Using a sensitive RIA for collagenous proteins, we have applied a series of consecutive selection steps to construct a hybridoma that produces antibody against native human Type II collagen. The selection steps consisted of: 1) hybrid cell line selection; 2) co-selection of IgG class immunoglobulin reactive to the immunizing antigen; 3) selection for anticollagen antibody by complete inhibition of antibody binding to Type II collagen-coated plates by prior specific collagenase digestion; 4) selection for anti-helical antibody by complete inhibition of antibody binding to heat-denatured Type II collagen-coated plates; 5) clonal selection; 6) selection for absence of crossreactivity to 5–7 additional native collagen types; and finally 7) selection for antibody yielding indirect immunofluorescent staining of human cartilage matrix, but not adjacent bone or perichondrium. Crossreactivity assays against other native human collagen types revealed little crossreactivity. In addition, we have demonstrated that native Type II collagen inhibits antibody binding to Type II collagen-coated plates; that Type II collagen can be recovered from antigen-antibody complexes; that a sensitive radio immunoassay for Type II collagen can be developed; and that the antibody is useful for indirect immunofluorescent studies of human cartilage matrix.
- Dulbecco's-modified Eagle media
- Tween® 20
- Type V
- collagen is used to denote AB collagen
- diethylaminoethyl cellulose
- hypoxanthine, aminopterin, thymidine
- hypoxanthine, thymidine
- monoclonal antibody
- phosphate-buffered saline
- polyoxyethylene (20) sorbitan monolaurate
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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