IMMUNIZATION WITH UNDENATURED BOVINE ZONULAR FIBRILS RESULTS IN MONOCLONAL-ANTIBODIES TO FIBRILLIN

Microfibrils were dissected from the zonular apparatus of the bovine e ye, homogenized and used as an immunogen to prepare monoclonal antibod ies. Initial screening of hybridomas was performed by immunoblotting t o a sonicate of zonular fibrils and by immunolocalization to frozen se ctions of the zonular apparatus. Subsequently, monoclonal antibodies w ith strong immunoreactivity to zonular fibrils were shown to recognize microfibrils in a wide range of connective tissues both by immunofluo rescent staining and by electron microscopic immunolocalization. All a ntibodies were found to recognize a single protein of 350 kDa on Weste rn blotting of the proteins secreted by bovine aortic smooth muscle ce lls. A protein of the same molecular weight and properties was recogni zed by an antibody previously prepared by another group against fibril lin. A member of the fibrillin family therefore represents the major i mmunogen of intact zonular fibrils, and the results support previous e vidence for a close relationship between zonular fibrils and other con nective tissue microfibrils. The zonular apparatus is a suitable syste m to obtain purified preparations of microfibrils in order to investig ate their composition and structural organization.