Immunization with undenatured bovine zonular fibrils results in monoclonal antibodies to fibrillin.
Microfibrils were dissected from the zonular apparatus of the bovine eye, homogenized and used as an immunogen to prepare monoclonal antibodies. Initial screening of hybridomas was performed by immunoblotting to a sonicate of zonular fibrils and by immunolocalization to frozen sections of the zonular apparatus. Subsequently, monoclonal antibodies with strong immunoreactivity to zonular fibrils were shown to recognize microfibrils in a wide range of connective tissues both by immunofluorescent staining and by electron microscopic immunolocalization. All antibodies were found to recognize a single protein of 350 kDa on Western blotting of the proteins secreted by bovine aortic smooth muscle cells. A protein of the same molecular weight and properties was recognized by an antibody previously prepared by another group against fibrillin. A member of the fibrillin family therefore represents the major immunogen of intact zonular fibrils, and the results support previous evidence for a close relationship between zonular fibrils and other connective tissue microfibrils. The zonular apparatus is a suitable system to obtain purified preparations of microfibrils in order to investigate their composition and structural organization.
Wright
D W
DW
Department of Cell Biology, University of Alabama, Birmingham 35294.
McDaniels
C N
CN
Swasdison
S
S
Accavitti
M A
MA
Mayne
P M
PM
Mayne
R
R
D008841:Actin Cytoskeleton / Q000276:immunology* / Q000648:ultrastructure; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000917:Antibody Formation; D001011:Aorta / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D005123:Eye / Q000276:immunology*; D000071837:Fibrillins; D005455:Fluorescent Antibody Technique; D008840:Microfilament Proteins / Q000032:analysis* / Q000276:immunology*; D016253:Microscopy, Immunoelectron; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000071837:Fibrillins; D008840:Microfilament Proteins