Anti-Drug Antibodies (ADA) Immunogenicity

Methods for Detecting ADA Immunogenicity

A range of detection technologies for ADA emerged recently because researchers acknowledge their vital role in both drug effectiveness and safety.

1. Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA remains a popular choice for ADA detection because of its superior sensitivity and simple procedure combined with wide-ranging application capabilities. The primary ELISA techniques used for detecting ADA include sandwich ELISA and bridging ELISA which feature unique characteristics alongside their respective benefits and limitations.

• Sandwich ELISA

Researchers utilize this testing method to determine ADA presence by evaluating its binding affinity to the Fab or Fab2 fragment of anti-TNF drugs. It is cost-effective and easy to operate. The adsorption of drugs on plastic surfaces produces epitope masking and non-specific binding which leads to higher chances of false-positive results. Insufficient washing steps lead to cross-binding between the Fc fragment of antibodies and immobilized drugs which results in an increase of false-positive results.

• Bridging ELISA

This detection method uses the drug to function as both the capture and detection reagent which creates an antigen-antibody bridge to identify ADA. Bridging ELISA demonstrates high sensitivity toward bivalent and multivalent ADAs while maintaining effectiveness in samples with high drug concentrations. This method encounters interference from rheumatoid factors and anti-isotype antibodies along with low-affinity antibodies that can produce false positive results. The bridging ELISA fails to detect IgG4-type ADA because IgG4 antibodies remain functionally monovalent and incapable of forming the required bridge. This testing method reacts strongly to drug presence which may cause inaccurate negative results. Clinical estimates show that half of patients could experience this condition during their treatment process.

Figure 2. A Newly Developed Assay Configuration for Determining the Neutralization Index of ADA in Patient Serum and the Competitive ELISA Between ADA and rhTNFα (Source: Vaisman-Mentesh A, et al. 2019)

2. Cell-Based Assays

Cell-based assays become essential when biologics work by targeting cellular components. These techniques measure how drugs function within cells to identify their ability to neutralize ADA molecules. Researchers employ reporter gene cell lines to evaluate drug effectiveness in activating signaling pathways. Regulatory bodies suggest that cell-based assays should be the primary method used to identify neutralizing ADA reactions in therapeutic proteins. A TNF-induced cytotoxicity assay serves as the standard method to measure neutralizing ADA when testing anti-TNF drugs. The standardization of these assays proves difficult because they need long testing durations which might extend to several days and their results can be influenced by serum matrix effects. Results can be affected by substances present in patient serum that interfere with the measurements.

3. Electrochemiluminescence (ECL) Assay

The ECL assay functions as an exceptionally sensitive and precise tool to detect ADAs. The technique generates a luminescent signal through electrochemical reactions to detect ADAs at low concentrations. ECL receives broad application in ADA analysis because of its exceptional sensitivity.

4. Immunogenicity Testing for Biosimilars

Clinical trial validation requires establishing specific methods for ADA detection during biosimilar development. The process involves direct comparisons of immunogenic responses between biosimilars and reference drugs to confirm their equivalence. The safety and efficacy of biosimilars depend on the establishment and validation of detection methods.

The thorough validation of results remains essential no matter which detection method is chosen. Researchers evaluate both sensitivity and specificity along with precision and drug tolerance when conducting their assessments. Sample pre-treatment techniques like adsorption or acid dissociation can be utilized to increase sensitivity.