Apc conjugated anti human fcγ specific secondary antibody

Example 1

Binding of DR5-targeting fusion proteins was assessed by flow cytometry, using a CHO cell line stably transfected with cDNA encoding full length DR5 or cancer cell lines that endogenously express DR5. A titration series of the fusion protein was incubated with the DR5-expressing cell lines (approx. 2.5-5×10⁴ cells/well) for 30 minutes at 4° C. in FACS Buffer (PBS 1% BSA, 0.1% NaN₃ pH 7.4) in 96 well plates. Following 3 wash steps in FACS buffer, an APC-conjugated anti-human Fcγ specific secondary antibody (Jackson ImmunoResearch) was added and incubated for 30 minutes at 4° C. Following three additional wash steps in FACS buffer bound antibody was detected via flow cytometry (IQue Intellicyte). Binding of fusion proteins to cynomologus monkey DR5 (cynoDR5) was determined by ELISA wherein a recombinant protein corresponding to the extracellular domain (ECD) of cynoDR5 fused to a murine Fc region (mFc) was immobilized on Medisorp 96 well plates (Nunc). Following sufficient blocking and washing steps, bound fusion proteins were detected using an HRP-conjugated anti-human Fcγ specific secondary antibody (Jackson ImmunoResearch) and TMB reagent and absorbance read at A_{650 nm}.

Example 1

Binding of DR5-targeting fusion proteins was assessed by flow cytometry, using a CHO cell line stably transfected with cDNA encoding full length DR5 or cancer cell lines that endogenously express DR5. A titration series of the fusion protein was incubated with the DR5-expressing cell lines (approx. 2.5-5×10⁴ cells/well) for 30 minutes at 4° C. in FACS Buffer (PBS 1% BSA, 0.1% NaN₃ pH 7.4) in 96 well plates. Following 3 wash steps in FACS buffer, an APC-conjugated anti-human Fcγ specific secondary antibody (Jackson ImmunoResearch) was added and incubated for 30 minutes at 4° C. Following three additional wash steps in FACS buffer bound antibody was detected via flow cytometry (IQue Intellicyte). Binding of fusion proteins to cynomologus monkey DR5 (cynoDR5) was determined by ELISA wherein a recombinant protein corresponding to the extracellular domain (ECD) of cynoDR5 fused to a murine Fc region (mFc) was immobilized on Medisorp 96 well plates (Nunc). Following sufficient blocking and washing steps, bound fusion proteins were detected using an HRP-conjugated anti-human Fcγ specific secondary antibody (Jackson ImmunoResearch) and TMB reagent and absorbance read at A_650nm.