Description
Human MIP-5 (Macrophage Inflammatory Protein 5) ELISA Kit
The Human MIP-5 (Macrophage Inflammatory Protein 5) ELISA Kit is a powerful tool for the precise measurement of MIP-5 levels in human serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for various research purposes.MIP-5, also known as CCL15, is a chemokine that plays a key role in immune responses and inflammation. It is involved in recruiting and activating immune cells, particularly monocytes and macrophages, to sites of inflammation.
With its crucial role in inflammatory processes, MIP-5 serves as a valuable biomarker for studying inflammatory conditions and potentially developing therapeutic interventions.By utilizing the Human MIP-5 ELISA Kit, researchers can gain valuable insights into the role of MIP-5 in various diseases, such as autoimmune disorders, infectious diseases, and inflammatory conditions. With its high sensitivity and reliability, this kit is an essential tool for advancing research in the field of immunology and inflammation.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.16-10 ng/mL |
| Sensitivity: | 0.10 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human MIP-5 in samples. No significant cross-reactivity or interference between Human MIP-5 and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human MIP-5. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human MIP-5 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-5, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human MIP-5. The concentration of Human MIP-5 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CCL15: Chemotactic factor that attracts T-cells and monocytes, but not neutrophils, eosinophils, or B-cells. Acts mainly via CC chemokine receptor CCR1. Also binds to CCR3. CCL15(22-92), CCL15(25-92) and CCL15(29-92) are more potent chemoattractants than the small-inducible cytokine A15. Belongs to the intercrine beta (chemokine CC) family. |
| UniProt Protein Details: | Protein type:Secreted; Motility/polarity/chemotaxis; Secreted, signal peptide Chromosomal Location of Human Ortholog: 17q12 Cellular Component: extracellular space Molecular Function:heparin binding; chemokine activity; receptor binding; chemoattractant activity Biological Process: cellular calcium ion homeostasis; positive chemotaxis; cell-cell signaling; immune response; chemotaxis; signal transduction |
| NCBI Summary: | This gene is located in a cluster of similar genes in the same region of chromosome 17. These genes encode CC cytokines, which are secreted proteins characterized by two adjacent cysteines. The product of this gene is chemotactic for T cells and monocytes, and acts through C-C chemokine receptor type 1 (CCR1). The proprotein is further processed into numerous smaller functional peptides. Naturally-occurring readthrough transcripts occur from this gene into the downstream gene, CCL14 (chemokine (C-C motif) ligand 14). [provided by RefSeq, Jan 2013] |
| UniProt Code: | Q16663 |
| NCBI GenInfo Identifier: | 3915594 |
| NCBI Gene ID: | 6359 |
| NCBI Accession: | Q16663. 2 |
| UniProt Secondary Accession: | Q16663,Q9UM74, B2RU34, E1P651, |
| UniProt Related Accession: | Q16663 |
| Molecular Weight: | 12,248 Da |
| NCBI Full Name: | C-C motif chemokine 15 |
| NCBI Synonym Full Names: | chemokine (C-C motif) ligand 15 |
| NCBI Official Symbol: | CCL15 |
| NCBI Official Synonym Symbols: | LKN1; NCC3; SY15; HCC-2; LKN-1; MIP-5; NCC-3; SCYL3; MIP-1D; MRP-2B; SCYA15; HMRP-2B; MIP-1 delta |
| NCBI Protein Information: | C-C motif chemokine 15; leukotactin 1; chemokine CC-2; new CC chemokine 3; small-inducible cytokine A15; macrophage inflammatory protein 5; small inducible cytokine subfamily A (Cys-Cys), member 15 |
| UniProt Protein Name: | C-C motif chemokine 15 |
| UniProt Synonym Protein Names: | Chemokine CC-2; HCC-2; Leukotactin-1; LKN-1; MIP-1 delta; Macrophage inflammatory protein 5; MIP-5; Mrp-2b; NCC-3; Small-inducible cytokine A15Cleaved into the following 3 chains:CCL15(22-92); CCL15(25-92); CCL15(29-92) |
| Protein Family: | C-C motif chemokine |
| UniProt Gene Name: | CCL15 |
| UniProt Entry Name: | CCL15_HUMAN |
As the OD 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 (ng/mL) | O.D | Average | Corrected |
| 10 | 2.323 2.381 | 2.352 | 2.285 |
| 5 | 1.582 1.616 | 1.599 | 1.532 |
| 2.5 | 0.862 0.84 | 0.851 | 0.784 |
| 1.25 | 0.472 0.488 | 0.48 | 0.413 |
| 0.63 | 0.257 0.241 | 0.249 | 0.182 |
| 0.32 | 0.174 0.154 | 0.164 | 0.097 |
| 0.16 | 0.112 0.122 | 0.117 | 0.05 |
| 0 | 0.063 0.071 | 0.067 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MIP-5 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-5 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 (ng/mL) | 0.52 | 0.99 | 3.43 | 0.48 | 0.99 | 3.58 |
| Standard deviation | 0.03 | 0.05 | 0.16 | 0.03 | 0.05 | 0.12 |
| C V (%) | 5.77 | 5.05 | 4.66 | 6.25 | 5.05 | 3.35 |
Recovery
The recovery of Human MIP-5 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) | 94-106 | 100 |
| EDTA plasma (n=5) | 89-103 | 94 |
| Cell culture media (n=5) | 87-99 | 93 |
Linearity
Samples were spiked with high concentrations of Human MIP-5 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 (%) | 91-106 | 95-106 | 89-104 |
| Average (%) | 99 | 100 | 96 | |
| 1:4 | Range (%) | 91-106 | 82-94 | 83-97 |
| Average (%) | 98 | 86 | 89 | |
| 1:8 | Range (%) | 94-110 | 81-96 | 86-102 |
| Average (%) | 100 | 88 | 93 | |
| 1:16 | Range (%) | 87-102 | 86-100 | 87-98 |
| Average (%) | 94 | 93 | 93 |
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 ELISA 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 | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| 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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
