Radioimmunoassay of Human Collagenase: SPECIFICITY OF THE ASSAY AND QUANTITATIVE DETERMINATION OF IN VIVO AND IN VITRO HUMAN SKIN COLLAGENASE

Abstract

Abstract A specific, highly sensitive radioimmunoassay has been developed for human skin collagenase. The assay is based upon competition of purified collagenase, or collagenase of tissue extracts, with isotopically labeled purified enzyme for specific antibody binding sites after which a second antibody precipitation is used to separate bound 125I-labeled human skin collagenase from free enzyme. The radioimmunoassay provides a range of 2 to 40 ng for determining enzyme concentration and has permitted quantitation of collagenase in crude tissue extracts of human skin under conditions in which enzymatic activity cannot be detected. In addition, other human collagenases can also be measured with 200 to 300 times greater sensitivity than can be achieved by enzymatic techniques. In comparative studies, the ability of a variety of human and animal collagenases to compete with 125I-labeled human skin collagenase for antibody binding sites has been assessed as a measure of the similarity between these enzymes. Human synovial, gingival, and granulocytic collagenases are identical in their cross-reactivity with human skin collagenase in the immunoassay, supporting the concept that extensive homologies probably exist among collagenases from various human organs. Although collagenase from another primate, the baboon, shows marked cross-reactivity with the human enzymes, collagenases from more distantly related species cross-react only at protein concentrations which are 20 to 30 times greater than those needed to detect the human enzymes. These findings suggest that fewer similarities to the human enzymes will be found when purified preparations of these animal collagenases become available.