Human Cell Biology ELISA Kits 5
Human MMP-8 (Matrix Metalloproteinase 8) CLIA Kit (HUES00827)
- SKU:
- HUES00827
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- MMP8, CLG1, HNC, PMNL-CL, Neutrophil Collagenase
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human MMP-8 in samples. No significant cross-reactivity or interference between Human MMP-8 and analogues was observed. |
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 MMP-8. 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 MMP-8 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 MMP-8, 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 MMP-8. The concentration of Human MMP-8 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | MMP8: Can degrade fibrillar type I, II, and III collagens. Belongs to the peptidase M10A family. |
| UniProt Protein Details: | Protein type:Protease; EC 3. 4. 24. 34; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 11q22. 3 Cellular Component: extracellular space; proteinaceous extracellular matrix; extracellular region Molecular Function:zinc ion binding; serine-type endopeptidase activity; metalloendopeptidase activity; calcium ion binding Biological Process: extracellular matrix disassembly; collagen catabolic process; extracellular matrix organization and biogenesis; proteolysis |
| NCBI Summary: | Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. However, the enzyme encoded by this gene is stored in secondary granules within neutrophils and is activated by autolytic cleavage. Its function is degradation of type I, II and III collagens. The gene is part of a cluster of MMP genes which localize to chromosome 11q22. 3. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P22894 |
| NCBI GenInfo Identifier: | 116862 |
| NCBI Gene ID: | 4317 |
| NCBI Accession: | P22894. 1 |
| UniProt Secondary Accession: | P22894,Q45F99, |
| UniProt Related Accession: | P22894 |
| Molecular Weight: | 53,412 Da |
| NCBI Full Name: | Neutrophil collagenase |
| NCBI Synonym Full Names: | matrix metallopeptidase 8 (neutrophil collagenase) |
| NCBI Official Symbol: | MMP8 |
| NCBI Official Synonym Symbols: | HNC; CLG1; MMP-8; PMNL-CL |
| NCBI Protein Information: | neutrophil collagenase; PMNL collagenase; matrix metalloproteinase-8; matrix metalloproteinase 8 (neutrophil collagenase) |
| UniProt Protein Name: | Neutrophil collagenase |
| UniProt Synonym Protein Names: | Matrix metalloproteinase-8; MMP-8; PMNL collagenase; PMNL-CL |
| Protein Family: | Neutrophil collagenase |
| UniProt Gene Name: | MMP8 |
| UniProt Entry Name: | MMP8_HUMAN |
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 |
| 2000 | 53074 58368 | 55721 | 55693 |
| 1000 | 23344 24604 | 23974 | 23946 |
| 500 | 11211 10853 | 11032 | 11004 |
| 250 | 4842 5746 | 5294 | 5266 |
| 125 | 2792 2424 | 2608 | 2580 |
| 62.5 | 1366 1256 | 1311 | 1283 |
| 31.25 | 669 679 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MMP-8 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MMP-8 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 98.45 | 171.13 | 670.93 | 102.85 | 179.85 | 713.09 |
| Standard deviation | 8.59 | 12.49 | 53.41 | 10.23 | 13.06 | 64.96 |
| C V (%) | 8.73 | 7.30 | 7.96 | 9.95 | 7.26 | 9.11 |
Recovery
The recovery of Human MMP-8 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) | 92-104 | 98 |
| EDTA plasma (n=5) | 94-105 | 100 |
| Cell culture media (n=5) | 92-109 | 99 |
Linearity
Samples were spiked with high concentrations of Human MMP-8 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) | ||
| 1:2 | Range (%) | 94-107 | 95-108 | 94-110 |
| Average (%) | 101 | 102 | 100 | |
| 1:4 | Range (%) | 98-116 | 94-107 | 84-96 |
| Average (%) | 106 | 101 | 90 | |
| 1:8 | Range (%) | 94-104 | 99-113 | 99-115 |
| Average (%) | 99 | 105 | 107 | |
| 1:16 | Range (%) | 89-101 | 86-97 | 95-113 |
| Average (%) | 94 | 91 | 103 |
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 ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent A | 1 vial, 5 mL | 4°C (shading light) |
| Substrate Reagent B | 1 vial, 5 mL | 4°C (shading light) |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Determine the RLU value of each well immediately.
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