title: Characterization of monoclonal antibodies against the glaucoma-associated protein myocilin.
abstract: Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well-characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33-214), two antibodies recognize the middle third of the protein (amino acids 215-368), and one antibody recognizes the C-terminus (amino acids 369-504). Isotyping revealed that all antibodies are of the IgG1 kappa class except one, which is IgG2b kappa. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells.
authors.affiliation: Department of Ophthalmology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
authors.forename: Mohamed-Karim
authors.initials: MK
authors.lastname: Ezzat
authors.forename: Kyle G
authors.initials: KG
authors.lastname: Howell
authors.forename: Cindy K
authors.initials: CK
authors.lastname: Bahler
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Beito
authors.forename: Nils
authors.initials: N
authors.lastname: Loewen
authors.forename: Eric M
authors.initials: EM
authors.lastname: Poeschla
authors.forename: Michael P
authors.initials: MP
authors.lastname: Fautsch
mesh_terms: D000369:Aged, 80 and over; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D001082:Aqueous Humor / Q000378:metabolism; D001666:Binding Sites, Antibody; D015153:Blotting, Western / Q000379:methods; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins / Q000276:immunology* / Q000302:isolation & purification; D005123:Eye / Q000378:metabolism; D005136:Eye Proteins / Q000276:immunology* / Q000302:isolation & purification; D005260:Female; D006023:Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D006825:Hybridomas; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000302:isolation & purification; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism; D000099179:Myocilin
chemical_list: D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D000099179:Myocilin