment at physiological temperature (e.g., 25–37°C) for accurate kinetic data.
  • Concentration Range: Ensure that the range of nivolumab concentrations spans both above and below the expected Kd to obtain robust data.
  • Mass Transport Limitation: Keep the ligand density low on the sensor chip to reduce mass transport effects, which can interfere with accurate kinetic measurements.

Summary

SPR provides precise real-time measurements of nivolumab’s affinity to PD-1 by determining both association and dissociation rates, enabling the calculation of the affinity constant. This technique is essential for developing and confirming nivolumab’s strong target specificity and favorable binding kinetics for therapeutic efficacy.

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