Description
Mouse MIP-1 beta (Macrophage Inflammatory Protein 1 Beta) ELISA Kit
The Mouse MIP-1 beta (Macrophage Inflammatory Protein-1 beta) ELISA Kit is specifically designed for the accurate measurement of MIP-1 beta levels in mouse serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit delivers reliable and reproducible results, making it an essential tool for various research applications.MIP-1 beta is a key cytokine involved in the inflammatory response, playing a crucial role in immune cell recruitment and activation.
Its dysregulation has been implicated in various inflammatory diseases and conditions, making it a valuable biomarker for studying and understanding these pathologies.This ELISA kit provides researchers with a powerful tool for quantifying MIP-1 beta levels in mouse samples, allowing for precise and accurate measurements that can aid in the investigation of inflammatory processes and potential therapeutic interventions.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Mouse |
| Detection Method: | Colormetric |
| Detection Range: | 15.63-1000 pg/mL |
| Sensitivity: | 9.38 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Mouse MIP-1 beta in samples. No significant cross-reactivity or interference between Mouse MIP-1 beta 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 Mouse MIP-1 beta. 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 Mouse MIP-1 beta 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 MIP-1 beta, 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 Mouse MIP-1 beta. The concentration of Mouse MIP-1 beta in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CCL4: Monokine with inflammatory and chemokinetic properties. Binds to CCR5. One of the major HIV-suppressive factors produced by CD8+ T-cells. Recombinant MIP-1-beta induces a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). The processed form MIP-1-beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5- mediated entry of HIV-1 in T-cells. MIP-1-beta(3-69) is also a ligand for CCR1 and CCR2 isoform B. Belongs to the intercrine beta (chemokine CC) family. |
| UniProt Protein Details: | Protein type:Secreted; Chemokine; Motility/polarity/chemotaxis; Secreted, signal peptide Cellular Component: extracellular space; extracellular region Molecular Function:identical protein binding; protein binding; CCR1 chemokine receptor binding; chemokine activity; cytokine activity; CCR5 chemokine receptor binding Biological Process: leukocyte chemotaxis; response to toxin; immune response; positive regulation of calcium-mediated signaling; positive regulation of tumor necrosis factor production; inflammatory response; chemotaxis; positive regulation of calcium ion transport; positive regulation of vascular permeability |
| UniProt Code: | P14097 |
| NCBI GenInfo Identifier: | 1346534 |
| NCBI Gene ID: | 20303 |
| NCBI Accession: | P14097. 3 |
| UniProt Related Accession: | P14097 |
| Molecular Weight: | 10,168 Da |
| NCBI Full Name: | C-C motif chemokine 4 |
| NCBI Synonym Full Names: | chemokine (C-C motif) ligand 4 |
| NCBI Official Symbol: | Ccl4 |
| NCBI Official Synonym Symbols: | Act-2; Mip1b; Scya4; MIP-1B; AT744. 1 |
| NCBI Protein Information: | C-C motif chemokine 4; ACT2; SIS-gamma; MIP-1 beta; MIP-1-beta; immune activation protein 2; small inducible cytokine A4; small-inducible cytokine A4; macrophage inflammatory protein 1-beta |
| UniProt Protein Name: | C-C motif chemokine 4 |
| UniProt Synonym Protein Names: | Immune activation protein 2; ACT-2; ACT2; Macrophage inflammatory protein 1-beta; MIP-1-beta; Protein H400; SIS-gamma; Small-inducible cytokine A4 |
| Protein Family: | C-C motif chemokine |
| UniProt Gene Name: | Ccl4 |
| UniProt Entry Name: | CCL4_MOUSE |
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 (pg/mL) | O.D | Average | Corrected |
| 1000 | 2.345 2.357 | 2.351 | 2.276 |
| 500 | 1.66 1.684 | 1.672 | 1.597 |
| 250 | 0.929 0.899 | 0.914 | 0.839 |
| 125 | 0.491 0.511 | 0.501 | 0.426 |
| 62.5 | 0.257 0.237 | 0.247 | 0.172 |
| 31.25 | 0.182 0.17 | 0.176 | 0.101 |
| 15.63 | 0.118 0.136 | 0.127 | 0.052 |
| 0 | 0.07 0.08 | 0.075 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse MIP-1 beta were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse MIP-1 beta 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) | 48.30 | 123.10 | 451.10 | 44.50 | 127.80 | 418.70 |
| Standard deviation | 2.50 | 6.80 | 17.60 | 2.20 | 7.40 | 20.90 |
| C V (%) | 5.18 | 5.52 | 3.90 | 4.94 | 5.79 | 4.99 |
Recovery
The recovery of Mouse MIP-1 beta 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) | 85-100 | 92 |
| EDTA plasma (n=5) | 94-108 | 100 |
| Cell culture media (n=5) | 95-110 | 100 |
Linearity
Samples were spiked with high concentrations of Mouse MIP-1 beta 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 | 98-113 | 91-103 |
| Average (%) | 102 | 103 | 97 | |
| 1:4 | Range (%) | 95-112 | 86-98 | 95-110 |
| Average (%) | 102 | 93 | 100 | |
| 1:8 | Range (%) | 95-107 | 89-102 | 97-110 |
| Average (%) | 101 | 94 | 102 | |
| 1:16 | Range (%) | 96-111 | 86-97 | 95-110 |
| Average (%) | 102 | 92 | 101 |
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.
