Rat Signaling ELISA Kits 2
Rat EGF (Epidermal Growth Factor) CLIA Kit (RTES00202)
- SKU:
- RTES00202
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 4.69pg/mL
- Range:
- 7.81-500pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- URG, HOMG4, Beta-Urogastrone
- Reactivity:
- Rat
- Sample Type:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| 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 Rat EGF in samples. No significant cross-reactivity or interference between Rat EGF 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 Rat EGF. 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 Rat EGF 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 Rat EGF, 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 Rat EGF. The concentration of Rat EGF in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | EGF: EGF stimulates the growth of various epidermal and epithelial tissues in vivo and in vitro and of some fibroblasts in cell culture. Magnesiotropic hormone that stimulates magnesium reabsorption in the renal distal convoluted tubule via engagement of EGFR and activation of the magnesium channel TRPM6. Defects in EGF are the cause of hypomagnesemia type 4 (HOMG4); also known as renal hypomagnesemia normocalciuric. HOMG4 is a disorder characterized by massive renal hypomagnesemia and normal levels of serum calcium and calcium excretion. Clinical features include seizures, mild-to mederate psychomotor retardation, and brisk tendon reflexes. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Ligand, receptor tyrosine kinase; Membrane protein, integral; Hormone Cellular Component: extracellular space; lysosomal membrane; plasma membrane; integral to membrane; intracellular Molecular Function:growth factor activity; calcium ion binding; epidermal growth factor receptor binding Biological Process: epidermal growth factor receptor signaling pathway; peptidyl-tyrosine phosphorylation; positive regulation of mitosis; activation of MAPKK activity; positive regulation of transcription, DNA-dependent; regulation of protein transport; positive regulation of MAP kinase activity; regulation of protein secretion; positive regulation of epidermal growth factor receptor activity; branching morphogenesis of a tube; positive regulation of cell proliferation; negative regulation of secretion; angiogenesis; regulation of peptidyl-tyrosine phosphorylation; positive regulation of phosphorylation; positive regulation of DNA binding; STAT protein nuclear translocation; positive regulation of granule cell precursor proliferation |
| NCBI Summary: | binds the epidermal growth factor receptor; may play a role in MAP kinase mediated signaling pathways [RGD, Feb 2006] |
| UniProt Code: | P07522 |
| NCBI GenInfo Identifier: | 1352360 |
| NCBI Gene ID: | 25313 |
| NCBI Accession: | P07522. 2 |
| UniProt Secondary Accession: | P07522,Q63183, |
| UniProt Related Accession: | P07522 |
| Molecular Weight: | 124,126 Da |
| NCBI Full Name: | Pro-epidermal growth factor |
| NCBI Synonym Full Names: | epidermal growth factor |
| NCBI Official Symbol: | Egf |
| NCBI Protein Information: | pro-epidermal growth factor |
| UniProt Protein Name: | Pro-epidermal growth factor |
| Protein Family: | EGF-like domain containing protein |
| UniProt Gene Name: | Egf |
| UniProt Entry Name: | EGF_RAT |
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 | 46526 56548 | 51537 | 51508 |
| 250 | 20818 21178 | 20998 | 20969 |
| 125 | 9533 9259 | 9396 | 9367 |
| 62.5 | 4468 4556 | 4512 | 4483 |
| 31.25 | 2306 2292 | 2299 | 2270 |
| 15.63 | 1317 1183 | 1250 | 1221 |
| 7.81 | 734 746 | 740 | 711 |
| 0 | 28 30 | 29 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat EGF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat EGF 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) | 24.73 | 66.18 | 173.33 | 22.29 | 68.29 | 169.81 |
| Standard deviation | 2.18 | 5.92 | 19.19 | 2.17 | 5.73 | 17.93 |
| C V (%) | 8.82 | 8.95 | 11.07 | 9.74 | 8.39 | 10.56 |
Recovery
The recovery of Rat EGF 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) | 95-110 | 102 |
| EDTA plasma (n=5) | 95-111 | 103 |
| Cell culture media (n=5) | 91-105 | 96 |
Linearity
Samples were spiked with high concentrations of Rat EGF 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 (%) | 101-112 | 98-112 | 98-115 |
| Average (%) | 106 | 105 | 105 | |
| 1:4 | Range (%) | 86-96 | 102-116 | 101-120 |
| Average (%) | 91 | 109 | 109 | |
| 1:8 | Range (%) | 90-105 | 95-107 | 91-104 |
| Average (%) | 98 | 102 | 97 | |
| 1:16 | Range (%) | 100-112 | 97-109 | 100-113 |
| Average (%) | 106 | 103 | 106 |
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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