MMP9 ELISA: Matrix Metalloproteinase-9
INSTRUCTION FOR USE
For quantification of MMP-9 in serum samples
FLS-BP09
This manual must be read entirely before using this product.
For research use only (RUO). Do not use for diagnostic procedures.
Version: 1.0
Firalis S.A. 35 rue du Fort 68330 – Huningue FRANCE |
Phone: +00 33 389 910 111 Email: contact@firalis.com Web: www.firalis.com |
Table of contents |
Background and Significance |
Materials provided & Storage conditions |
Other material required |
Sample collection, Storage and Preparation |
Reagent preparation |
Assay procedure |
Intended Use
Firalis MMP-9 ELISA kit is intended to be used for the in vitro quantification of human MMP-9 in serum samples.
- The assay is intended for research use only.
- The total assay time is less than 5 hours.
- The assay measures MMP-9 in serum samples.
- The format is 96 wells.
- Components of the kit are provided ready to use, lyophilized, concentrated or to reconstitute (see table of MATERIALS PROVIDED).
Background and Significance
Matrix Metelloproteinase-9 (MMP-9) is part of the matrix metalloproteinase family, a group of structurally related zinc-containing proteolytic enzymes. These enzymes play an important role in tissue remodeling of extracellular matrix. MMP-9 is a 92 kDa protein with a sequence length of 707 amino acids that is produced by epithelial cells and is stored in the secretory granules of neutrophils and eosinophils. MMP-9 is involved in vascular leakage, migration of inflammatory cells and wound repair. Several pre-clinical studies suggest that MMP-9 has the potential to predict, diagnose and prognosticate cardiac remodeling and related events. In fact, it has been shown that MMP-9 null mice that suffered from myocardial infarction (MI), were protected against cardiac rupture and demonstrated a decrease in collagen and macrophage infiltration in cardiac tissue that resulted eventually in a decrease in left ventricular (LV) dimensions, a hallmark of cardiac remodeling1, 2, 3, 4, 5, 6.
Principle of Assay
The Firalis Assay MMP-9 is a sandwich ELISA (Enzyme Linked Immunosorbent Assay) plate-based assay. The 96-wells plate is already coated with the monoclonal capture antibody specific for the soluble form of “MMP-9”. Standards, Quality controls (QC) and diluted samples are incubated in the pre-coated wells. MMP-9 present in the solutions will bind to the capture antibody while unbound and/or weakly bound substances are removed in washing steps. Then, a biotinylated secondary monoclonal antibody is added to each well, followed by another washing step to remove unbound antibody. Next, the streptavidin conjugate horseradish peroxidase (HRP) is added in each well and allowed to form a complex with the bound biotinylated antibodies during the incubation time. A third washing step is performed to remove unbound HRP-streptavidin and the enzyme substrate TMB (Tetramethylbenzidine) is added into the wells. The TMB is oxidized by HRP in the presence of hydrogen peroxide, turning TMB blue. The intensity of the colour produced is proportional to the MMP-9 amount present in each well. Finally, sulfuric acid is added in each well to stop the enzymatic reaction, which will cause the oxidized TMB to turn yellow. The absorbance of this yellow product is measured at 450 nm and the signal is correlated to the concentration of MMP-9. A standard curve is constructed by plotting absorbance values against concentrations of standards. The concentration of the biomarker in analysed samples are determined using this standard curve.
Materials provided & Storage conditions
The APO-Easy® Genotyping kit includes consumables to perform the qPCR amplification and fluorogenic detection of APOE mutations using ThermoFisher QS5-Dx (equipment not provided with the kit). The equipment needed for the assay but not provided with the kit and their references are given in Table 1.
