Hrp labeled goat anti mouse igg

IgE F127 was coated. Isolated anti-IgE IgG or anti-IgE(PNG) were applied at 2μg/ml and were further serial diluted (1:3). After 1h incubation, the plates were washed 3 times for 5min either with 7M urea in PBST (PBS mixed with 0.05% Tween20) or PBST only. To detect mouse IgG antibodies, HRP-labeled goat anti-mouse IgG (The Jackson Laboratory) was used. The formula used to determine the avidity index (AI) was AIx = OD (dilution x) + urea/OD (dilution x)–urea.

Plates were coated with mouse IgE F127, IgE(PNG), or Fel d 1. Serial dilution of sera (1:25, then serially 1:3) was added. HRP-labeled goat anti-mouse IgG (The Jackson Laboratory) antibodies were used to detect IgG (1:2’000). To detect IgG subclasses, HRP labeled rat anti-mouse IgG1 (BD Pharmingen), goat anti-mouse IgG2a (Bio-Rad), goat anti-mouse IgG2b (Invitrogen, Carlsbad, CA, USA), and goat anti-mouse IgG3 (SouthernBiotech, Birmingham, AL, USA) were used in a 1:2’000 dilution.

DCs were lysed in cell lysis buffer (Millipore, USA) with protease inhibitor cocktail (Sigma, USA). Protein concentration was determined by the Bradford method. Equal amounts of proteins were separated by 9% SDS-PAGE and then transferred to nitrocellulose membranes (Millipore). Primary antibodies Rhob (anti-mouse, Cell Signaling, USA), Rhoa (anti-rabbit, Cell Signaling), Rac-1 (anti-rabbit, Cell Signaling), CDC42 (anti-mouse, Santa Cruz, USA), MR (anti-goat, Abcam, UK), and GAPDH (anti-mouse, Santa Cruz, USA) and secondary antibodies HRP-labeled goat anti-mouse IgG (Jackson ImmunoResearch, USA), HRP-labeled goat anti-rabbit IgG (Jackson ImmunoResearch, USA), and HRP-labeled rabbit anti-IgG (Jackson ImmunoResearch) were all used in the Western blot. Protein detection was performed using a chemiluminescence system (SuperSignal West Pico; Pierce, Rockford, IL, USA).

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).