Mouse Cell Signalling ELISA Kits 2
Mouse Hepc (Hepcidin) CLIA Kit (MOES00365)
- SKU:
- MOES00365
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- Hepcidin Antimicrobial Peptide
- Reactivity:
- Mouse
- Sample Type:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| 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 Mouse Hepc in samples. No significant cross-reactivity or interference between Mouse Hepc 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 Mouse Hepc. 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 Mouse Hepc 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 Mouse Hepc, 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 Mouse Hepc. The concentration of Mouse Hepc in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | HAMP: Seems to act as a signaling molecule involved in the maintenance of iron homeostasis. Seems to be required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages. Defects in HAMP are the cause of hemochromatosis type 2B (HFE2B); also known as juvenile hemochromatosis (JH). HFE2B is a disorder of iron metabolism with excess deposition of iron in the tissues, bronze skin pigmentation, hepatic cirrhosis, arthropathy and diabetes. The most common symptoms of hemochromatosis type 2 at presentation are hypogonadism and cardiomyopathy. Belongs to the hepcidin family. |
| UniProt Protein Details: | Protein type:Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 7 B1|7 19. 27 cM Cellular Component: extracellular region; extracellular space; nucleus Molecular Function:copper ion binding; hormone activity Biological Process: cellular iron ion homeostasis; cellular response to extracellular stimulus; defense response to bacterium; defense response to fungus; iron ion homeostasis; JAK-STAT cascade; myeloid cell homeostasis; negative regulation of bone resorption; negative regulation of inflammatory response; negative regulation of transcription from RNA polymerase II promoter; positive regulation of macrophage activation; positive regulation of transcription from RNA polymerase II promoter |
| NCBI Summary: | This gene encodes hepcidin, an antimicrobial peptide and master hormonal regulator of systemic iron metabolism. The encoded preproprotein is synthesized in the hepatocytes where it undergoes proteolytic processing to generate disulfide-linked mature peptides that are secreted into the bloodstream. Mice lacking the encoded protein develop multivisceral iron overlaod, with sparing of the spleen macrophages. Certain mutations in the human ortholog of this gene cause hemochromatosis type 2B, also known as juvenile hemochromatosis. This gene is located adjacent to a related hepcidin gene on chromosome 7. [provided by RefSeq, Aug 2016] |
| UniProt Code: | Q9EQ21 |
| NCBI GenInfo Identifier: | 14211542 |
| NCBI Gene ID: | 84506 |
| NCBI Accession: | NP_115930. 1 |
| UniProt Related Accession: | Q9EQ21 |
| Molecular Weight: | |
| NCBI Full Name: | hepcidin preproprotein |
| NCBI Synonym Full Names: | hepcidin antimicrobial peptide |
| NCBI Official Symbol: | Hamp |
| NCBI Official Synonym Symbols: | Hepc; Hamp1; Hepc1 |
| NCBI Protein Information: | hepcidin |
| UniProt Protein Name: | Hepcidin |
| Protein Family: | Heparin-sulfate lyase |
| UniProt Gene Name: | Hamp |
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 | 53817 57625 | 55721 | 55693 |
| 1000 | 22927 25021 | 23974 | 23946 |
| 500 | 11945 10119 | 11032 | 11004 |
| 250 | 5058 5530 | 5294 | 5266 |
| 125 | 2682 2534 | 2608 | 2580 |
| 62.5 | 1371 1251 | 1311 | 1283 |
| 31.25 | 663 685 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse Hepc were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse Hepc 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) | 110.04 | 302.39 | 988.50 | 100.07 | 298.43 | 904.19 |
| Standard deviation | 10.27 | 26.19 | 79.48 | 10.59 | 34.32 | 81.20 |
| C V (%) | 9.33 | 8.66 | 8.04 | 10.58 | 11.50 | 8.98 |
Recovery
The recovery of Mouse Hepc 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) | 90-101 | 95 |
| EDTA plasma (n=5) | 86-99 | 92 |
| Cell culture media (n=5) | 83-96 | 90 |
Linearity
Samples were spiked with high concentrations of Mouse Hepc 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 (%) | 90-102 | 92-108 | 97-114 |
| Average (%) | 96 | 99 | 104 | |
| 1:4 | Range (%) | 103-117 | 97-112 | 88-100 |
| Average (%) | 109 | 104 | 95 | |
| 1:8 | Range (%) | 90-104 | 89-100 | 90-102 |
| Average (%) | 96 | 94 | 96 | |
| 1:16 | Range (%) | 92-104 | 90-103 | 93-106 |
| Average (%) | 97 | 95 | 99 |
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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