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Production and characterization of antibody blocking epidermal growth factor:receptor interactions
Abstract:
Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes. The immunoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5 degrees C). Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.
Insights
Antibodies against A-431 cell membranes block epidermal growth factor (EGF) binding to its receptors. These antibodies also inhibit EGF-induced DNA synthesis in fibroblasts, demonstrating their role in blocking EGF signaling pathways.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- A-431 cells possess a high density of epidermal growth factor receptors (EGFR).
- Membrane preparations from A-431 cells retain high EGFR binding activity.
Purpose of the Study:
- To generate antibodies against A-431 cell membrane components, specifically targeting EGFR.
- To investigate the functional role of these antibodies in inhibiting EGF binding and downstream signaling.
Main Methods:
- Production of antisera in rabbits using A-431 cell membranes as antigen.
- Double-immunodiffusion to identify antibodies against membrane components.
- Inhibition assays measuring 125I-labeled EGF binding to intact cells, membrane preparations, and solubilized membranes.
- Assessment of antibody effects on EGF-induced DNA synthesis in quiescent fibroblasts.
Main Results:
- Immune serum contained precipitating antibodies against multiple A-431 membrane components.
- Immunoglobulin G (IgG) fraction and its fragments inhibited EGF binding to EGFR on various cell types and preparations.
- Antibodies blocked EGF binding at low temperatures, indicating specific receptor interaction.
- Anti-A-431 antibodies mimicked anti-EGF antibodies in blocking EGF-induced DNA synthesis, with reversal possible within 5 hours of EGF addition.
Conclusions:
- Antibodies generated against A-431 cell membranes effectively target EGFR and inhibit EGF binding.
- These antibodies can block the mitogenic effects of EGF by interfering with its signaling pathway.
- The findings highlight the potential of anti-EGFR antibodies in modulating cellular responses to growth factors.