|
DESCRIPTION |
Storage |
Ref. |
Qty |
① |
MMP-9 Microplate: 96 well microplate (12 strips of 8 wells) coated with a mouse monoclonal antibody against MMP-9. |
|
CTP-BP-02 |
96-w microtiter plate |
② |
Human MMP-9 standard: 180 ng of recombinant, lyophilized human MMP-9 / vial. |
STD-BP-02 |
1 vial |
|
③ |
Mouse monoclonal antibody (Detection Antibody): 125 µl (80-fold concentrated solution)/ vial |
DTA-BP-02 |
1 vial |
|
④ |
Streptavidin-HRP: 80 µL of HRP (100-fold concentrated solution) /vial. |
FLS-HRP03-02 |
1 vial |
|
⑤ |
10x Wash Buffer: 50mL |
FLS-WSB-03 |
1 vial |
|
⑥ |
TMB substrate: 12 mL of tetramethylbenzidine/vial |
FLS-TMB01 |
1 vial |
|
⑦ |
Stop Solution: 6mL of 2N sulfuric acid/vial |
FLS-SPS01 |
1 vial |
|
⑧ |
QC high Human recombinant MMP9 |
QCH-BP-02 |
1 vial |
|
⑨ |
QC low Human recombinant MMP9 |
QCL-BP-02 |
1 vial |
|
/ |
Plate Sealers 12 strips |
|
N/A |
5 units |
Other material required
- Deionized or distilled water
- Test tubes for dilution of standards and samples
- Appropriate glassware for dilutions
- Precision pipettes and appropriate tips
- Multichannel pipette
- Reservoirs
- Paper towels and aluminium foil
- Microplate washer (Optional)
- Orbital microplate shaker.
- Vortex
- Microplate reader to measure absorbance at 450 nm
Sample collection, Storage and Preparation
Sample preparation:
Use a serum separator tube and allow samples to clot for 30 minutes at room temperature before centrifuging for 15 minutes at 1000 g. Isolate serum and use it immediately or aliquot and store serum samples at ≤ -80 °C. Avoid repeated freeze-thaw cycles. Serum samples require a 180-fold dilution in 1X Wash Buffer before measurement. Sample stability has not been evaluated.
Reagent preparation
- Bring all reagents to room temperature.
- Prepare the quantity of reagents needed for the test.
- Do not use components after the expiration date marked on their label.
10X Wash Buffer ⑤: is supplied as a 10X concentrated solution. Before the test, warm to room temperature and mix gently until crystals have completely dissolved. To prepare 1X Wash Buffer dilute the concentrated solution 10X ⑤ in deionized water. (e.g. dilute 50 mL of 10X Wash Buffer in deionized water to obtain a final volume of 500 mL). The diluted wash buffer is stable for 1 year when stored at 2-8 °C. Opened 10X Wash buffer is stable until its expiration date (see label on the bottle) when stored at 2-8°C.
Standard ②: the MMP-9 Standard should be reconstituted in 1 mL of distilled water at least 15 min prior the assay with occasional shaking. Harsh mixing and pipetting may be harmful for the integrity of the solution and should be avoided. The resulting concentration of MMP-9 in the stock solution is 180 ng/mL. Do not store the reconstituted vial. Sealed lyophilized vial is stable until its expiration date (see label on the vial) when stored at 2-8°C.
Proceed with the preparation of the 7 calibrators by serial dilutions with freshly prepared 1X Wash Buffer. The 180 ng/mL calibrator serves as the highest calibrator (stock solution). 1X Wash Buffer serves as the zero calibrator (0 ng/mL). The table below explains how to construct the calibration curve in duplicate.
Volume of standard |
Volume of 1X Wash Buffer |
Concentration |
50 µL of stock solution |
250 µL |
30 ng/mL |
150 µL of 30 ng/mL |
150 µL |
15 ng/mL |
150 µL of 15 ng/mL |
150 µL |
7.50 ng/mL |
150 µL of 7.50 ng/mL |
150 µL |
3.75 ng/mL |
150 µL of 3.75 ng/mL |
150 µL |
1.875 ng/mL |
150 µL of 1.875 ng/mL |
150 µL |
0.9375 ng/mL |
150 µL of 0.9375 ng/mL |
150 µL |
0.47 ng/mL |
Note: It is recommended to use a precision pipette and a careful technique to perform the dilutions in order to get precise results.
Mix each tube before the next transfer. Prepared calibrators are ready to use, do not further dilute them. Do not store diluted solutions.
Coated Microplate ①, TMB substrate ⑥ and Stop solution ⑦ are provided ready to use. Opened TMB substrate and Stop solution are stable until their expiration date (see label on bottles) when stored at 2-8°C. Be careful to return the unused strips of the plate to the aluminium zip-sealed bag with desiccant and seal. Remaining strips are stable 1 month when stored at 2-8°C and protected from moisture. Sealed components are stable until their expiration date (see label on the considered reagent) when stored at 2-8°C.
