Immunofluorescent localization of type V collagen in the chick embryo with monoclonal antibodies.

C3H mice were immunized with native, pepsin-extracted chick type V collagen, and their spleen cells were fused with myeloma cells from the cell line X63 Ag 8653. Several hybridoma cell clones were obtained and grown to mass culture which produced monoclonal antibodies reacting with native, but not or very little with denatured type V collagen, or isolated alpha 1(V) and alpha 2(V) chains in a radio-binding assay. No cross-reaction was found with native collagen types I, III and IV collagen; some clones showed a slight cross-reaction with types II and M collagen, and all clones cross-reacted with native collagen molecules containing 1 alpha, 2 alpha, and 3 alpha chains. This indicated that this collagen type probably carries similar antigenic sites and is closely related to type V collagen. Two clones were further characterized by SDS gel electrophoresis and radial immunodiffusion; the secreted antibodies belong to the IgG1 subclass. For immunofluorescence studies monoclonal antibodies were purified from culture medium or ascites fluid by ammonium sulfate precipitation or affinity chromatography on type V collagen. Type V collagen was localized in corneal stroma, bone matrix, mesodermal layer of the skin, muscle connective tissue and perichondrium. Short pretreatment of the tissue sections with pepsin was necessary in order to reveal fluorescence reactions.