Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing.

Open Access
Publisher Website
Google Scholar
Abstract
The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the EM after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only 1 of the 3 pepsin-resistant fragments of the molecule. The 3 native fragments were designated (F1)2F2, F3 and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8, IIB12, and ID2. By EM, monoclonal antibody IA8 recognized as epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of 4 type IV molecules. Monoclonal antibody IIB12, in contrast, recognize an epitope located only 73 nm from the 7S domain. The F3 fragment apparently is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [.alpha.1(IV)]2.alpha.2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2, were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. This site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by .alpha.-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.