Human TIMP1 / EPA / CLGI ELISA Pair Set

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Target Name (Antigen)
  • TIMP-1
  • TIMP1
  • DKFZp468A0912
  • CLGI
  • EPA
  • EPO
  • HCI
  • TIMP
  • Timp
  • Clgi
  • TIMP metallopeptidase inhibitor 1
  • tissue inhibitor of metalloproteinase 1
  • TIMP1
  • Timp1
  • LOC100009047
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Purpose The ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utlizes monoclonal antibody specific for TIMP1 (EPA / CLGI) coated on a 96-well plate. Standards and samples are addedto the wells, and any TIMP1 (EPA / CLGI) present binds to the immobilized antibody. The wells are washed and abiotinylated rabbit anti- TIMP1 polyclonal antibody is then added, producing an antibody-antigen-antibody "sandwich".To produces color in proportion to the amount of TIMP1 (EPA / CLGI) present in the sample strepavidin-HRP andTMB substrate solution are loaded. The absorbances of the microwell are read at 450 nm.
Sensitivity The minimum detectable dose of human TIMP1 ( EPA / CLGI ) was determined to be approximately 3.125 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Characteristics Pair Set
Components Capture Antibody - 1.0 mg/mL of mouse anti-TIMP1 monoclonal antibody. Dilute to a workingconcentration of 2.0 μg/mL in CBS before coating.
Detection Antibody - 0.5 mg/mL of biotinylated rabbit anti-TIMP1 polyclonal antibody. Dilute to aworking concentration of 1.0 μg/mL in detection antibody dilution buffer before use.
Standard - Each vial contains 4.8 ng of recombinant TIMP1. Reconstitute standard powder with1mL detection antibody dilution buffer. After reconstitution, store at -20 °C to -70 °C in a manualdefrost freezer. A seven-point standard curve using 2-fold serial dilutions in sample dilution buffer,and a high standard of 200 pg/mL is recommended.
Streptavidin-HRP - 50 μL of streptavidin conjugated to horseradish-peroxidase. 1:2000 Dilution indetection antibody dilution buffer before use.
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name TIMP1
Background Metalloproteinase inhibitor 1, also known as Tissue inhibitor of metalloproteinases 1, Fibroblast collagenaseinhibitor, Collagenase inhibitor, CLGI and TIMP1, is a secreted protein which belongs to the proteaseinhibitor I35 (TIMP) family. TIMP1 complexes with metalloproteinases (such as collagenases) andirreversibly inactivates them by binding to their catalytic zinc cofactor. TIMP1 also mediates erythropoiesis. Unlike IL-3, it is species-specific, stimulating the growth and differentiation of only human and murineerythroid progenitors. TIMP1 is known to act on MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13 and MMP-16. TIMP1 does not act on MMP-14. Excessive TIMP1 wasdeleterious to ovulation and embryo development. Novel TIMP1 modulating therapies may be developed toalleviate infertility in women with endometriosis. Tissue inhibitors of metalloproteinases (TIMP) family are natural inhibitors of the matrix metalloproteinases(MMPs) , the zinc enzymes involved in extracellular matrix maintenance and remodeling. The TIMP familyencompasses four members ( TIMP1, TIMP2, TIMP3, TIMP4) , and the inducible form TIMP1 is aglycoprotein expressed by a variety of cell types. It inhibits most MMPs except membrane-type MMPsubfamily by forming non-covalent binary complex, but also interacts with the hemopexin-like (PEX) domainof pro MMP-9. TIMP1 has been identified as a multifunctional molecule with divergent functions. It exhibitskeratinocyte and fibroblast growth factor-like activity and acts as a cell survival factor, and additionallyparticipates in tumor metastasis, angiogenesis, inflammatory responses, as well as CNS homeostasis. Thus, TIMP1 overexpression may serve to help identify patients with particularly aggressive disease for adjuvanttreatments.
Research Area Proteolysis / Ubiquitin, Angiogenesis, Extracellular Matrix, Matrix Metalloproteinases, Cancer, Inflammation
Application Notes Optimal working dilution should be determined by the investigator.

The human TIMP1 ( EPA / CLGI ) ELISA Pair Set is for the quantitative determination of human TIMP1.This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.

Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
    4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 100 μL of Streptavidin-HRP to each well. Incubate for 1 hour at room temperature.
    6. Repeat the aspiration/wash as in step 2 of plate preparation.
    7. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    8. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

Restrictions For Research Use only
Format Lyophilized
Precaution of Use The Stop Solution suggested for use with this Pair Set is an acid solution. Wear eye, hand, face, andclothing protection when using this material.
Handling Advice Avoid repeated freeze-thaw cycles.
Storage 4 °C/-20 °C/-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Detection Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Streptavidin-HRP: Store at 4°C and protect it from prolonged exposure to light. DO NOT FREEZE! It is stable for up to 6 months from date of receipt.
Expiry Date 6 months
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