MMP-13 ELISA Kit

The MMP-13 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of latent and active human MMP-13 protein in body fluids such as serum and synovial fluids. Note: When analyzing pro-MMP-13 and active MMP-13 in parallel, the level of pro-MMP-13 must be ~10 times higher than the level of active MMP-13 in order to obtain a comparable signal.

The assay recognizes the activated form of MMP-13. It does not recognize the latent or activated forms of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, or the catalytic domain of MT-1 MMP, MT-2 MMP, MT-3 MMP, MT-4 MMP, or MT-5 MMP. High concentrations of latent MMP-13 may contribute to absorbance values measured in the assay. When present at equal concentrations, latent MMP-13 yields about 1/10 the absorbance value of active MMP-13.

Assay Time: ~5 hours

Serum:• Serum samples may be stored at -80°C.• Avoid freeze/thaw cycles.• When the serum is stored at -80°C, it is absolutely necessary to mix the samples thoroughly prior to measuring.• Dilute the serum samples 1:10 with Assay Buffer, depending on the possible concentration of the analyte. For measuring these dilutions use the standards prepared with Serum Standard Diluent.• If you need samples with a dilution of more than 1:10, initially prepare a 1:10 dilution with Assay Buffer and do further dilution steps using Serum Standard Diluent. This is necessary to maintain a serum concentration of 10% in the sample.Synovial Fluid:• Centrifuge the collected synovial fluid for 15 min at 4000 x g or higher.• The supernatant can be stored at -80°C.• Avoid freeze/thaw cycles.• When the synovial fluid was stored at -80°C, it is absolutely necessary to mix the samples thoroughly prior to measuring.• Dilute the samples 1:10 or more with Assay Buffer, depending on the possible concentration of the analyte. For measuring these samples, use the standards prepared with Serum Standard Diluent.• If you need samples with a dilution of more than 1:10, initially prepare a 1:10 dilution with Assay Buffer and do further dilution steps using Serum Standard Diluent.• It is important to note that the recovery of MMP-13 in synovial fluid is only 50% or less (see table 5).Tissue culture supernatant• Centrifuge the samples to remove any particles.• The supernatants can be stored at -80°C.• Avoid freeze/thaw cycles.• Dilute the samples 1:3 or more with Assay Buffer, depending on the possible concentration of the analyte. For measuring diluted samples use the standards prepared with Assay BufferNote: Fetal or neonatal calf serum may contain Collagenase 3. Always measure your culture media as background controls!

All components of the kit can be stored in the refrigerator (4°C). Once reconstituted, the standard solution should be used immediately or stored at -20°C. The diluted Biotinylated Detection Antibody and diluted Conjugate Solution should be prepared freshly directly before use. When running a partial plate, only a suitable aliquot should be activated.

It is absolutely necessary to equilibrate all reagents to room temperature (1 h incubation on the bench) prior to use in order to prevent margin effects. Use either distilled or deionized water for the reconstitution of the standard and for the dilution of the wash buffer concentrate. Always seal the plates with the provided foil during incubation.• Assay Buffer: After equilibration to room temperature, the buffer is ready to use.• Wash Buffer: Dilute the contents of the bottle (25 ml) to 500 ml with distilled or deionized water. Thoroughly wash the stock bottle to ensure that the entire contents are used.• Standard: Add 1 ml distilled or deionized water to the standard tube (yellow lid) and allow the contents to dissolve for 30 min. Gently mix, but avoid foaming of the reagent!• Serum Standard Diluent: After equilibration to room temperature, the reagent is ready to use.• Detection Buffer: After equilibration to room temperature, the reagent is ready to use.• Biotinylated Antibody: Dilute Biotinylated Antibody 100-fold with Detection Buffer. For the whole plate, add 120 µl from the antibody solution tube (red lid) to 12 ml Detection Buffer. When running half a plate, add 60 ml antibody solution to 6 ml Detection Buffer.• Conjugate Solution: Dilute the provided Conjugate Solution 40-fold with Detection Buffer. For the whole plate, add 300 µl of the conjugate tube (blue lid) to 12 ml Detection Buffer. When running half a plate, add 150 µl of the conjugate to 6 ml of Detection Buffer.• Substrate and Stop Solution: After equilibration to room temperature, the reagents are ready to use.Preparation of Standards with Assay Buffer(For tissue culture supernatants)1. Label 7 tubes with 32, 63, 125, 250, 500, 1000, and 2000 pg/ml.2. Pipette 900 µl of Assay Buffer into the 2000 pg/ml tube, the remaining tubes pipette 500 µl of Assay Buffer.3. Pipette 100 µl of the stock standard (20 ng/ml) into the 2000 pg/ml tube and mix thoroughly.4. Pipette 500 µl of the 2000 pg/ml standard into the tube labeled with 1000 pg/ml and mix thoroughly.5. Repeat this dilution procedure with the other standard tubes.6. The blank value (0 pg/ml) is obtained by using simple Assay Buffer.7. The stock solution is not part of the standard curve and can be stored at -20°C.Preparation of standards with Serum Standard Diluent(For the highly sensitive measurement of serum and synovial fluid samples)1. Label 7 tubes with 32, 63, 125, 250, 500, 1000, and 2000 pg/ml.2. Pipette 900 µl of Serum Standard Diluent into the 2000 pg/ml tube, in the remaining tubes pipette 500 µl of Serum Standard Diluent.3. Pipette 100 µl of the stock standard (20 ng/ml) into the 2000 pg/ml tube and mix thoroughly.4. Pipette 500 µl of the 2000 pg/ml standard into the tube labeled with 1000 pg/ml and mix thoroughly.5. Repeat this dilution procedure with the other standard tubes.6. The blank value (0 pg/ml) is obtained by using Serum Standard Diluent.7. The stock solution is not part of the standard curve and can be stored at -20°C.

Calculations and Evaluation of ResultsThe calculation is illustrated using representative data: the assay data should be similar to that shown in Table 1.1. Calculate the average absorbance for each set of standard wells.2. A standard curve is generated by plotting the mean absorbance (y axis) against pg/ml standard (x-axis, Figure 1).3. The pg/ml values of the samples can be read directly from the graph or calculated by the regression coefficients.4. Multiply the calculated pg/ml values by the dilution factor of the samples.

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Coated 96-Well Plate, Assay Buffer, Wash Buffer Concentrate, Standard, Serum Standard Diluent, Detection Buffer, Biotinylated Antibody, Conjugate Solution, TMB Substrate, Stop Solution, and a user protocol.

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

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