Hrp conjugated anti his antibody

A 96-well plate was coated with recombinant human CTLA-4 (7268-CT-100, R&D Systems) and blocked as above. Serial dilutions of test articles were added to the washed ELISA plates followed by addition of a 2.6 µg/mL solution of recombinant human CD80 or CD86 (9050-B1-100 or 9090-B2-100, R&D Systems). Plates were washed and HRP-conjugated anti-His antibody (652504, BioLegend) was added to the plate. Plates were washed followed by incubation with chemiluminescent substrate (37069, Thermo Fisher Scientific). After incubation, chemiluminescence was measured in Light Units by a plate reader. Data were fit by non-linear regression analysis. IC₅₀ values represent the concentration at which 50% of maximum CD80 or CD86 was bound to CTLA-4.

SDS-PAGE and immunoblotting were used to assess the size and integrity of purified mAbs and mutated BoNTs. Briefly, proteins were separated on a 4–15% polyacrylamide gel under reducing (125 mM Tris-HCl pH 6.8, 12% (w/v) SDS, 10% (v/v) glycerol, 22% (v/v) beta-mercaptoethanol, 0.001% (w/v) bromophenol blue) and non-reducing (125 mM Tris-HCl pH 6.8, 12% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue) conditions. Proteins were either stained with InstantBlue® dye or transferred to nitrocellulose membrane. The membrane was blocked with 5% (w/v) skim milk to prevent nonspecific binding, and then incubated with HRP-conjugated anti-human IgG antibody (2040–05, Southernbiotech, USA) diluted 1:10,000 and HRP-conjugated anti-human kappa antibody diluted 1:2,500 for mAb detection or HRP-conjugated anti-His antibody (652503, Biolegend, USA) diluted 1:10,000 for mBoNT/A1 and ciBoNT/B1 detection. Finally, the probed mAbs and recombinant BoNTs were treated with enhanced chemiluminescence (ECL) reagent (Promega, USA) and exposed to X-ray film (Carestream, USA).