Human Cell Biology ELISA Kits 3
Human MIP-3 alpha (Macrophage Inflammatory Protein 3 Alpha) CLIA Kit (HUES00027)
- SKU:
- HUES00027
- 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:
- 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: | 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 Human MIP-3 alpha in samples. No significant cross-reactivity or interference between Human 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 Human 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 Human 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 Human 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 Human MIP-3 alpha. The concentration of Human MIP-3 alpha in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | CCL20: Chemotactic factor that attracts lymphocytes and, slightly, neutrophils, but not monocytes. Inhibits proliferation of myeloid progenitors in colony formation assays. May be involved in formation and function of the mucosal lymphoid tissues by attracting lymphocytes and dendritic cells towards epithelial cells. C-terminal processed forms have been shown to be equally chemotactically active for leukocytes. Possesses antibacterial activity E. coli ATCC 25922 and S. aureus ATCC 29213. Belongs to the intercrine beta (chemokine CC) family. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Chemokine; Secreted; Secreted, signal peptide; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 2q36. 3 Cellular Component: extracellular space; extracellular region Molecular Function:protein binding; chemokine activity Biological Process: cell-cell signaling; wound healing; defense response to bacterium; immune response; chemotaxis; inflammatory response; signal transduction; positive regulation of interleukin-1 alpha biosynthetic process; positive regulation of nitric-oxide synthase biosynthetic process |
| NCBI Summary: | This antimicrobial gene belongs to the subfamily of small cytokine CC genes. Cytokines are a family of secreted proteins involved in immunoregulatory and inflammatory processes. The CC cytokines are proteins characterized by two adjacent cysteines. The protein encoded by this gene displays chemotactic activity for lymphocytes and can repress proliferation of myeloid progenitors. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2014] |
| UniProt Code: | P78556 |
| NCBI GenInfo Identifier: | 2829668 |
| NCBI Gene ID: | 6364 |
| NCBI Accession: | P78556. 1 |
| UniProt Secondary Accession: | P78556,Q53S51, Q99664, |
| UniProt Related Accession: | P78556 |
| Molecular Weight: | 10,691 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: | CKb4; LARC; ST38; MIP3A; Exodus; MIP-3a; SCYA20; MIP-3-alpha |
| NCBI Protein Information: | C-C motif chemokine 20; CC chemokine LARC; beta chemokine exodus-1; small-inducible cytokine A20; macrophage inflammatory protein 3 alpha; liver and activation-regulated chemokine; small inducible cytokine subfamily A (Cys-Cys), member 20 |
| UniProt Protein Name: | C-C motif chemokine 20 |
| UniProt Synonym Protein Names: | Beta-chemokine exodus-1; CC chemokine LARC; Liver and activation-regulated chemokine; Macrophage inflammatory protein 3 alpha; MIP-3-alpha; Small-inducible cytokine A20Cleaved into the following 3 chains:CCL20(1-67); CCL20(1-64); CCL20(2-70) |
| Protein Family: | C-C motif chemokine |
| UniProt Gene Name: | CCL20 |
| UniProt Entry Name: | CCL20_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 |
| 500 | 50838 52236 | 51537 | 51508 |
| 250 | 20795 21201 | 20998 | 20969 |
| 125 | 9846 8946 | 9396 | 9367 |
| 62.5 | 4349 4675 | 4512 | 4483 |
| 31.25 | 2361 2237 | 2299 | 2270 |
| 15.63 | 1339 1161 | 1250 | 1221 |
| 7.81 | 731 749 | 740 | 711 |
| 0 | 28 30 | 29 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human 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 Human 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) | 24.32 | 57.87 | 223.32 | 24.56 | 57.95 | 222.84 |
| Standard deviation | 2.15 | 4.35 | 14.14 | 2.95 | 5.20 | 16.31 |
| C V (%) | 8.84 | 7.52 | 6.33 | 12.01 | 8.97 | 7.32 |
Recovery
The recovery of Human 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) | 87-100 | 93 |
| EDTA plasma (n=5) | 85-96 | 91 |
| Cell culture media (n=5) | 89-103 | 95 |
Linearity
Samples were spiked with high concentrations of Human 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 (%) | 92-109 | 101-115 | 89-102 |
| Average (%) | 99 | 108 | 97 | |
| 1:4 | Range (%) | 93-108 | 90-103 | 95-107 |
| Average (%) | 100 | 96 | 101 | |
| 1:8 | Range (%) | 98-114 | 93-111 | 95-108 |
| Average (%) | 105 | 101 | 101 | |
| 1:16 | Range (%) | 84-98 | 92-104 | 99-111 |
| Average (%) | 90 | 98 | 105 |
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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