Description
Human ENA-78 (Epithelial Neutrophil Activating Peptide 78) ELISA Kit
The Human ENA-78 (Epithelial Neutrophil Activating Peptide-78) ELISA Kit is a cutting-edge tool designed for the precise detection of ENA-78 levels in human samples such as serum, plasma, and cell culture supernatants. This kit boasts exceptional sensitivity and specificity, ensuring accurate and consistent results to support a wide range of research endeavors.ENA-78, also known as CXCL5, is a key chemokine involved in inflammatory processes, particularly in recruiting neutrophils to sites of infection or injury.
Its dysregulation has been implicated in various diseases like asthma, COPD, and autoimmune disorders, highlighting its value as a biomarker for understanding and potentially treating these conditions.With the Human ENA-78 ELISA Kit, researchers can delve deeper into the role of this potent chemokine in health and disease, paving the way for innovative therapeutic interventions and clinical advancements.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human ENA-78 in samples. No significant cross-reactivity or interference between Human ENA-78 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 ENA-78. 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 ENA-78 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 ENA-78, 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 ENA-78. The concentration of Human ENA-78 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | CXCL5: Involved in neutrophil activation. In vitro, ENA-78(8- 78) and ENA-78(9-78) show a threefold higher chemotactic activity for neutrophil granulocytes. Belongs to the intercrine alpha (chemokine CxC) family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 4q13. 3 Cellular Component: extracellular space; extracellular region Molecular Function:chemokine activity; CXCR chemokine receptor binding Biological Process: G-protein coupled receptor protein signaling pathway; cell-cell signaling; positive regulation of cell proliferation; neutrophil mediated immunity; immune response; positive regulation of leukocyte chemotaxis; response to lipopolysaccharide; chemotaxis; inflammatory response; signal transduction |
| NCBI Summary: | This gene encodes a protein that is a member of the CXC subfamily of chemokines. Chemokines, which recruit and activate leukocytes, are classified by function (inflammatory or homeostatic) or by structure. This protein is proposed to bind the G-protein coupled receptor chemokine (C-X-C motif) receptor 2 to recruit neutrophils, to promote angiogenesis and to remodel connective tissues. This protein is thought to play a role in cancer cell proliferation, migration, and invasion. [provided by RefSeq, May 2013] |
| UniProt Code: | P42830 |
| NCBI GenInfo Identifier: | 1169525 |
| NCBI Gene ID: | 6374 |
| NCBI Accession: | P42830. 1 |
| UniProt Secondary Accession: | P42830,Q96QE1, |
| UniProt Related Accession: | P42830 |
| Molecular Weight: | 11,972 Da |
| NCBI Full Name: | C-X-C motif chemokine 5 |
| NCBI Synonym Full Names: | chemokine (C-X-C motif) ligand 5 |
| NCBI Official Symbol: | CXCL5 |
| NCBI Official Synonym Symbols: | SCYB5; ENA-78 |
| NCBI Protein Information: | C-X-C motif chemokine 5; neutrophil-activating protein 78; neutrophil-activating peptide ENA-78; epithelial-derived neutrophil-activating protein 78; small inducible cytokine subfamily B (Cys-X-Cys), member 5 (epithelial-derived neutrophil-activating peptide 78) |
| UniProt Protein Name: | C-X-C motif chemokine 5 |
| UniProt Synonym Protein Names: | ENA-78(1-78); Epithelial-derived neutrophil-activating protein 78; Neutrophil-activating peptide ENA-78; Small-inducible cytokine B5Cleaved into the following 2 chains:ENA-78(8-78); ENA-78(9-78) |
| Protein Family: | C-X-C motif chemokine |
| UniProt Gene Name: | CXCL5 |
| UniProt Entry Name: | CXCL5_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 (pg/mL) | O.D | Average | Corrected |
| 2000 | 2.475 2.489 | 2.482 | 2.395 |
| 1000 | 1.606 1.636 | 1.621 | 1.534 |
| 500 | 0.916 0.912 | 0.914 | 0.827 |
| 250 | 0.501 0.531 | 0.516 | 0.429 |
| 125 | 0.298 0.292 | 0.295 | 0.208 |
| 62.5 | 0.195 0.181 | 0.188 | 0.101 |
| 31.25 | 0.134 0.144 | 0.139 | 0.052 |
| 0 | 0.078 0.096 | 0.087 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ENA-78 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ENA-78 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) | 109.30 | 197.13 | 810.84 | 102.41 | 202.65 | 888.63 |
| Standard deviation | 7.37 | 11.49 | 38.51 | 5.78 | 11.73 | 44.52 |
| C V (%) | 6.74 | 5.83 | 4.75 | 5.64 | 5.79 | 5.01 |
Recovery
The recovery of Human ENA-78 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) | 92-108 | 100 |
| EDTA plasma (n=5) | 88-102 | 93 |
| Cell culture media (n=5) | 95-110 | 101 |
Linearity
Samples were spiked with high concentrations of Human ENA-78 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 | 99-111 | 96-113 |
| Average (%) | 99 | 105 | 103 | |
| 1:4 | Range (%) | 92-103 | 87-100 | 86-99 |
| Average (%) | 98 | 92 | 92 | |
| 1:8 | Range (%) | 92-106 | 83-96 | 88-99 |
| Average (%) | 98 | 90 | 94 | |
| 1:16 | Range (%) | 91-103 | 85-95 | 87-100 |
| Average (%) | 97 | 90 | 92 |
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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.
