GENERATION OF TYPE-VIII COLLAGEN-SPECIFIC ANTIBODIES

We generated a specific polyclonal antibody against the alpha 1 chain of type VIII collagen. The antibody detects type VIII collagen and is definitely free of crossreactivities with the closely related type X c ollagen. The antibody was generated against a dodecamer peptide chosen to satisfy the following requirements: (a) maximal homology between c ollagen type VIII molecules from different species; (b) maximal antige nicity as predicted by algorithms from Emini et al. (J. Virol. (1985) 55, 836), Hoop and Woods (Proc. Natl. Acad. Sci. USA (1981) 78, 3824), and Karplus and Schulz (Natunvis-senschaften (1985) 72, 212); and (c) maximal specificity, i.e. absence of this sequence in all other prote ins known so far. All three requirements were satisfied for a sequence fragment of 12 amino acids (100-111) in the alpha 1(VIII) NC2 domain. This peptide was produced synthetically. Polyclonal antibodies were r aised in rabbits and affinity purified on a peptide column. The antibo dy was tested in a quantitative EIA, immunoblots and in immunocytochem istry and found to be well-suited for all three types of application. The antibody did not crossreact with type X collagen and other extrace llular matrix molecules in the EIA. In immunoblots of affinity-purifie d extracts of the Descemet membrane, a major source of type VIII colla gen, the antibody detected several known forms of type VIII collagen. In immunocytochemistry the antibody stained endothelial and astrocytom a cells in monolayer cultures, and cells and extracellular matrix in c ryosections of the human Ewing sarcoma, arterial vessels and chicken e mbryonic heart, whereas the chicken tibiotarsus remained negative. Thi s distribution of immunoreactivity corresponds to the distribution of type VIII but not that of type X collagen. In conclusion this antibody may serve as a highly specific and sensitive tool for investigating t he appearance and regulation of type VIII collagen.