Rpe conjugated goat anti mouse igg

Dilutions of the various mAbs were mixed with DENV1 (MOI = 1), DENV2 (MOI = 1), DENV3 (MOI = 5), or DENV4 (MOI = 1) for 1 hour at 4°C; the mixtures were then used to infect K562 cells. After 3 days, the cells were fixed with 3.7% formaldehyde, permeabilized in 2% FBS PBS containing 0.1% saponin, and stained with 4G2. After washing, the cells were incubated with R-phycoerythrin (RPE)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), and analyzed by flow cytometry. Normal mouse IgG (NMIgG) was used as a negative control. In addition, DENV2 S221 (MOI = 5) was incubated with diluted mAbs for 1 hour at 4°C. Subsequently, the mixtures were added to infect K562 cells. After 3 days, the cells were fixed, permeabilized, and stained with 4G2. After washing, the cells were incubated with the RPE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), and analyzed by flow cytometry.

Antigen binding was assayed via ELISA. ELISA plate was coated with 0.5 μg/mL histidine-tagged TF-ECD for overnight, sequentially washed, blocked with 2% BSA in PBS and incubated with various doses of TF antibody, then detected with HRP-labeled goat anti-mouse IgG (Jackson ImmunoResearch, Cat#115-035-146). To measure binding to cell surface TF, MDA-MB-231 or BxPC3 cells (3×10⁵) were incubated with various doses of TF antibody in 200 μL serum-free medium at 4°C for 1 h, washed with PBS and bound antibody was detected with R-PE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Cat#115-116-071) by FACS analysis (BD FACSArialTM IIU).
To analyze the internalization of antibody into cancer cells, MDA-MB-231 cells were plated at 50% confluence in glass bottom cell culture dish (NEST, Cat#801002) and incubated with 10 μg/mL of TF antibody at 37°C or 4°C for the indicated time. The cells were fixed with 4% formaldehyde, permeablized and detected with Alexa Fluor 647-conjugated AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch, Cat#715-605-150). The lysosomes were visualized via incubating cells with a rabbit anti-human LAMP-2 (Abcam, Cat#ab125068) and detected with Alexa Fluor 488-conjugated AffiniPure donkey anti-Rabbit IgG (Jackson ImmunoResearch, Cat#711-545-152). Images were acquired under confocal microscope (Zeiss, LSM710).

The pCBD1, pCBD2-2J-2-9-1, pCBD3, and pCBD4 constructs express prM/E proteins of DENV1-4 [30 (link)]. Site-directed PCR mutagenesis of the selected amino acid residue was performed. After mutagenesis, the constructs were sequenced to confirm that there were no unintended mutations at other sites. The BHK-21 cells were transfected with vector expressing the wild-type or mutant E proteins of DENV1-4 using polyjet in vitro DNA transfection reagent (SignGen Laboratories). After 2 days, the cells were collected, fixed, and permeabilized. For staining, cells were incubated with mAbs (DD11-4, DD18-5, or a mixture of mAbs, all at concentrations of 1 μg/ml) at 4°C for 30 min. After washing, cells were incubated with RPE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), and analyzed by flow cytometry. The mixed mAbs against DENV1 EDIII (DA6-7 and DA11-3)[28 (link)], DENV2 EDIII (DB32-6, DB25-2, and 3H5)[18 (link)], DENV3 EDIII (DC9-6, DC12-3, and DC14-3) (generated by our laboratory), and DENV4 EDIII (DD17-4 and DD27-8) were used as positive controls for the relevant experiments. The binding index of a mAb to a mutant E protein was measured using the following formula: (intensity of the mutant E/intensity of wild-type E [by mAb])/(intensity of mutant E/intensity of wild-type E [by mixed mAbs]) [31 (link)].