Immunogenecity assay to measure the ADA

ADAs to the human antibodies were measured in NHP serum and Sprague Dawley rat serum samples using the Universal Indirect Species-Specific Assay (UNISA) as previously described [22]. Briefly, the 96-well standard binding plate (MSD, Gaithersburg,MD, USA) was coated overnight with 1 μg/ml human IgG in PBS (35 μL/well). The positive controls consisted of 100 and 500 ng/ml of NHP or rat anti-human IgG Fc chimeric antibody spiked into pooled normal NHP or rat serum, respectively. The assay positive controls and serum samples were diluted 1:200 in an assay buffer (5× milk diluent/block (Kirkegaard and Perry Laboratories “KPL,”Gaithersburg, MD, USA)). The coated and blocked plates (blocked with assay buffer, 200 μl/well overnight) were washed on day two with 1× wash buffer (KPL, Gaithersburg, MD, USA) and then the diluted assay controls and serum sample were added (100 μl/well) to the plate and incubated for approximately 3 hours. Plates were washed, and ruthenylated mouse anti-NHP IgG Fc or rabbit anti-rat IgG Fc antibody was added (0.5 μg/ml, 35 μl/well) and incubated for approximately 30 minutes. Following another wash, 2× T read buffer (MSD, Gaithersburg, MD, USA) was added (150 μl/well). The plates were read using the SECTOR Imager 6000 Instrument (MSD, Gaithersburg, MD, USA) and analyzed utilizing Discovery Workbench software (v2. 0 7.3). The resulting ECL was measured and reported in ECL units. The ECL response of the sample over the ECL response of the background of the assay was captured as signal to noise (S/N). The NHP assay has a sensitivity of 5.8 ng/ml, and the Sprague Dawley rat assay has a sensitivity of 64 ng/ml based on a species specific positive control antibody diluted in neat, negative control NHP or Sprague Dawley rat sera.