Detection antibody ③ is supplied as a 80X concentrated solution. To prepare the working solution of detection antibody, dilute the concentrated solution 80X ③ in 1X Wash Buffer. For example, dilute 75 µL of 80X concentrated Detection Antibody in 1X Wash Buffer to a final volume of 6 mL. Opened concentrated antibody is stable until its expiration date (see label on the bottle) when stored at 2-8°C. Do not store the diluted detection antibody.
Streptavidin-HRP ④ is supplied as a 100X concentrated solution. To prepare the working solution of Streptavidin-HRP, dilute the concentrated solution 100X ④ in 1X Wash Buffer. For example, dilute 60 µL of 100X concentrated Streptavidin-HRP in 1X Wash Buffer to a final volume of 6 mL. Opened concentrated Streptavidin-HRP is stable until its expiration date (see label on the bottle) when stored at 2-8°C. Do not store the diluted streptavidin-HRP.
Quality controls High ⑧ and Low ⑨ are provided as lyophilized. Reconstitute each vial with 1 mL of 1X Wash Buffer. Let it dissolve at least 15 min with occasional shaking. Harsh mixing and pipetting may be harmful for the integrity of the solution and should be avoided. The resulting concentration of MMP-9 in the QC High and QC Low solutions are respectively within [7.2-10.8] ng/mL and [1.9-2.8] ng/mL. Do not store the reconstituted vial. Otherwise, sealed lyophilized vials are stable until their expiration date (see label on the vial) when stored at 2-8°C.
Each testing laboratory should establish a quality control program to monitor the performance of the human MMP-9 ELISA test.
Assay procedure
Required time: 4 hours.
Bring all reagents and samples to room temperature before use. It is recommended that all standards and controls be assayed in duplicate.
- Prepare all reagents, working standards and samples as indicated in the previous section.
- Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
- Wash the wells once with wash buffer (250 µL per well).
- Pipet 50 µL of standards, quality controls and diluted samples into the appropriate wells. Cover the plate with provided sealer.
- Incubate the plate for 2 hours at room temperature (20-25°C), at 600 rpm on an orbital microplate shaker.
- Wash the wells 3 times with wash buffer (250 µL per well). After the last wash, invert the plate and blot it against clean paper towels.
- Add 50 µL of diluted detection antibody into each well. Cover the plate with provided sealer.
- Incubate the plate for 1 hour at room temperature (20-25°C), at 600 rpm on an orbital microplate shaker.
- Wash three times as in step 6.
- Add 50 µL of diluted streptavidin-HRP into each well. Cover the plate with provided sealer.
- Incubate the plate for 30 minutes at room temperature (20-25°C), at 600 rpm on an orbital microplate shaker.
- Wash three times as in step 6.
- Add 100 µL of TMB substrate to each well. Protect from light.
- Incubate the plate in the dark for 10 min, at room temperature (20-25°C).
- Stop the reaction by adding 50 µL of stop solution. The colour in wells changes from blue to yellow. Gently tap the plate to ensure correct mixing.
- Read the optical density of each well using a microplate reader set to 450 nm. The optical density should be read within 5 min after step 15.
If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Note: For manual washing, aspirate carefully the contents of the wells, avoiding to touch the bottom of the well with the tip, and pipet 250 µL wash buffer into each well. Repeat two times more. After final washing step, invert and tap the plate against paper towel. Make sure that wash buffer has been completely removed.
Note: If samples generate values higher than the highest standard, further dilute the samples and repeat the assay.
Note: High concentrations of MMP-9 are found in saliva. Use a face mask and gloves to protect kit reagents from contamination.
Calculation and Interpreatationof results
Calculate the average of duplicate readings, if applied, for each standard, control, and sample. The standard curve is constructed by plotting the absorbance (Y) of standards against the known concentration (X) of standards on a logarithmic scale, using a four-parameter algorithm. For sample analysis, raw absorbance values are interpolated and the concentrations are calculated. Results are reported as concentration of MMP-9 (ng/mL) in samples. The measured concentration in samples calculated from the standard curve must be multiplied by their respective dilution factor (DF).
Technical Advice
Symbol legends used in the IFU and labels of the kit
Thank you for choosing Firalis!
For further information, MSDS or Instructions in other languages, please contact us at contact@firalis.com