Rat Signaling ELISA Kits 3
Rat MIP-3 alpha (Macrophage Inflammatory Protein 3 Alpha) CLIA Kit (RTES00372)
- SKU:
- RTES00372
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 4.69pg/mL
- Range:
- 7.81-500pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- CCL20, CKb4, LARC, MIP3A, SCYA20, ST38
- Reactivity:
- Rat
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| Detection method: | Chemiluminescence |
| Detection range: | 7.81-500 pg/mL |
| Sensitivity: | 4.69 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Rat MIP-3 alpha in samples. No significant cross-reactivity or interference between Rat MIP-3 alpha 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 Rat MIP-3 alpha. 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 Rat MIP-3 alpha 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 Rat MIP-3 alpha, 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 Rat MIP-3 alpha. The concentration of Rat MIP-3 alpha in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | Acts as a ligand for C-C chemokine receptor CCR6. Signals through binding and activation of CCR6 and induces a strong chemotactic response and mobilization of intracellular calcium ions. The ligand-receptor pair CCL20-CCR6 is responsible for the chemotaxis of dendritic cells (DC), effector/memory T-cells and B-cells and plays an important role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and autoimmune diseases. CCL20 acts as a chemotactic factor that attracts lymphocytes and, slightly, neutrophils, but not monocytes. Involved in the recruitment of both the proinflammatory IL17 producing helper T-cells (Th17) and the regulatory T-cells (Treg) to sites of inflammation. Required for optimal migration of thymic natural regulatory T cells (nTregs) and DN1 early thymocyte progenitor cells. Positively regulates sperm motility and chemotaxis via its binding to CCR6 which triggers Ca2+ mobilization in the sperm which is important for its motility. May be involved in formation and function of the mucosal lymphoid tissues by attracting lymphocytes and dendritic cells towards epithelial cells. |
| NCBI Summary: | chemokine upregulated in ischemic brain tissue [RGD, Feb 2006] |
| UniProt Code: | P97884 |
| NCBI GenInfo Identifier: | 6094385 |
| NCBI Gene ID: | 29538 |
| NCBI Accession: | P97884. 1 |
| UniProt Related Accession: | P97884 |
| Molecular Weight: | 10,875 Da |
| NCBI Full Name: | C-C motif chemokine 20 |
| NCBI Synonym Full Names: | chemokine (C-C motif) ligand 20 |
| NCBI Official Symbol: | Ccl20 |
| NCBI Official Synonym Symbols: | ST38; Scya20 |
| NCBI Protein Information: | C-C motif chemokine 20; MIP-3-alpha; CC chemokine LARC; CC chemokine ST38; beta chemokine exodus-1; beta-chemokine exodus-1; small-inducible cytokine A20; small inducible cytokine subfamily A20; macrophage inflammatory protein 3 alpha; liver and activation-regulated chemokine |
| UniProt Protein Name: | C-C motif chemokine 20 |
| UniProt Synonym Protein Names: | Beta-chemokine exodus-1; CC chemokine LARC; CC chemokine ST38; Liver and activation-regulated chemokine; Macrophage inflammatory protein 3 alpha; MIP-3-alpha; Small-inducible cytokine A20 |
| Protein Family: | C-C motif chemokine |
| UniProt Gene Name: | Ccl20 |
| UniProt Entry Name: | CCL20_RAT |
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 |
| 500 | 50759 52315 | 51537 | 51508 |
| 250 | 19625 22371 | 20998 | 20969 |
| 125 | 9892 8900 | 9396 | 9367 |
| 62.5 | 4397 4627 | 4512 | 4483 |
| 31.25 | 2442 2156 | 2299 | 2270 |
| 15.63 | 1345 1155 | 1250 | 1221 |
| 7.81 | 692 788 | 740 | 711 |
| 0 | 28 30 | 29 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat MIP-3 alpha were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat MIP-3 alpha 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) | 24.91 | 77.49 | 245.73 | 23.74 | 82.08 | 242.11 |
| Standard deviation | 3.04 | 5.51 | 20.69 | 2.92 | 9.24 | 22.81 |
| C V (%) | 12.20 | 7.11 | 8.42 | 12.30 | 11.26 | 9.42 |
Recovery
The recovery of Rat MIP-3 alpha 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) | 95-108 | 102 |
| EDTA plasma (n=5) | 98-113 | 106 |
| Cell culture media (n=5) | 97-110 | 103 |
Linearity
Samples were spiked with high concentrations of Rat MIP-3 alpha 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 (%) | 88-105 | 90-101 | 99-117 |
| Average (%) | 96 | 95 | 107 | |
| 1:4 | Range (%) | 96-107 | 96-111 | 86-96 |
| Average (%) | 102 | 103 | 91 | |
| 1:8 | Range (%) | 99-110 | 97-114 | 87-99 |
| Average (%) | 104 | 105 | 94 | |
| 1:16 | Range (%) | 100-118 | 86-97 | 89-102 |
| Average (%) | 108 | 91 | 94 |
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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