Assay Development Guidelines – ADA

Anti-Drug Antibodies (ADA) can be formed in a patient or experimental animal upon administration of a protein drug. The ADA can be a safety concern, may neutralize the effect of the drug and/or may mask epitopes on the drugs used by the PK assay, resulting in lower than expected exposure values. In all of these instances, it is important to monitor the ADA and an ADA assay needs to be developed.

The assay format we recommend for ADA assays on Gyrolab system is a homogeneous bridging format, which detects ADA of all isotypes and is not species dependent. This means that it can be used both for pre-clinical and clinical studies. In this assay format, the drug is used as both capture (biotinylated) and detection (fluorescently labeled) reagent and the sample and the reagents are mixed before addition to the streptavidin columns on the CD.

A number of combinations will be formed in this mixture. What will be detected in the assay are complexes where ADA or positive control form a bridge between a biotinylated drug molecule and a drug molecule labeled with Alexa Fluor 647.

Bridging assay for ADA analysis

A general problem with ADA assays is that free drug forms complexes with the ADA and prevent it from being detected by the ADA assay. There are two approaches to solving this problem in Gyrolab system:

  • Automated acid dissociation using Gyrolab Mixing CD 96
  • Offline incubation of the ADA assay reagents with the sample before analysis using Gyrolab Bioaffy CD. The incubation time will depend on dissociation rate and the requirements for drug tolerance

The high binding capacity of the particles in the Gyrolab CD allows high concentrations of labeled reagents that can compete with the free drug and give a stoichiometric advantage over free drug interference.

You can download ‘Gyrolab ADA assay protocol’, which gives detailed guidelines on how to develop ADA assays, here: Download

You can download the Methods and Experiments described in ‘Gyrolab ADA assay protocol’ here:

Methods: (Zipped ZML files)
Method 200-1W-004-A
Method Mixing96-1W-003-A

Experiments:

Gyrolab Mixing CD 96

ADA Exp 1 – Initial Screen             Download zml, Download pdf
ADA Exp 2 – Optimization            Download zml, Download pdf
ADA Exp 3 – Screening cut point  Download zml, Download pdf
ADA Exp 4 – Drug Tolerance         Download zml, Download pdf

Gyrolab Bioaffy 200

ADA Exp 1 – Initial Screen              Download zml, Download pdf
ADA Exp 2 – Optimization             Download zml, Download pdf 
ADA Exp 3 – Screening cut point   Download zml, Download pdf 
ADA Exp 4 – Drug Tolerance         Download zml, Download pdf