CR3022 F(ab')2 Fragment Production

The monoclonal antibody CR3022 was purchased from Absolute Antibody (#Ab01680-10.0) as an IgG1. F(ab’)2 was produced using the Genovis FragIT kit (#A2-FR2-100, Genovis) according to manufacturer’s protocol. Briefly, 1-5 mg of IgG1 was added to the FragIT column after equilibration and allowed to rock at room temperature for 45 minutes, the column was then centrifuged to elute IgG fragments. Fc fragments were removed by passing through a CaptureSelect Fc Column. F(ab’)2 fragments were eluted and collected. F(ab’)2 production was checked through SDS-PAGE. The resulting gel was first imaged under white light to show the fluorophore labeling of the F(ab’)2 before staining with SimplyBlue SafeStain (Thermo-Fisher, #LC6060). For labeling, 3 mgs of purified CR3022-F(ab’)2 was combined with 60ug of sulfo-Cy5 NHS ester (#53320, Lumiprobe), sulfo-Cy3 NHS ester (#51320, Lumiprobe), or AF647-NHS ester (#A20106, Thermo-Fisher) in PBS with 100mM sodium bicarbonate and gently rocked at room temperature for 1 hour as previously described (43 (link)). Solutions were then passed through a Zeba column (Thermo-Fisher, #89882) to remove free dye. Labeled F(ab’)2 was filtered through a 0.22-μm filter and stored at 4°C in the dark. Labeled F(ab’)2 to be infused into macaques was tested using the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo-Fisher, #88282).