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|Antigen||Amphiregulin (AREG) ELISA Kits|
|Reactivity||Mouse (Murine) Alternatives|
Kits with alternative reactivity to:
|Method Type||Sandwich ELISA|
|Detection Range||8-600 pg/mL|
|Minimum Detection Limit||8 pg/mL|
|1 reference available|
|Supplier||Log in to see|
Product Details Amphiregulin ELISA KitTarget details Application Details Handling References for Amphiregulin Kit (ABIN625111) Images
|Purpose||Mouse Amphiregulin ELISA Kit for cell culture supernatants, plasma, and serum samples.|
|Sample Type||Serum, Plasma, Cell Culture Supernatant|
|Specificity||This ELISA kit shows no cross-reactivity with following cytokines tested: Mouse CD30, L CD30, T CD40, CRG-2, CTACK, CXCL16, Eotaxin , Eotaxin-2, Fas Ligand, Fractalkine, GCSF, GM-CFS, IFN- gamma, IGFBP-3, IGFBP-5, IGFBP-6, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-3 Rb, IL-4, IL-5, IL-9, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-17, KC, Leptin R, LEPTIN(OB), LIX, L-Selectin, Lymphotactin, MCP-1, MCP-5, M-CSF, MIG, MIP-1 alpha, MIP-1 gamma, MIP-2, MIP-3 beta, MIP-3 alpha, PF-4, P-Selectin, RANTES, SCF, SDF-1 alpha, TARC, TCA-3, TECK, TIMP-1, TNF-alpha, TNF RI, TNF RII, TPO, VCAM-1, VEGF.|
|Material not included||
Target detailsProduct Details Amphiregulin ELISA Kit Application Details Handling References for Amphiregulin Kit (ABIN625111) Images back to top
|Alternative Name||Amphiregulin (AREG ELISA Kit Abstract)|
|Background||The Mouse Amphiregulin ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of mouse Amphiregulin in serum, plasma and cell culture supernatants. This assay employs an antibody specific for mouse Amphiregulin coated on a 96-well plate. Standards and samples are pipetted into the wells and Amphiregulin present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-mouse Amphiregulin antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Amphiregulin bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.|
|Research Area||Cancer, Growth Factors|
|Pathways||RTK Signaling, EGFR Signaling Pathway|
Application DetailsProduct Details Amphiregulin ELISA Kit Target details Handling References for Amphiregulin Kit (ABIN625111) Images back to top
|Application Notes||Recommended Dilution for serum and plasma samples2 fold|
|Sample Volume||100 μL|
1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
2. Sample dilution: If your samples need to be diluted, 1xAssay Diluent (Item E) should be used for dilution of serum/plasma/culture supernatants. Suggested dilution for normal serum/plasma: 2-fold*. * Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
3. Assay Diluent (Item E) should be diluted 5-fold with deionized or distilled water before use.
4. Preparation of standard: Briefly spin the vial of Item C. Add 400 µL 1x Assay Diluent (Item E) into Item C vial to prepare a 50 ng/mL standard solution. Dissolve the powder thoroughly by a gentle mix. Add 8 µL Amphiregulin standard solution from the vial of Item C, into a tube with 492 µL 1x Assay Diluent to prepare 800 pg/mL standard solution. Pipette 400myl 1x Assay Diluent into each tube. Use the 800 pg/mL standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Assay Diluent serves as the zero standard (0 pg/mL). 200 µL 8 µL standard + 492 µL 200myl 200 µL 200 µL 200 µL 200 µL 800 266.7 88.89 29.63 9.88 3.29 1.10 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure.
7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 200-fold with 1x Assay Diluent. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 50 µL of HRP-Streptavidin concentrate into a tube with 10 ml 1x Assay Diluent to prepare a 200-fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well.
1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
5. Discard the solution. Repeat the wash as in step
6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
7. Discard the solution. Repeat the wash as in step
8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.
|Calculation of Results||
Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent Mouse AR concentration (pg/mL) 0.1 1 10 100 1000 O D =4 50 n m 0.001 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of Amphiregulin is typically less than 2 pg/mL.
Recovery: Recovery was determined by spiking various levels of Amphiregulin into normal mouse serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 130.1 120-138 Plasma 123.4 112-130 Cell culture media 85.25 76-95
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 113.2 114.0 101.4 Range ( %) 102-122 107-121 92-118 1:4 Average % of Expected 114.4 92.82 113.2 Range ( %) 103-124 83-101 103-121
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %
|Assay Precision||Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %|
|Restrictions||For Research Use only|
HandlingProduct Details Amphiregulin ELISA Kit Target details Application Details References for Amphiregulin Kit (ABIN625111) Images back to top
|Handling Advice||Avoid repeated freeze-thaw cycles.|
|Storage Comment||The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.|
|Expiry Date||6 months|
References for Amphiregulin Kit (ABIN625111)Product Details Amphiregulin ELISA Kit Target details Application Details Handling Images back to top
|Product cited in:||
Miyabayashi, Ijichi, Mohri, Tada, Yamamoto, Asaoka, Ikenoue, Tateishi, Nakai, Isayama, Morishita, Omata, Moses, Koike: "Erlotinib prolongs survival in pancreatic cancer by blocking gemcitabine-induced MAPK signals." in: Cancer research, Vol. 73, Issue 7, pp. 2221-34, 2013
ImagesProduct Details Amphiregulin ELISA Kit Target details Application Details Handling References for Amphiregulin Kit (ABIN625111) back to top