Matrix Metallopeptidase 1 (Interstitial Collagenase) (MMP1) ELISA Kit

Antigen: Matrix Metallopeptidase 1 (Interstitial Collagenase) (MMP1)

Synonyms: CLG, CLGN, Dm1-MMP, l(2)k04809, mmp1, DmelCG4859, CG4859, MMP1, Mmp1a, Mmp1

Reactivity: Human

Application: ELISA

Catalog no.: ABIN625053

Additional Information

Characteristics: Human MMP-1 ELISA Kit For Serum, Plasma, Cell Culture Supernatant and Urine

Alternative name: MMP-1

Sample Type: Serum, Plasma, Cell Culture Supernatant, Urine

Description: The Human MMP-1 ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human MMP-1 pro and active forms in serum, plasma (using heparin as an anticoagulant, EDTA and Citrate are not recommended ), cell culture supernatants and urine. This assay employs an antibody specific for human MMP-1 coated on a 96-well plate. Standards and samples are pipetted into the wells and MMP-1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human MMP-1 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of MMP-1 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Specificity: This ELISA kit shows no cross-reactivity with any of the cytokines tested (e.g., human Angiogenin, BDNF, BLC, ENA-78, FGF-4, IL-1 alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, IL-309, IP-10, G-CSF, GM-CSF, IFN- gamma, Leptin (OB), MCP-1, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1delta, MMP-2, - 3, -9, -10, PARC, RANTES, SCF, TARC, TGF- beta, TIMP-1, TIMP-2, TNF-alpha, TNF- beta, TPO, VEGF).

Sensitivity: The minimum detectable dose of MMP-1 is typically less than 8 pg/ml.

Application Details

Reagent Preparation: 1. Bring all reagents and samples to room temperature (18 - 25°C) before use. 2. Sample dilution: If your samples need to be diluted, Assay Diluent (Item E) is used for dilution of serum/plasma/culture supernatants/urine. 3. Assay Diluent (Item E) should be diluted 5-fold with deionized or distilled water before use. 4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µl 1x Assay Diluent (Item E) into Item C vial to prepare a 0.1 µg/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 120 µl MMP-1 standard from the vial of item C, into a tube with 546.7 µl 1x Assay Diluent Buffer (for serum/plasma/cell culture medium/urine) to prepare a 18000 pg/ml stock standard solution. Pipette 400 µl 1x Assay Diluent into each tube. Use the stock standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. Gently vortex to mix. 1x Assay Diluent serves as the zero standard (0 pg/ml). 5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer. 6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µl of 1x Assay Diluent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4°C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure. 7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 22,000-fold with 1x Assay Diluent. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 2 µl of HRP-Streptavidin concentrate into a tube with 198.0 µl 1x Assay Diluent to prepare a 100-fold diluted HRP-Streptavidin solution (do not store the diluted solution for next day use). Mix through and then pipette 50 µl of prepared 100-fold diluted solution into a tube with 11 ml 1x Assay Diluent to prepare a final 22,000 fold diluted HRP-Streptavidin solution.

Assay Procedure: 1. Bring all reagents and samples to room temperature (18 - 25°C) before use. It is recommended that all standards and samples be run at least in duplicate. 2. Add 100 µl of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4°C with gentle shaking. 3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 4. Add 100 µl of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking. 5. Discard the solution. Repeat the wash as in step 3. 6. Add 100 µl of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking. 7. Discard the solution. Repeat the wash as in step 3. 8. Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking. 9. Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calculation of Results: Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.

Components: 1. MMP-1 Microplate (Item A): 96 wells (12 strips x 8 wells) coated with anti-human MMP-1. 2. Wash Buffer Concentrate (20x) (Item B): 25 ml of 20x concentrated solution 3. Standards (Item C): 2 vials, recombinant human MMP-1. 4. Assay Diluent (Item E): 15 ml of 5x concentrated buffered. For Standard/Sample (serum/plasma/cell culture medium/urine) diluent. 5. Detection Antibody MMP-1 (Item F): 2 vial of biotinylated anti-human MMP-1 (each vial is enough to assay half microplate).. 6. HRP-Streptavidin Concentrate (Item G): 8 µl 22,000x concentrated HRP-conjugated streptavidin. 7. TMB One-Step Substrate Reagent (Item H): 12 ml of 3,3™,5,5™-tetramethylbenzidine (TMB) in buffered solution. 8. Stop Solution (Item I): 8 ml of 2 M sulfuric acid.

Material not included: 1. Microplate reader capable of measuring absorbance at 450 nm. 2. Precision pipettes to deliver 2 µl to 1 ml volumes. 3. Adjustable 1-25 ml pipettes for reagent preparation. 4. 100 ml and 1 liter graduated cylinders. 5. Absorbent paper. 6. Distilled or deionized water. 7. Log-log graph paper or computer and software for ELISA data analysis. 8. Tubes to prepare standard or sample dilutions.

Storage: May be stored for up to 6 months at 2° to 8°C from the date of shipment. Standard (recombinant protein) should be stored at -20°C or -80°C (recommended at -80°C) after reconstitution. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge. Note: the kit can be used within one year if the whole kit is stored at -20°C . Avoid repeated freeze-thaw cycles.

Restrictions: For Research Use only

Publications

Publications: Chen, Wong, Tam et al.: "Activation of human fibroblast-like synoviocytes by uric acid crystals in rheumatoid arthritis." in: Cellular & molecular immunology, 2011 (PubMed).

Kraus, Winter, Jepsen et al.: "Interactions of Adiponectin and Lipopolysaccharide from Porphyromonas gingivalis on Human Oral Epithelial Cells." in: PLoS ONE, Vol. 7, Issue 2, pp. e30716, 2012 (PubMed). Further details: The concentrations of IL1?, IL8 and MMP1 in culture supernatants were analyzed at 24 h and 72 h.