Comparison of techniques for measurement of gelatinases/type IV collagenases: enzyme-linked immunoassays versus substrate degradation assays

Abstract

Radiolabeled substrate degradation assays and gelatin zymography are routinely employed to assay 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) in biological fluids. Enzyme-linked immunosorbent assays (ELISA) have recently been developed for the quantitation of these matrix metalloproteinases (MMP). In this study, we have compared ELISA to standard substrate degradation assays for measurement of MMP-2 and MMP-9 in human plasma and tumor-conditioned media. Gelatin Sepharose chromatography and gel filtration chromatography were employed as partial purification procedures for MMP-2 and MMP-9. The ELISA data for MMP-2 and MMP-9 are linear on a log:log regression curve over a wide range of MMP concentrations and are specific for the designated gelatinase, with no overlap detected with related metalloproteinases. The minimum detectable concentrations of MMP-2 and MMP-9 were approximately 0.5 ng/ml and 0.2 ng/ml, respectively, in the ELISA as compared to 4 ng/ml and 3 ng/ml, respectively, in gelatin zymography. The [3H]gelatin degradation assay required a combination of > 50 ng/ml of MMP-2 and MMP-9 for detection. Although gelatin zymography was less sensitive than ELISA (primarily due to the smaller sample volume employed) and was more difficult to quantitate, this procedure offers the important advantage of being able to distinguish between latent and activated gelatinases.

Calcitonin increases invasiveness of prostate cancer cells: Role for cyclic AMP-dependent protein kinase A in calcitonin action

“…The conditioned media from T47D cell culture (RPMI 1640 without serum), which contains high levels of MMP-2 and -9, were used as positive controls. 21 The results presented in Figure 5a reveal detectable MMP-2 (72 kDa) and MMP-9 (88-90 kDa) gelatinolytic activity in the conditioned media of vehicle-treated cells cultured in serum-free media (lane 1 of Fig. 5a,b).…”

“…High-affinity CT receptors have been identified in membrane preparations of primary tumors as well as androgen-responsive LNCaP and highly metastatic but androgen refractory PC-3M prostate cancer cells. 10 Exogenously added CT stimulates the proliferation of LNCaP cells by stimulating cAMP accumulation and increasing cytoplasmic Ca 21 transients, and CT also increases chemotaxis of these cells by unidentified mechanisms. 10,11 These results, when combined with our other results that the expression of CT and CTR is upregulated in prostate carcinomas (PCs), suggest an important paracrine/autocrine role for ''CT System'' in prostate cancer progression.…”

Increased Matrix Metalloproteinase-9 Activity in Unstable Carotid Plaques

“…The product of the optical density and area of the band was compared directly with a standardized positive control (media conditioned by HT-1080 human fibrosarcoma cells) to obtain a ratio comparable between gels, as previously described. 5 The identities of the lytic zones on the gelatin zymograms were confirmed as MMP-2 and MMP-9 (72 and 92 kDa, respectively) by immunoblotting.…”

Advances in assays of matrix metalloproteinases (MMPs) and their inhibitors

“…Western blotting requires knowledge of the MMPs native substrate and the availability of anti-MMPs antibodies. And what is more, it is expensive and time-consuming [111]. Zymography is more suitable for screening of MMP inhibitors; however, zymography gives a measure of total potential enzymatic activity but does not allow determination of the net level of activity present in a sample.…”