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ELISA (enzyme-linked immunosorbent assay) for target binding affinity of monoclonal antibodies

ELISA (enzyme-linked immunosorbent assay) is commonly used in early-stage monoclonal antibody (mAb) development to assess target binding affinity and cross-reactivity with other proteins. For nivolumab, which targets the PD-1 receptor, ELISA can quantify its binding to PD-1 and evaluate any unintended binding to non-target proteins, helping establish specificity and minimize potential off-target effects.

Here’s a step-by-step procedure for conducting an ELISA to measure nivolumab’s target binding and cross-reactivity:

1. Materials and Reagents

  • Nivolumab (primary antibody)
  • Recombinant human PD-1 protein (target antigen)
  • Control proteins for cross-reactivity testing (e.g., other immune checkpoint proteins like CTLA-4, PD-L1, and unrelated proteins to confirm specificity)
  • Secondary antibody (anti-human IgG conjugated to horseradish peroxidase (HRP))
  • Blocking buffer (e.g., 1% BSA in PBS)
  • Washing buffer (e.g., PBS with 0.05% Tween-20)
  • TMB substrate solution (for HRP detection)
  • Stop solution (e.g., 1 M sulfuric acid)
  • Microtiter ELISA plates

2. Coating the Plate with Antigen

  • Dilute the recombinant PD-1 protein in coating buffer (e.g., 0.1 M carbonate-bicarbonate buffer, pH 9.6) to a concentration of approximately 1–10 μg/mL.
  • Add 100 µL of the antigen solution to each well of the microtiter plate and incubate overnight at 4°C, allowing the PD-1 to adhere to the plate.
  • For cross-reactivity testing, add 100 µL of control proteins (at the same concentration as PD-1) to separate wells.

3. Blocking Non-Specific Binding Sites

  • Remove the coating solution and wash the plate 3 times with washing buffer.
  • Add 200 µL of blocking buffer to each well and incubate for 1 hour at room temperature. This step minimizes non-specific binding of nivolumab to the plate.
  • Discard the blocking buffer and wash the plate again 3 times with washing buffer.

4. Incubating with Nivolumab

  • Prepare a range of dilutions of nivolumab in blocking buffer (e.g., from 0.1 to 10 µg/mL) to create a binding curve.
  • Add 100 µL of each dilution to the appropriate wells. Use separate wells for each concentration for PD-1, as well as the control proteins for cross-reactivity.
  • Incubate the plate for 1–2 hours at room temperature to allow binding to the antigen.
  • After incubation, wash the plate 3–5 times with washing buffer to remove unbound nivolumab.

5. Adding Secondary Antibody

  • Dilute the HRP-conjugated secondary antibody in blocking buffer according to the manufacturer’s instructions.
  • Add 100 µL of the diluted secondary antibody to each well and incubate for 1 hour at room temperature.
  • Wash the plate 3–5 times with washing buffer to remove unbound secondary antibody.

6. Developing the Signal

  • Add 100 µL of TMB substrate solution to each well. Incubate the plate in the dark at room temperature for 10–15 minutes, allowing the color to develop. The intensity of the color is proportional to the amount of nivolumab bound to the antigen.
  • Monitor the color development visually or using a plate reader to avoid overdevelopment.

7. Stopping the Reaction and Reading the Plate

  • Add 50 µL of stop solution to each well to terminate the reaction. This changes the color from blue to yellow.
  • Measure the absorbance at 450 nm (with a reference at 620–650 nm, if available) using a microplate reader.

8. Data Analysis

  • Target Binding (PD-1 Wells): Generate a dose-response curve for nivolumab binding to PD-1 by plotting absorbance against nivolumab concentration. Calculate the binding affinity (Kd) if necessary using curve-fitting software.
  • Cross-Reactivity (Control Protein Wells): Compare absorbance values of nivolumab bound to control proteins with the values from PD-1 wells. A low or negligible absorbance in the control wells indicates high specificity for PD-1.

Key Considerations

  • Controls: Include wells with no nivolumab as negative controls to account for background signal. Also, consider an IgG isotype control to confirm that binding is specific to nivolumab rather than non-specific binding by antibodies in general.
  • Replicates: Perform the assay in duplicate or triplicate for each concentration to ensure reproducibility.
  • Sensitivity: Ensure the assay’s sensitivity by optimizing nivolumab concentrations, incubation times, and secondary antibody dilutions, especially for low-level cross-reactivity detection.

Summary

This ELISA assay evaluates nivolumab’s binding to its intended target, PD-1, and confirms the lack of binding to other proteins, ensuring its specificity and reducing the risk of off-target effects. The data from this assay provide essential validation for nivolumab’s specificity before advancing to in vivo studies and clinical trials.

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