Carbonate bicarbonate buffer

Nunc Immuno 96 well plates (Fisher Scientific) were coated with 1μg/ml (50 μl/well) HA protein (Sino Biological) diluted in carbonate/bicarbonate buffer (Fisher Scientific) overnight at 4°C or 1 hour at 37 °C. After washing and blocking with TBS for 1 hour the serum samples were diluted and added to the plate. Serially diluted HA-specific monoclonal antibody (Sino Biological) served as standard. Anti-mouse IgG-HRP (1:20,000; Fisher Scientific) in combination with TMB (Fisher Scientific) solution was used for detection. The signals were read at 450 nm using accuSkan FC microplate photometer (Fisher Scientific).

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.

Serum concentrations of BsAb-1 and BsAb-2 in mouse serum were determined using an antigen capture ELISA. All washes and dilutions were performed with freshly made TBS-T (Accuris). All volumes should be assumed to be 100 µL/well except for coating, blocking and washing, which are at 200 µL/well. The night before the assay, Nunc MaxiSorp™ flat-bottom plates (Invitrogen) were coated with recombinant human IL2Rγ protein diluted to 1 µg/mL in carbonate-bicarbonate buffer (Thermo Scientific) and left at 4 °C. The next day, the plates were washed 5 times and then blocked with 1% BSA for 30 min. The plates were washed once and then multiple dilutions of the serum samples were added, along with a reference standard. Stocks of known concentration for BsAb-1 and BsAb-2 were used to make the standard curve. After 1 h at room temp, the plate was washed 8 times and then biotinylated anti-human IgG4-Fc (MABTECH) diluted to 3 µg/mL was added. The plates were incubated at room temp for another 30 min and then washed again 8 times. Next, the plates were incubated for 30 min with HRP-Streptavidin (Thermo Scientific) diluted 1:4000. Following an additional 8 washes, the plates were incubated in the dark for 6 min with room-temperature 1-Step Ultra TMB (Thermo Scientific). The reaction was stopped with 100 µL/well 2 N sulfuric acid. Absorbance was assessed at 450 nm and 570 nm.

A microtiter 96-well plate (Nunc MaxiSorp, Thermo Fisher Scientific) was coated with 350 ng/well of recombinant 16E6 [19 (link)] or BSA in 50 mM carbonate/bicarbonate buffer, pH 9.4 (Thermo Fisher Scientific). The plate was saturated with 200 μl/well of 2% Non-Fat Dry Milk (NFDM, Bio-Rad) PBS buffer at 37°C for 2 h and incubated 1 h at 37°C with 100 μl/well of scFvI7 or scFvI7nuc in PBS (2.5 μg/ml). An in-house anti-16E6 mouse polyclonal IgG 1 : 1000 in 2% NFDM-PBS was used as a positive control [19 (link)]. The wells were rinsed with PBS for 3 times and incubated with 50 μl/well of mouse anti-V5 tag monoclonal antibody (mAb, Life Technologies, USA) 1 : 2500 in 2% NFDM-PBS 1 h at 37°C. After repeated washes, wells were incubated with 100 μl of anti-mouse horseradish peroxidase- (HRP-) conjugated polyclonal IgG (Sigma-Aldrich, USA) 1 : 20000 in 2% NFDM-PBS, 1 h at 37°C. After a final wash, color was developed using the TMB substrate kit (Vector Laboratories, Burlingame, CA). The reaction was stopped after 2 h with 100 μl of 2 M H₂SO₄ and OD₄₅₀ determined in a microtiter plate reader (iMark, Bio-Rad).

Clear Flat-Bottom Immuno Nonsterile 96-well plates (Thermo USA 442404) were coated with 0.1 μg/mL recombinant cynoPVRIG protein (Acro Biosystems, Inc., Newark, Delaware, USA) in carbonate–bicarbonate buffer (Thermo Fisher Scientific, USA). After overnight incubation at 4 °C, the plates were blocked with PBS containing 1% BSA and 0.05% Tween-20 for 1 h. Serially diluted antibody and 2 μg/mL cynomolgus PVRL2 His protein (#90,206-C08H SinoBiological) were added to the plates and incubated for 3 h at room temperature. After washing with PBS containing 0.05% Tween-20, the protein expression of human PVRL2 was detected using an anti-6X His tag antibody (HRP) (Abcam, AB1187). The color reactions were initiated by adding TMB (Solarbio, Beijing #PR1200) and stopped with ELISA stop buffer (Solarbio, Beijing). The absorbance at 450 and 620 nm was measured with a microplate reader (Thermo, USA, Multiskan Fc).