Solid-phase radioimmunoassay of rat skin collagenase comparative studies and in vivo measurement of the enzyme

Abstract

The solid-phase antibody method has been employed to develop a sensitive radioimmunoassay for the measurement of collagenase in rat skin. The solid-phase radioimmunoassay, in which approximately 1.0 μg of functionally monospecific anticollagenase antibody globulin is bound to polypropylene tubes, determines 0.3–20 ng of enzyme. This assay permits the measurement of collagenase either in crude tissue extracts of rat skin, which contain no detectable enzyme activity, or in enzymatically active preparations obtained from tissue culture medium. The immunoassay is at least 1000 times more sensitive than the enzymatic assay. The similarities between rat skin and rat uterus collagenases, as well as collagenases from a variety of other animal sources, were evaluated by assessing their relative binding capabilities in the solid-phase radioimmunoassay. Rat uterus collagenase was found to be identical in its cross-reactivity with rat skin collagenase in the immunoassay indicating that these rat collagenases are structurally closely related. Of particular interest was the observation that the cross-reactivity between the collagenase from mouse skin and the rat skin collagenase was one of complete immunologic identity although this was not the case for the skin collagenase from another rodent, the guinea pig. This indicates that the solid-phase radioimmunoassay can be used to measure collagenase levels in mouse skin and perhaps other mouse tissues as well. Collagenases from phylogenetically more distant species cross-react much less strongly, syggesting the existence of fewer similarities to the rat enzymes. Quantitative determination of collagenase in rat skin using the solid-phase radioimmunoassay indicates that the enzyme content reaches a maximum during the first 24 h after birth and by one week of age has declined to a level found in the mature animal.