Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.78-50 ng/mL |
| Sensitivity: | 0.47 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human CXCR2 in samples. No significant cross-reactivity or interference between Human CXCR2 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 CXCR2. 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 CXCR2 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 CXCR2, 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 CXCR2. The concentration of Human CXCR2 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | IL8RB: Receptor for interleukin-8 which is a powerful neutrophil chemotactic factor. Binding of IL-8 to the receptor causes activation of neutrophils. This response is mediated via a G-protein that activates a phosphatidylinositol-calcium second messenger system. Binds to IL-8 with high affinity. Also binds with high affinity to CXCL3, GRO/MGSA and NAP-2. Belongs to the G-protein coupled receptor 1 family. |
| UniProt Protein Details: | Protein type:GPCR, family 1; Receptor, cytokine; Membrane protein, multi-pass; Membrane protein, integral; Receptor, GPCR Chromosomal Location of Human Ortholog: 2q35 Cellular Component: cell surface; integral to plasma membrane; intracellular; mast cell granule; membrane; plasma membrane Molecular Function:C-X-C chemokine receptor activity; interleukin-8 binding; interleukin-8 receptor activity; protein binding; signal transducer activity Biological Process: cell surface receptor linked signal transduction; cellular defense response; chemotaxis; dendritic cell chemotaxis; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); inflammatory response; neutrophil activation; neutrophil chemotaxis; positive regulation of cell proliferation; receptor internalization; signal transduction |
| NCBI Summary: | The protein encoded by this gene is a member of the G-protein-coupled receptor family. This protein is a receptor for interleukin 8 (IL8). It binds to IL8 with high affinity, and transduces the signal through a G-protein activated second messenger system. This receptor also binds to chemokine (C-X-C motif) ligand 1 (CXCL1/MGSA), a protein with melanoma growth stimulating activity, and has been shown to be a major component required for serum-dependent melanoma cell growth. This receptor mediates neutrophil migration to sites of inflammation. The angiogenic effects of IL8 in intestinal microvascular endothelial cells are found to be mediated by this receptor. Knockout studies in mice suggested that this receptor controls the positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration. This gene, IL8RA, a gene encoding another high affinity IL8 receptor, as well as IL8RBP, a pseudogene of IL8RB, form a gene cluster in a region mapped to chromosome 2q33-q36. Alternatively spliced variants, encoding the same protein, have been identified. [provided by RefSeq, Nov 2009] |
| UniProt Code: | P25025 |
| NCBI GenInfo Identifier: | 1352454 |
| NCBI Gene ID: | 3579 |
| NCBI Accession: | P25025. 2 |
| UniProt Secondary Accession: | P25025,Q8IUZ1, Q9P2T6, Q9P2T7, |
| UniProt Related Accession: | P25025,AAB24742,AAA15417 |
| Molecular Weight: | 40,759 Da |
| NCBI Full Name: | C-X-C chemokine receptor type 2 |
| NCBI Synonym Full Names: | C-X-C motif chemokine receptor 2 |
| NCBI Official Symbol: | CXCR2 |
| NCBI Official Synonym Symbols: | CD182; IL8R2; IL8RA; IL8RB; CMKAR2; CDw128b |
| NCBI Protein Information: | C-X-C chemokine receptor type 2 |
| UniProt Protein Name: | C-X-C chemokine receptor type 2 |
| UniProt Synonym Protein Names: | CDw128b; GRO/MGSA receptor; High affinity interleukin-8 receptor B; IL-8R B; IL-8 receptor type 2; CD_antigen: CD182 |
| Protein Family: | C-X-C chemokine receptor |
| UniProt Gene Name: | CXCR2 |
| UniProt Entry Name: | CXCR2_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 |
| 50 | 2.359 2.395 | 2.377 | 2.314 |
| 25 | 1.637 1.639 | 1.638 | 1.575 |
| 12.5 | 0.874 0.848 | 0.861 | 0.798 |
| 6.25 | 0.394 0.408 | 0.401 | 0.338 |
| 3.13 | 0.261 0.253 | 0.257 | 0.194 |
| 1.57 | 0.16 0.158 | 0.159 | 0.096 |
| 0.78 | 0.108 0.116 | 0.112 | 0.049 |
| 0 | 0.054 0.072 | 0.063 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CXCR2 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CXCR2 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) | 2.76 | 5.72 | 24.54 | 2.57 | 5.86 | 23.93 |
| Standard deviation | 0.15 | 0.27 | 0.87 | 0.13 | 0.26 | 0.87 |
| C V (%) | 5.43 | 4.72 | 3.55 | 5.06 | 4.44 | 3.64 |
Recovery
The recovery of Human CXCR2 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) | 93-105 | 99 |
| EDTA plasma (n=5) | 93-109 | 99 |
| Cell culture media (n=5) | 89-100 | 94 |
Linearity
Samples were spiked with high concentrations of Human CXCR2 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 (%) | 87-98 | 98-110 | 94-107 |
| Average (%) | 92 | 103 | 101 | |
| 1:4 | Range (%) | 93-104 | 84-96 | 88-103 |
| Average (%) | 99 | 90 | 94 | |
| 1:8 | Range (%) | 87-100 | 85-97 | 88-102 |
| Average (%) | 92 | 90 | 94 | |
| 1:16 | Range (%) | 94-105 | 81-93 | 84-98 |
| Average (%) | 99 | 87 | 90 |
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.
