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Production and characterization of hybridoma antibody to native human type II collagen
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. Cross-reactivity assays against other native human collagen types revealed little cross-reactivity. 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 radioimmunoassay for Type II collagen can be developed; and that the antibody is useful for indirect immunofluorescent studies of human cartilage matrix.
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. Cross-reactivity assays against other native human collagen types revealed little cross-reactivity. 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 radioimmunoassay for Type II collagen can be developed; and that the antibody is useful for indirect immunofluorescent studies of human cartilage matrix.