HBEGF Kit ELISA

N° du produit ABIN625303

Kit ELISA HBEGF Humain, Colorimetric test pour la quantification de Humain HBEGF et a été mentionné dans 4+ publications.

Aperçu rapide pour HBEGF Kit ELISA (ABIN625303)

Antigène: HBEGF (Heparin-Binding EGF-Like Growth Factor (HBEGF))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 20-4000 pg/mL

Application: ELISA

Type d'échantillon: Plasma, Cell Culture Supernatant, Serum

Détail du produit HBEGF Kit ELISA

Seuil minimal de détection: 20 pg/mL

Fonction: Human HB-EGF ELISA Kit for cell culture supernatants, plasma, and serum samples.

Analytical Method: Quantitative

Specificité: Cross Reactivity: This ELISA kit shows no cross-reactivity with the following cytokines tested: human Angiogenin, BDNF, BLC, ENA-78, FGF-4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin (OB), MCP-1, MCP-2, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.

Sensibilité: < 20 pg/mL

Attributs du produit:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Ingrédients:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Matériel non inclus:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Information d'application

Indications d'application: Recommended Dilution for serum and plasma samples2 fold

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Préparation des réactifs:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: If your samples need to be diluted, 1x Assay Diluent D (Item K) should be used for dilution of serum/plasma samples. Assay Diluent C should be used for dilution of cell culture supernates/urine. 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 B and Assay Diluent D should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL 1x Assay Diluent D (for serum/plasma samples) or Assay Diluent C (for cell culture supernates/urine) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 40 µL HB-EGF standard (50 ng/mL) from the vial of Item C, into a tube with 460 µL 1x Assay Diluent D or Assay Diluent C to prepare a 4,000 pg/mL standard solution. Pipette 300 µL 1x Assay Diluent D or Assay Diluent C into each tube. Use the 4,000 pg/mL standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Assay Diluent D or Assay Diluent C serves as the zero standard (0 pg/mL). 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 40 µL standard (50 ng/mL) + 460 µL 4,000 1,600 640 256 102.4 40.96 16.38 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 B 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 B 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 300-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 40 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a final 300 fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Procédure de l'essai:

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.

Calcul des résultats:

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 D HB-EGF concentration (pg/mL) 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10 Assay Diluent C HB-EGF concentration (pg/mL) 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of HB-EGF is typically less than 20 pg/mL.
Recovery: Recovery was determined by spiking human HB-EGF into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 99.18 89-108 Plasma 93.73 84-103 Cell culture media 81.22 73-90
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 81.24 89.03 104.7 Range ( %) 72-90 80-97 96-111 1:4 Average % of Expected 80.64 85.31 102.7 Range ( %) 70-90 74-93 91-113
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Précision du teste: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: Avoid repeated freeze-thaw cycles.

Stock: -20 °C

Stockage commentaire: 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.

Date de péremption: 6 months

Références pour HBEGF Kit ELISA (ABIN625303)

Lim, Yoo, Kim, Hur, Lee, Hur, Lee, Lee, Park, Lee, Chang, Kim, Kang, Hong, Kim, Kim, Yoon, Nam, Yang, Kim, Cho, Won: "GC1118, an Anti-EGFR Antibody with a Distinct Binding Epitope and Superior Inhibitory Activity against High-Affinity EGFR Ligands." dans: Molecular cancer therapeutics, Vol. 15, Issue 2, pp. 251-63, (2016) (PubMed).

David, Sahay, Mege, Descotes, Leblanc, Ribeiro, Clézardin, Peyruchaud: "Identification of heparin-binding EGF-like growth factor (HB-EGF) as a biomarker for lysophosphatidic acid receptor type 1 (LPA1) activation in human breast and prostate cancers." dans: PLoS ONE, Vol. 9, Issue 5, pp. e97771, (2015) (PubMed).

Koob, Lim, Massee, Zabek, Rennert, Gurtner, Li: "Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration." dans: Vascular cell, Vol. 6, pp. 10, (2014) (PubMed).

Xu, Gurski, Zhang, Harrington, Farach-Carson, Jia: "Recreating the tumor microenvironment in a bilayer, hyaluronic acid hydrogel construct for the growth of prostate cancer spheroids." dans: Biomaterials, Vol. 33, Issue 35, pp. 9049-60, (2012) (PubMed).

Détail du antigène

Antigène: HBEGF (Heparin-Binding EGF-Like Growth Factor (HBEGF))

Autre désignation: HB-EGF

Sujet: The Human HB-EGF (Heparin-binding EGF-like Growth Factor) ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human HB-EGF in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human HB-EGF coated on a 96-well plate. Standards and samples are pipetted into the wells and HB-EGF present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human HB-EGF 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 HB-EGF 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%.

ID gène: 1839

UniProt: Q99075

Pathways: Signalisation RTK, Fc-epsilon Receptor Signaling Pathway, EGFR Signaling Pathway, Neurotrophin Signaling Pathway