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M. K. Ezzat, K. G. Howell, T. G. Beito, M. P. Fautsch; Characterization of Monoclonal Antibodies Generated Against Human Myocilin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1617.
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© ARVO (1962-2015); The Authors (2016-present)
Myocilin is one of the most studied glaucoma-associated proteins however its function is unknown. Determination of myocilin’s function will partly depend on an accurate and reliable panel of antibodies to verify binding interactions with associated proteins. The aim of this study was to generate and characterize monoclonal antibodies against human myocilin.
Secreted full-length recombinant myocilin was purified from conditioned media isolated from cultured human trabecular meshwork cells overexpressing myocilin. BALB/c mice were immunized with purified recombinant myocilin. Splenocytes from 3 mice were individually fused with myeloma cell line (F/O) and resulting hybridomas were screened by Western blot analysis. Antibody binding was assessed using conditioned media from human TM5 and 293 cells over expressing myocilin, and human 293 cell lysate obtained from cells expressing myocilin mutant Gly368Stop. Human aqueous humor was also analyzed via Western blots. Immunohistochemistry was performed on primary monolayer trabecular cells and fresh trabecular meshwork tissue to verify specificity.
Six monoclonal antibodies were generated to human recombinant myocilin. All antibodies are of the IgG1Κ class except one, which has an IgG2bΚ isotype. Two antibodies recognize the N-terminus of myocilin (amino acids 33-214), 2 antibodies recognize the middle third of the protein (amino acids 215-368), and 2 antibodies recognize the C-terminus (amino acids 369-504). Purified myocilin monoclonal antibodies recognized myocilin in human aqueous humor and trabecular meshwork lysate by Western blot. All of the monoclonal antibodies recognized myocilin by immunohistochemistry, showing perinuclear staining consistent with an endoplasmic reticulum/Golgi apparatus distribution. A light diffuse cellular staining was also detected with several of the antibodies.
Six myocilin monoclonal antibodies have been established. Generation of antibodies against various parts of the myocilin molecule will help to delineate myocilin function in normal and glaucomatous pathologies. These antibodies are available to research laboratories interested in myocilin.
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