Figure 6

Recombinantly expressed monoclonal antibodies are immunoreactive and functional.

(A–B) Flow cytometric histograms showing fluorescent intensity of cells incubated with purified IgG/IgE antibodies plus secondary FITC-labelled goat anti-human IgG/IgE (open) and cells with secondary antibody only (filled) against the number of cells. (A) Flow cytometric assessment of anti-CSPG4 IgG₁/IgE antibodies shows specific binding to native CSPG4 antigen present on the cell surface of A375 melanoma cells and no binding above background to primary melanocytes. (B) Fc regions of anti-CSPG4 IgG₁ and 102.1F10 IgG₄ isotypes demonstrate effector-binding to U937 monocytic cell line, expressing human Fcγ receptors. The IgE antibody isotypes also bind similarly to RBL SX38 mast cells, expressing human FcεR1 receptor. (C) Immunofluorescence staining of A375 cells confirms specific binding of anti-CSPG4 IgG₁/IgE and no background binding with isotype control hapten specific NIP-IgG₁/IgE detected by goat anti-human IgG/IgE-FITC. (D) Grass pollen allergen specificity is confirmed by anti-human sandwich ELISA, showing specific binding of recombinant 102.1F10 IgG₄/IgE and original patient serum to the ELISA plate-bound Phl p 7 allergen and no binding above background with unspecific human myeloma IgG₄ and MOv18 IgE isotype controls.