COMPARISON OF TECHNIQUES FOR MEASUREMENT OF GELATINASES TYPE-IV COLLAGENASES - ENZYME-LINKED IMMUNOASSAYS VERSUS SUBSTRATE DEGRADATION ASSAYS/

Radiolabeled substrate degradation assays and gelatin zymography are r outinely employed to assay 72 kDa gelatinase A (MMP-2) and 92 kDa gela tinase B (MMP-9) in biological fluids. Enzyme-linked immunosorbent ass ays (ELISA) have recently been developed for the quantitation of these matrix metalloproteinases (MMP). In this study, we have compared ELIS A to standard substrate degradation assays for measurement of MMP-2 an d MMP-9 in human plasma and tumor-conditioned media. Gelatin Sepharose chromatography and gel filtration chromatography were employed as par tial purification procedures for MMP-2 and MMP-9, The ELISA data for M MP-2 and MMP-9 are linear on a log:log regression curve over a wide ra nge of MMP concentrations and are specific for the designated gelatina se, with no overlap detected with related metalloproteinases. The mini mum detectable concentrations of MMP-2 and MMP-9 were approximately 0. 5ng/ml and 0.2ng/ml, respectively, in the ELISA as compared to 4ng/ml and 3ng/ml, respectively, in gelatin zymography. The [H-3]gelatin degr adation assay required a combination of >50 ng/ml of MMP-2 and MMP-9 f or detection. Although gelatin zymography was less sensitive than ELIS A (primarily due to the smaller sample volume employed) and was more d ifficult to quantitate, this procedure offers the important advantage of being able to distinguish between latent and activated gelatinases.