Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 78.13-5000 pg/mL |
| Sensitivity: | 46.88 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human EGFR in samples. No significant cross-reactivity or interference between Human EGFR 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 Human EGFR. 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 Human EGFR 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 EGFR, 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 Human EGFR. The concentration of Human EGFR in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | EGFR: a receptor tyrosine kinase. This is a receptor for epidermal growth factor (EGF) and related growth factors including TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30, and vaccinia virus growth factor. EGFR is involved in the control of cell growth and differentiation. It is a single-pass transmembrane tyrosine kinase. Ligand binding to this receptor results in receptor dimerization, autophosphorylation (in trans), activation of various downstream signaling molecules and lysosomal degradation. It can be phosphorylated and activated by Src. Activated EGFR binds the SH2 domain of phospholipase C-gamma (PLC-gamma), activating PLC-gamma-mediated downstream signaling. Phosphorylated EGFR binds Cbl, leading to its ubiquitination and degradation. Grb2 and SHC bind to phospho-EGFR and are involved in the activation of MAP kinase signaling pathways. Phosphorylation on Ser and Thr residues is thought to represent a mechanism for attenuation of EGFR kinase activity. EGFR is overexpressed in breast, head and neck cancers, correlating with poor survival. Activating somatic mutations are seen in lung cancer, corresponding to the minority of patients with strong responses to the EGFR inhibitor Iressa (gefitinib). Mutations and amplifications are also seen in glioblastoma, and upregulation is seen in colon cancer and neoplasms. In xenografts, inhibitors synergize with cytotoxic drugs in the inhibition of many tumor types. Inhibitors include: Iressa/ZD1839, Erbitux, Tarceva, and lapatinib. Four alternatively spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:Tumor suppressor; Protein kinase, tyrosine (receptor); Protein kinase, TK; Kinase, protein; Membrane protein, integral; EC 2. 7. 10. 1; TK group; EGFR family Chromosomal Location of Human Ortholog: 7p12 Cellular Component: extracellular space; endoplasmic reticulum membrane; nuclear membrane; cell surface; focal adhesion; basolateral plasma membrane; integral to membrane; lipid raft; Golgi membrane; membrane; perinuclear region of cytoplasm; cytoplasm; apical plasma membrane; plasma membrane; AP-2 adaptor complex; endosome membrane; nucleus; receptor complex; endosome Molecular Function:identical protein binding; epidermal growth factor receptor activity; epidermal growth factor binding; nitric-oxide synthase regulator activity; transmembrane receptor protein tyrosine kinase activity; receptor signaling protein tyrosine kinase activity; protein phosphatase binding; protein kinase binding; actin filament binding; integrin binding; protein binding; transmembrane receptor activity; enzyme binding; MAP kinase kinase kinase activity; protein heterodimerization activity; ubiquitin protein ligase binding; protein-tyrosine kinase activity; double-stranded DNA binding; chromatin binding; glycoprotein binding; ATP binding Biological Process: circadian rhythm; diterpenoid metabolic process; positive regulation of nitric oxide biosynthetic process; nerve growth factor receptor signaling pathway; activation of MAPKK activity; alkanesulfonate metabolic process; protein insertion into membrane; positive regulation of vasodilation; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; positive regulation of MAP kinase activity; positive regulation of fibroblast proliferation; cell-cell adhesion; ovulation cycle; cell surface receptor linked signal transduction; hair follicle development; positive regulation of superoxide release; negative regulation of mitotic cell cycle; positive regulation of DNA repair; fibroblast growth factor receptor signaling pathway; digestive tract morphogenesis; response to osmotic stress; phospholipase C activation; response to hydroxyisoflavone; hydrogen peroxide metabolic process; positive regulation of transcription