Human TIMP-1 (Tissue Inhibitors of Metalloproteinase 1) CLIA Kit (HUES00158)

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HUES00158
€599

Description

ELISA Kit Technical ManualMSDS

Human TIMP-1 (Tissue Inhibitors of Metalloproteinase 1) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human TIMP-1 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human TIMP-1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human TIMP-1, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human TIMP-1. The concentration of Human TIMP-1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human TIMP-1 in samples. No significant cross-reactivity or interference between Human TIMP-1 and analogues was observed.

Typical Data

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
RLU Average Corrected
200055606
55836
5572155693
100023151
24797
2397423946
50011223
10841
1103211004
2504866
5722
52945266
1252730
2486
26082580
62.51363
1259
13111283
31.25672
676
674646
028
28
28--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TIMP-1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TIMP-1 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)103.06280.80875.1592.92285.71946.10
Standard deviation8.2733.3955.059.2328.8098.96
C V (%)8.0211.896.299.9310.0810.46

Recovery

The recovery of Human TIMP-1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)90-10295
EDTA plasma (n=5)96-114104
Cell culture media (n=5)87-10192

Linearity

Samples were spiked with high concentrations of Human TIMP-1 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

    Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)88-10294-11087-102
Average (%)9410193
1:4Range (%)92-10694-10596-110
Average (%)9899103
1:8Range (%)96-107102-11590-101
Average (%)10210996
1:16Range (%)97-112101-11993-108
Average (%)103109101

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human TIMP-1 (Tissue Inhibitors of Metalloproteinase 1) CLIA Kit (HUES00158) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Human TIMP-1 (Tissue Inhibitors of Metalloproteinase 1) CLIA Kit (HUES00158) Protein Information

UniProt Protein Function:TIMP1: Complexes with metalloproteinases (such as collagenases) and irreversibly inactivates them by binding to their catalytic zinc cofactor. Also mediates erythropoiesis in vitro; but, unlike IL-3, it is species-specific, stimulating the growth and differentiation of only human and murine erythroid progenitors. 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. Does not act on MMP-14. Belongs to the protease inhibitor I35 (TIMP) family.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Secreted, signal peptide; Secreted

Chromosomal Location of Human Ortholog: Xp11.3-p11.23

Cellular Component: extracellular space; proteinaceous extracellular matrix; extracellular region; basement membrane

Molecular Function:metalloendopeptidase inhibitor activity; protein binding; growth factor activity; protease binding; metal ion binding; cytokine activity

Biological Process: response to peptide hormone stimulus; platelet activation; extracellular matrix organization and biogenesis; negative regulation of metalloenzyme activity; response to hormone stimulus; negative regulation of membrane protein ectodomain proteolysis; extracellular matrix disassembly; platelet degranulation; response to cytokine stimulus; positive regulation of cell proliferation; blood coagulation; aging; negative regulation of apoptosis

NCBI Summary:This gene belongs to the TIMP gene family. The proteins encoded by this gene family are natural inhibitors of the matrix metalloproteinases (MMPs), a group of peptidases involved in degradation of the extracellular matrix. In addition to its inhibitory role against most of the known MMPs, the encoded protein is able to promote cell proliferation in a wide range of cell types, and may also have an anti-apoptotic function. Transcription of this gene is highly inducible in response to many cytokines and hormones. In addition, the expression from some but not all inactive X chromosomes suggests that this gene inactivation is polymorphic in human females. This gene is located within intron 6 of the synapsin I gene and is transcribed in the opposite direction. [provided by RefSeq, Jul 2008]
UniProt Code:P01033
NCBI GenInfo Identifier:135850
NCBI Gene ID:7076
NCBI Accession:P01033.1
UniProt Secondary Accession:P01033,Q14252, Q9UCU1,
UniProt Related Accession:P01033
Molecular Weight:23,171 Da
NCBI Full Name:Metalloproteinase inhibitor 1
NCBI Synonym Full Names:TIMP metallopeptidase inhibitor 1
NCBI Official Symbol:TIMP1  
NCBI Official Synonym Symbols:EPA; EPO; HCI; CLGI; TIMP  
NCBI Protein Information:metalloproteinase inhibitor 1; TIMP-1; collagenase inhibitor; erythroid potentiating activity; erythroid-potentiating activity; fibroblast collagenase inhibitor; tissue inhibitor of metalloproteinases 1
UniProt Protein Name:Metalloproteinase inhibitor 1
UniProt Synonym Protein Names:Erythroid-potentiating activity; EPA; Fibroblast collagenase inhibitor; Collagenase inhibitor; Tissue inhibitor of metalloproteinases 1; TIMP-1
Protein Family:Metalloproteinase inhibitor
UniProt Gene Name:TIMP1  
UniProt Entry Name:TIMP1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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