Introduction

The assessment of immunogenicity of drugs is crucial in evaluating treatment options, particularly in clinical studies involving therapeutic antibodies. The formation of anti-drug antibodies (ADAs) induced by treatment has been associated with loss of response, hypersensitivity reactions, and severe therapy-limiting side effects. Therefore, measuring ADAs is increasingly important in evaluating a patient's response to therapy.

ADA bridging ELISA represents an innovative assay format for measuring the immunogenicity of therapeutic drugs, including proteins, monoclonal antibodies, and other biologics. In this assay, the biotinylated drug is captured on streptavidin-coated plates, and anti-drug antibodies in the sample bind to the captured drug. For the detection of bivalent anti-drug antibodies, a dye or HRP-labeled drug is used. The advantages of this technique include its high sensitivity and ability to allow high-throughput sample screening. However, the specificity of bridging ELISA assays in the complex matrix of human serum may be limited due to matrix components, soluble target molecules, or drug components that can challenge the assay. Therefore, the use of high-quality assay reagents and blocking solutions is crucial to obtaining meaningful results.

Note:The bridging ELISA protocol serves as a general recommendation for using streptavidin high binding capacity coated plates for anti-drug antibody (ADA) assays. Each laboratory should customize and implement it according to their specific requirements.

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Materials

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Protocol

Note: When using concentrated solutions (e.g., 20X TBS Tween 20 Buffer or 20X PBS Tween 20 Buffer), ensure they are diluted to 1X with sterile, distilled water. The protocol allows flexibility in using either colorimetric or chemiluminescent substrates. For colorimetric assays, use TMB substrate solution along with a stop solution. For chemiluminescent assays, a chemiluminescent substrate is required, but a stop solution is not necessary.

  1. Biotinylate the drug using EZ-Link Sulfo-NHS-Biotinylation Kit as per the manufacturer’s instructions.
  1. Add 100 µL of biotinylated drug (1 µg/mL) to each well of the Streptavidin Coated High Capacity Plate.
  1. Incubate the plate at room temperature for 1 hour.
  1. Aspirate the biotinylated drug solution and wash the plate three times with >300 µL of Wash Buffer. After the last wash, invert the plate to remove residual liquid and blot on paper towel.
  1. Add 300 µL of Blocking buffer to each well of the Streptavidin Coated High Capacity Plate.
  1. Incubate the plate at room temperature for 1 hour.
  1. Remove the blocking buffer solution and wash the plate three times using Wash Buffer (same as step 4).
  1. Add 100 µL of test sample to its respective well of the Streptavidin Coated High Capacity Plate.
  1. Incubate the plate at room temperature for 1 hour.
  1. Remove the sample solution and wash the plate three times with Wash Buffer (same as step 4).
  1. Add 100 µL of Goat anti-Human IgG (H+L) Secondary Antibody, HRP to each well.
  1. Incubate the plate at room temperature for 1 hour.
  1. Remove the detection antibody and wash the plate three times with Wash Buffer (same as step 4).
  1. Follow the manufacturer’s instructions for the specific detection system (colorimetric or chemiluminescent).

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References
  1. Bivi N., Swearingen C.A., Shockley T.E., et al. Development and validation of a novel immunogenicity assay to detect anti-drug and anti-PEG antibodies simultaneously with high sensitivity. J Immunol Methods, 2020. 486: 112856.
  2. Mikulskis A., Yeung D., Subramanyam M., et al. Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development. J Immunol Methods, 2011. 365(1-2): 38–49.
  3. Alleyn M., Closson K., Gentile A., et al. Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira®. AAPS J, 2020. 22(5): 104
  4. Sinha A., Rinker S., Souliotis M.R., et al. Critical reagent considerations for immunogenicity assay development for bispecific biotherapeutic candidates. Bioanalysis, 2024. 16(15): 781–790.
  5. Deng X., Hou Y., Yuan W., et al. Eliminating drug target interference with specific antibody or its F(ab')2 fragment in the bridging immunogenicity assay. Bioanalysis, 2024. 16(7): 135–148.
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Stylesheet for Classic Wide Template adjustments

仅供科研使用，不可用于诊断目的。