from RNA polymerase II promoter; response to oxidative stress; regulation of nitric-oxide synthase activity; response to calcium ion; negative regulation of protein catabolic process; positive regulation of epithelial cell proliferation; negative regulation of apoptosis; negative regulation of epidermal growth factor receptor signaling pathway; axon guidance; tongue development; embryonic placenta development; peptidyl-tyrosine phosphorylation; translation; protein amino acid autophosphorylation; positive regulation of smooth muscle cell proliferation; signal transduction; positive regulation of synaptic transmission, glutamatergic; learning and/or memory; positive regulation of cell proliferation; salivary gland morphogenesis; response to stress; regulation of peptidyl-tyrosine phosphorylation; epidermal growth factor receptor signaling pathway; ossification; phosphoinositide-mediated signaling; MAPKKK cascade; liver development; cell proliferation; positive regulation of protein kinase B signaling cascade; cerebral cortex cell migration; calcium-dependent phospholipase A2 activation; positive regulation of vasoconstriction; innate immune response; positive regulation of protein amino acid phosphorylation; astrocyte activation; positive regulation of DNA replication; positive regulation of phosphorylation; response to cobalamin; positive regulation of cell migration; lung development; positive regulation of inflammatory response Disease: Lung Cancer |
| NCBI Summary: | The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor. Binding of the protein to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation. Mutations in this gene are associated with lung cancer. Multiple alternatively spliced transcript variants that encode different protein isoforms have been found for this gene. [provided by RefSeq, Jul 2010] |
| UniProt Code: | P00533 |
| NCBI GenInfo Identifier: | 2811086 |
| NCBI Gene ID: | 1956 |
| NCBI Accession: | P00533. 2 |
| UniProt Secondary Accession: | P00533,O00688, O00732, P06268, Q14225, Q68GS5, Q92795 Q9BZS2, Q9GZX1, Q9H2C9, Q9H3C9, Q9UMD7, |
| UniProt Related Accession: | P00533 |
| Molecular Weight: | 69,228 Da |
| NCBI Full Name: | Epidermal growth factor receptor |
| NCBI Synonym Full Names: | epidermal growth factor receptor |
| NCBI Official Symbol: | EGFR |
| NCBI Official Synonym Symbols: | ERBB; HER1; mENA; ERBB1; PIG61; NISBD2 |
| NCBI Protein Information: | epidermal growth factor receptor; proto-oncogene c-ErbB-1; cell growth inhibiting protein 40; cell proliferation-inducing protein 61; receptor tyrosine-protein kinase erbB-1; avian erythroblastic leukemia viral (v-erb-b) oncogene homolog |
| UniProt Protein Name: | Epidermal growth factor receptor |
| UniProt Synonym Protein Names: | Proto-oncogene c-ErbB-1; Receptor tyrosine-protein kinase erbB-1 |
| Protein Family: | Pro-epidermal growth factor |
| UniProt Gene Name: | EGFR |
| UniProt Entry Name: | EGFR_HUMAN |
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 |
| 5000 | 60898 61614 | 61256 | 61233 |
| 2500 | 33955 34753 | 34354 | 34331 |
| 1250 | 19768 16570 | 18169 | 18146 |
| 625 | 8759 10025 | 9392 | 9369 |
| 312.5 | 5105 4561 | 4833 | 4810 |
| 156.25 | 2561 2461 | 2511 | 2488 |
| 78.13 | 1339 1339 | 1339 | 1316 |
| 0 | 23 23 | 23 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human EGFR were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human EGFR 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) | 273.36 | 667.43 | 1782.29 | 293.44 | 716.88 | 1928.34 |
| Standard deviation | 31.79 | 74.22 | 186.96 | 30.43 | 85.81 | 214.43 |
| C V (%) | 11.63 | 11.12 | 10.49 | 10.37 | 11.97 | 11.12 |
Recovery
The recovery of Human EGFR 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) | 90-104 | 98 |
| EDTA plasma (n=5) | 86-99 | 92 |
| Cell culture media (n=5) | 92-106 | 98 |
Linearity
Samples were spiked with high concentrations of Human EGFR 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 (%) | 95-111 | 99-111 | 92-107 |
| Average (%) | 102 | 105 | 100 | |
| 1:4 | Range (%) | 95-112 | 87-102 | 87-99 |
| Average (%) | 103 | 94 | 92 | |
| 1:8 | Range (%) | 99-114 | 102-116 | 84-96 |
| Average (%) | 105 | 107 | 91 | |
| 1:16 | Range (%) | 88-102 | 92-105 | 96-110 |
| Average (%) | 93 | 100 | 103 |
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.
