MMP-9 ELISA Kit

The Calbiochem MMP-9 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of human MMP-9 protein in tissue culture medium, serum, and plasma.

The MMP-9 ELISA Kit detects free and TIMP-1 bound MMP-9 in human serum and plasma samples and cell culture supernatants. Specificity was demonstrated by immunoaffinity extraction (inhibition of assay signal) of MMP-9 positive samples by a specific MMP-9 antibody, a specific TIMP-1 antibody, a specific TIMP-2 antibody and a specific TIMP-3 antibody. The MMP-9 antibody, which is not a component of the ELISA, extracted almost all MMP-9 (almost the entire signal was lost), while the control antibody (non MMP-9 antibody) did not affect the signal of the MMP-9 positive samples. The TIMP-1 antibody extracted most of the pro-MMP-9 activity detected by this immunoassay. Addition of TIMP-1 and TIMP-2 recombinant proteins to both positive and negative samples does not change the level of MMP-9 detected demonstrating that the assay has the same specificity for the free and the TIMP bound form of MMP-9.



It is known that 2-aminophenylmercuric acetate (AMPA) treatment promotes the autocatalytic cleavage of the N terminal propeptide of the latent 92-kDa enzyme to yield an 84-kDa enzyme. Further treatment causes the autocatalytic cleavage of the carboxyl terminus to generate the 68-kDa form of MMP-9. Both tissue culture supernatants and sera samples were tested in the MMP-9 ELISA after APMA treatment. In every case almost all the signal disappeared after APMA treatment. Analysis of the samples by zymography showed both cleavage of the proenzyme by APMA and a good correlation with the levels of the proenzyme determined with the MMP-9 ELISA. In addition the assay recognizes pure recombinant pro-MMP-9 protein, but not active MMP-9 recombinant protein.

Assay Time: 3.5 h

• Suspension Cells: Pellet suspension cells by centrifugation at 1000 x g for 10 min, 4°C. Remove supernatant for testing. Samples may be stored at -20°C.• Adherent Cells: Remove tissue culture medium and centrifuge at 1000 x g for 10 min. Remove supernatant for testing. Samples may be store at -20°C.• Serum and Plasma: Serum and plasma samples should be diluted with Sample Diluent 1:10 (normal samples) or 1:40 (most cancer samples) or greater as needed.Samples found to contain greater than 20 ng/ml MMP-9 (i.e., outside the range of the standard curve) must be diluted with Sample Diluent (provided), so that the MMP-9 concentration falls within the range spanned by the standard curve, and assayed again.

• 1X Wash Buffer: Prepare 1X Wash Buffer by adding 25 ml 20X Wash Buffer Concentrate to 475 ml of dH₂O. Mix well.• MMP-9 Standard and Standard Curve: Each time an assay is performed, reconstitute a Lyophilized Standard by carefully and accurately pipetting dH₂O and sample diluent, as described on the lyophilized MMP-9 Standard vial label to give a concentration of 20 ng/ml. Let the reconstituted standard sit for 15 min at room temperature, with occasional swirling. Avoid excessive agitation of the standard. After reconstituting the MMP-9 Standard it should be diluted with Sample Diluent. Obtain seven tubes and label them 20, 10, 5, 2.5, 1.25, 0.625 and 0 ng/ml. Add 250 µl of Sample Diluent into each tube except the 20 ng/ml tube (first tube) that gets "undiluted" reconstituted standard. Remove 500 µl from the original vial of lyophilized material and add it to the first tube. Remove 250 µl from the first tube (20 ng/ml) and add it to the second tube (10 ng/ml) and mix gently. Repeat this procedure until you reach the sixth tube (0.625 ng/ml). The last tube (0 ng/ml) should just be Sample Diluent. Reconstituted standards should be discarded after one use.• 1X Conjugate: Just prior to use, dilute a sufficient volume of 400X Conjugate 1:400 with Conjugate Diluent to provide 100 µl 1X Conjugate for each sample and standard well. For example add 30 µl to 12 ml of Conjugate Diluent, mix gently. Filter with a 0.2 µm syringe filter prior to use. Discard any unused 1X Conjugate.

Upon arrival, store the entire contents at -20°C. Avoid multiple freeze/thaw cycles. Do not expose reagents to excessive light.

1. Average the duplicate absorbance values for each standard, including the zero and all sample values. 2. On graph paper, plot the mean absorbance values for each of the standards on the Y axis, versus the concentration of each standard (ng/ml) on the X axis.3. Determine the concentration of unknowns by interpolation from the standard curve. There are a variety of plate reader software packages available (Softmax, Molecular Devices Corporation, Menlo Park, CA; KinetiCalc, BioTek Instruments, Inc. Winooski, VT) for analysis of data, which simplifies this process. 4. For samples that have been diluted, the MMP-9 concentration must be multiplied by the dilution factor (ie., if the sample was diluted five-fold, then the MMP-9 value obtained from the standard curve must be multiplied by five).

These results demonstrate that the assay specifically recognizes free and TIMP bound zymogen form of MMP-9.

96-Well Coated Plate, MMP-9 Standard, Biotinylated Detector Antibody, 400X Streptavidin Peroxidase Conjugate, Diluent, Substrate, Assay Buffers, Stop Solution, Plate Sealer, and a user protocol.

Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.

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Toxicity: Multiple Toxicity Values, refer to MSDS (O)