Goat anti mouse igg pe

Example 12

HEK293 cells transfected respectively with pcDNA3.1-hCXCR7 and pcDNA3.1-mCXCR7 were used to test cross-reactive binding of Nanobodies to mouse CXCR7 in FACS analysis. Cells were incubated with 32 nM Mab 11G8 (R&D), Mab 9C4 (MBL), Mab 8F11 (Biolegend) or with 800 nM Nanobody for 2 h at 4° C. Nanobody binding was detected using mouse anti-myc (Serotec) and anti-mouse IgG-PE (Jackson Immunoresearch) and Mab binding by goat anti-mouse IgG-PE (Jackson Immunoresearch). Nanobodies 08A10, 14G03, 07B11 and Mab9C4 are not cross-reactive to mouse CXCR7, Nanobodies 08A05 and 07C03 are partially cross-reactive with mouse CXCR7 and Mab 8F11, Mab 11G8 and 01C10 are cross-reactive with mouse CXCR7 (Table B-11).

Cross-reactive binding to cynomolgus CXCR7 was assessed in the same way. Nanobodies 08A10, 14G03, 07B11, 08A05, 07C03, 01C10 and Mab 9C4, Mab 8F11 and Mab 11G8 are all cross-reactive to cynomolgus CXCR7 (Table B-11).

Example 4

The murine 11A1 antibody mcMMAF conjugate was evaluated for its ability to internalize in the NTB-A⁺ cell line U-266 (FIG. 1).

U-266 antigen-positive cells were incubated with 5 μg/mL anti-human NTB-A antibody drug conjugate, 11A1-mcMMAF (the antimitotic agent MMAF conjugated via a maleimidocaproyl linker (mc) to a stoichiometry of 4 drugs per antibody via cysteine linkages) for 30 minutes on a shaker at 4° C. Cells were washed three times with RPMI 1640 media+10% fetal bovine serum and then plated out at 5×105 cells/100 μL, per well into two identical 96-well U-bottom plates (BioSciences, San Jose, Calif.). One plate was placed at 37° C. and the other at 4° C. One sample was immediately washed and stained for time zero. Cells were collected from both plates at 0.5, 1, 2, 8, and 24 hour time points, washed two times with cold wash buffer (PBS+2.5% fetal bovine serum), and stained with 10 μg/mL goat-anti-mouse IgG-PE (Jackson ImmunoResearch, West Grove, Pa.) for 30 minutes, on ice and protected from light. Cells were again washed twice with wash buffer and fixed with 500 μL of PBS+1% paraformaldehyde. Once all samples were collected and stained, they were analyzed on the FACs Calibur flow cytometer (BD BioSciences, San Jose, Calif.), and data was expressed as a percentage of time zero MFI.