Clinical Utility of the MMP-2 Immunoassay. The assay has a 30% percent clinical sensitivity (ability to recognize affected individuals) at a 95% clinical specificity (ability to recognize unaffected individuals).

Detection of MMP-2 Following APMA Treatment. Levels of MMP-2 detected by the MMP-2 ELISA after 2-aminophenylmercuric acetate (APMA) treatment. APMA promotes the autocatalytic cleavage of the N-terminal prosequence of the latent 72-kDa enzyme to yield the active form of the enzyme. HT1080 and astrocytoma tissue culture supernatants, recombinant proMMP-2 and normal human serum samples were either untreated (-APMA) or incubated for four hours at 37°C with 2 mM APMA (+APMA) or a volume of DMSO to control for the DMSO base of APMA (Vehicle Ctrl).

Parallelism. The study tested dilution-recovery of 28 positive samples. Dilutions were run in the MMP-2 ELISA and the "found" doses were plotted against the "expected" doses to determine linearity of dilution. The slope is very close to one and the intercept is not significantly different from zero. These studies are consistent with the absence of cross-reacting and matrix effects such as pH, salts, and endogenous binders that interfere with the reagents used in the assay. Note: Ca = Cancer; NHS = Normal Human Sera; TC= Tissue Culture.

Precision. The pooled coefficients of variation (according to the formula of Henry et. al., 1974) and between assay coefficients of variation are plotted against MMP-2 concentrations. The pooled data were collected from samples run eight times using two different lots of plates and two different lots of detector antibody in replicates of three on two separate occasions.

Sensitivity. The lower limit of detection (LLD), commonly used to define sensitivity, was measured by assaying four replicates of zero eight times using two different lots of plates and two different lots of detector antibody. The grand mean signal and pooled standard deviation of zero was calculated. The grand mean of each standard (run in replicates of four in the eight assays) was used for the standard curve, and the response, mean signal of zero plus two standard deviations, read in dose from the standard curve is the LLD; that is, the smallest dose that is not zero with 95% confidence.

Sensitivity Using 96-Well Format. Astrocytoma and HS578T cells were plated out at various cell densities into 96 plate wells. Cells were incubated at 37°C for 24 h. Supernatant was measured in the MMP-2 ELISA. MMP-2 levels were detectable at cell concentrations of 4 X 10³ Astrocytoma cells/well and 0.6 X 10^{4 HS578T cells/well and above.}

Specificity for Active MMP-2. Levels of MMP-2 detected by the ELISA after immunoaffinity extraction of MMP-2 positive samples with a MMP-2 antibody that is not used in the ELISA and a control antibody.

Specificity for Active MMP-2. Levels of MMP-2 detected by the ELISA after immunoaffinity extraction of MMP-2 positive samples with a MMP-2 antibody that is not used in the ELISA and a control antibody.

Specificity for the MMP-2:TIMP-2 Complex. Levels of a highly purified 72 kDa MMP-2 that exists in a stable, but non-covalent 1:1complex with TIMP-2 detected after neutralization with a MMP-2 antibody that is not used in the ELISA.