ELISA was used to detect ADA against Leronlimab in plasma as previously described [19 (link)]. Briefly, 2 μg/mL Leronlimab (Cytodyn, Vancouver, WA) was diluted in carbonate-bicarbonate buffer (ThermoFisher) and coated into half-area 96-well Costar Assay Plates (Corning) overnight at 4°C. The next day, plates were washed three times with PBS-T and blocked with Blocking Buffer for at least two hours in RT. Afterward, blocked plates were washed three times with PBS-T. Heat-inactivated plasma samples were serially diluted in Blocking Buffer with six serial dilutions and plated onto blocked plates in duplicates for 30 minutes in RT. After this incubation step, plates were washed three times with 0.5 M NaCl in PBS and incubated with 5,000-fold diluted secondary antibody that recognized rhesus macaque IgG and conjugated to HRP (anti-rhesus IgG1/3[1B3]-HRP; NHP Reagent Resource; 0.5 mg/mL) in Blocking Buffer. Plates were incubated at RT for 30 minutes, with the remaining steps following the quantification ELISA described above. ADA titers were defined as the reciprocal of the highest dilution of the sample that yielded a positive result (e.g., dilution of 1/2460 = titer of 2460). A positive result was defined as twice that of background value for each individual macaque.
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