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  • 產(chǎn)品名稱：TIMP1 ELISA Kit(TIMPMetallopeptidaseInhibitor1)

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TIMP1 ELISA Kit(TIMPMetallopeptidaseInhibitor1)
詳情介紹：
Purpose This immunoassay kit allows for the in vitro quantitative determination of human tissue inhibitors of metalloproteinase 1,TIMP-1 concentrations in cell culture supernates, serum, plasma and other biological fluids.
Sample Type Cell Culture Supernatant, Serum, Plasma, Biological Fluids
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay recognizes recombinant and natural human TIMP-1.
Cross-Reactivity (Details) No significant cross-reactivity or interference was observed.
Sensitivity < 0.078 ng/mL
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
Characteristics Homo sapiens,Human,Metalloproteinase inhibitor 1,Erythroid-potentiating activity,EPA,Fibroblast collagenase inhibitor,Collagenase inhibitor,Tissue inhibitor of metalloproteinases 1,TIMP-1,TIMP1,CLGI,
Components Reagent (Quantity): Assay plate (1×20ml), Standard (2), Sample Diluent (1×20ml), Assay Diluent A (1×10ml), Assay Diluent B (1×10ml), Detection Reagent A (1×120 μl), Detection Reagent B (1×120 μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instruction (1)
Material not included Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.
Alternative Name TIMP1 (TIMP1 ELISA Kit Abstract)
Background Matrix MetalloProteinases (MMPs) are zinc-dependent endopeptidases that catalyze degradation of extracellular matrix proteins, thereby controlling such processes as development, tissue remodeling, wound healing and tumor metastasis. The activity of MMPs is controlled by regulation of expression and secretion, by proteolytic activation of pro-enzymes and by the Tissue Inhibitors of MetalloProteinases (TIMPs) . TIMPs form 1:1, non-covalent complexes with MMPs, blocking access of substrates to the MMP catalytic site. TIMPs are highly specific for MMPs in general but not for any particular MMP. Functional specificity is conferred by other characteristics. TIMP-1 is an inducible protein and TIMP-2 is a constitutive protein and both are soluble and widely distributed. TIMP-3 is restricted to the extracellular matrix and TIMP-4 is largely restricted to cardiac tissue. TIMP-1 is a 184 amino acid residue glycosylated protein, though glycosylation is not necessary for activity . It has 12 cysteines (conserved among all TIMPs) that form disulfide bonds in a pattern that gives distinct N- and C-terminal domains . The N-terminal domain contains sites that bind to the MMP substrate- . Binding of TIMP-1 does not leave a peptide bond in position for proteolysis and is not cleaved . The TIMP/MMP complex can dissociate to yield enzyme and active TIMP-1. The C-terminal domain binds to an external site on MMPs, increasing overall affinity .TIMP-1 binds with high affinity to the inactive pro-MMP-9, forming a complex in which TIMP-1 retains its ability to inhibit the activity of another active MMP via its N-terminal domain . TIMP-1 is widely synthesized by many cells and tissues. Transcription of the TIMP-1 gene is induced by pro-inflammatory cytokines (IL-1, IL-6, OSM, LIF and TNF- alpha ), TGF-1 and phorbol esters . Many physiological functions of TIMP-1 are closely tied to the functions of MMPs, and an improper balance of MMP and TIMP production correlates with pathological conditions such as arthritis, tumor growth and metastasis. On the other hand, TIMP-1 was independently discovered as an erythroid potentiating activity , an activity that 2 appears to be functionally distinct from MMP inhibitory activity . TIMP-1 binds to certain cell lines and is translocated to the nucleus .It inhibits apoptosis in B-cells ,further suggesting that TIMP-1 independently functions in multiple ways to support survival and growth of cells in contrast to its function of inhibition MMP.
Sample Volume 100 μL
Plate Pre-coated
Protocol The microtiter plate provided in this kit has been pre-coated with an antibody specific to TIMP-1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for TIMP-1 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain TIMP-1, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of TIMP-1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Reagent Preparation

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 10 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (10 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). ng/mL 10 5 2.5 1.25 0.625 0.312 0.156 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A 4 and B (1:100), respectively.
Sample Collection Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20 C or -80 C . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8 C within 30 minutes of collection. Store samples at 3 -20 C or -80 C . Avoid repeated freeze-thaw cycles. Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles. Note: Serum, plasma, and cell culture supernatant samples to be used within 7 days may be stored at 2-8 C , otherwise samples must stored at -20 C ( ≤ 1 months) or -80 C ( ≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. Sample preparation - Serum/plasma samples require a 20-30 fold dilution. Sample should be diluted by 0.1 M PBS ( PH=7.0-7.2 ) .
Assay Procedure

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
1. Add 100 μ l of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 C .
2. Remove the liquid of each well, don ’ t wash.
3. Add 100 μ l of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 C . Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μ l) using a squirt bottle, multi-channel pipette, manifold dispenser 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.
5. Add 100 μ l of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 C .
6. Repeat the aspiration/wash five times as in step
4. 7. Add 90 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37 C . Protect from light.
8. Add 50 μ l of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
Important Note:
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by 5 pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.
Calculation of Results

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the TIMP-1 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Restrictions For Research Use only
Handling Advice 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Storage 4 °C/-20 °C
Storage Comment The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.