Double-antibody enzyme-linked immunosorbent microassay for quantification of collagen types I and II

Specific quantification of collagen types I and II by sandwich ELISA or double-sandwich ELISA is described. Specific anticollagen antibodies are linked to polystyrene microplates. The collagen to be measured binds to the coating antibodies. Collagen type-specific second antibodies react with the immobilized antigen. The second antibodies are either labeled with peroxidase or are detected by anti-IgG antibodies conjugated with peroxidase. Bound peroxidase is estimated by the color reaction produced with the substrate 5-aminosalicylic acid. Optimization of the test procedure was achieved by varying the conditions for coating, antigen and second antibody incubations. Sensitivity, precision, specificity and recovery of the method have been analyzed. The assay is compared with inhibition ELISA and hydroxyproline determination.