Human EGFR ELISA Kit (HUFI00010)- High Sensitivity

Product Name:	Human EGFR ELISA Kit
Product Code:	HUFI00010
Size:	96 Assays
Alias:	EGFR, ErbB-1, ErbB1, ERBB, HER1, Mena
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human EGFRRR concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human EGFRRR and the recovery rates were calculated by comparing the measured value to the expected amount of Human EGFRRR in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-105	100
EDTA plasma(n=5)	86-103	95
UFH plasma(n=5)	86-94	90

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human EGFRRR and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-100%	85-99%	85-102%
EDTA plasma(n=5)	84-97%	83-101%	84-100%
UFH plasma(n=5)	80-90%	80-98%	83-99%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P00533

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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.