Methods
The purpose of this study was to develop and qualify a method for detection of anti-drug antibodies (ADA) against therapeutic peptide with nanocarrier in dog plasma using a Meso Scale Diagnostics (MSD) Electrochemiluminescence Immunoassay. The qualification plan was approved by the Sponsor. MSD utilizes electrochemiluminescence detection to detect binding events on patterned arrays by converting electric energy into light energy. Electrochemiluminescence detection offers a unique combination of sensitivity, dynamic range and convenience.
The MSD assay measures ADA against the therapeutic nanomaterial in dog plasma in an MSD 96-well-standard plate. In brief, the assay employs a sandwich immunoassay format where wells were coated with the drug on the standard MSD plate containing high-binding electrodes. Samples in serial dilutions and a solution containing the labeled secondary detection antibody are added to the plate. The ADA in the sample binds to the drugs immobilized on the working electrode surface; recruitment of the labeled detection antibody to the ADA completes the sandwich. Then an MSD read buffer is added to the plate that provides the appropriate chemical environment for electrochemiluminescence and the plate is loaded into an MSD SECTOR instrument for the analysis, see Figure 1.
The ADA from dog plasma will be detected using the immobilized Test Article (drug) for capture with anti-dog secondary antibody linked with sulfo-TAG streptavidin for development of the assay. The system must be qualified for different species (i.e. rat, dog, human). In order to qualify the ADA assay, we designed an assay utilizing ADA raised in rabbits immunized with the drug (the only available specific antibody to the drug), see Figure 2.
Results
The method was qualified for accurately measuring induction of Ab to the test article in dog plasma. The screen cut-off point was determined for each dilution of plasma. The data showed good precision (%CV) and good accuracy (%RE) for both intra- and inter-assay calculations confirming acceptable selectivity of the assay.
The range of quantitation during sample analysis was 0.49 ng/L – 500 ng/mL. The limit of detection was 0.456 ng/mL. The lowest limit of qualification (LLOQ) was 0.98 ng/mL. The minimum required dilution (MRD) of samples was 1:50 (2% plasma) for this assay. This allow to use as low as 2.5 uL of plasma for ADA analysis. Using of this method confirmed that there were no ADA induced in dogs due to the drug administration.