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Figure 1 | BMC Immunology

Figure 1

From: A novel assay for monitoring internalization of nanocarrier coupled antibodies

Figure 1

A. Effect of liposomal Ni-NTA-lipid concentration on target specific uptake into SKBR3 cells. Liposomes were formulated with entrapped 35 mM HPTS (fluorescent marker) and 0.5, 2 or 5 % Ni-NTA-lipid (% of phospholipid content) and tested for internalization into SKBR3 tumor cells using 20 μg/ml of anti-ErbB2-scFv-F5-(His)6. Liposomes and antibody were not pre-mixed but added sequencially to the media. Liposome concentration, μM liposome phospholipids; fluorescence, relative units. Fluorescence represents a measure of internalization of anti-ErbB2-scFv-F5-(His)6 into SKBR3 cells. B. Specificity of the CLIA assay with respect to the presence of (His)6-tagged, internalizing ligand, and fluorescently labeled Ni-NTA-derivatized liposomes.SKBR3 tumor cells were incubated with 500 μM Ni-NTA-liposomes (Ni-NTA = 5% of phospholipid content) and anti-ErbB2-scFv-F5 (without a (His)6-tag), or an irrelevant control scFv antibody (anti-VEGRF2-scFv-4G7-(His)6) or liposomes without scFv (Ni-NTA liposomes (no scFv). Alternatively, anti-ErbB2-scFv-F5-(His)6 was co-incubated with liposomes formulated without the Ni-NTA-DOGS lipid (anti-ErbB2-scFv- F5-(His)6 (no NTA)). All antibody concentrations were 20 μg/mL. The fluorescence was read as a measure of liposome internalization. C. Assaying internalization of monoclonal IgG antibodies without (His)6-tag by CLIA using a Protein A-(His)6 chemical conjugate. SKBR3 cells were incubated with anti-ErbB2-scFv-F5-(His)6 (20 μg/mL), with anti-ErbB2 mAb Herceptin (20 μg/mL), Protein A-(His)6 alone (also 20 μg/mL), or mixture of anti-ErbB2 mAb Herceptin and Protein A-(His)6 in the presence of 500 μM fluorescently labeled Ni-NTA-liposomes (Ni-NTA = 5% of phospholipid content). Immunoliposomes were allowed to internalize for four hours. The cells were lysed in base and the fluorescence was read as a measure of internalization. D. Co-localization of internalized liposomes with transferrin-PE. Ni-NTA-liposomes, anti-ErbB2-scFv-F5-(His)6 (20 μg/mL), and transferrin-phycoerythrin were co-incubated for two hours with SKBR3 cells before observing cellular localization by fluorescence microscopy using a dual-pass filter. E. Temperature dependence of liposome internalization. Ni-NTA liposomes (Ni-NTA = 5% of phospholipid content) were incubated in the presence or absence of ErbB2-scFv-F5-(His)6 (20 μg/mL) at either 4 C or at 37 C for 4 hours. The cells were then washed with either PBS or immidazole and lysed in base and the fluorescence was read as a measure of internalization.

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