We selected the ligands EGF, TGF-α, AREG, EREG, NRG, HGF, and IGF-1, which were previously reported to be associated with the activation and cross-talk of the EGFR downstream signaling pathway in solid tumors. We used ELISA kits to measure serum levels of ligands as follow: Human HGF Quantikine ELISA Kit (DHG00; R&D Systems, Minneapolis, MN, USA), Human Epiregulin ELISA kit (CSB-EL007779HU; CUSABIO, Wuhan, China), Human Amphiregulin ELISA kit (E90006Hu; USCN Life Science, Wuhan, China), Human EGF Quantikine ELISA kit (DEG00; R&D Systems), Human TGF-α Quantikine ELISA kit (DTGA00; R&D Systems), Human Neureglin-1 ELISA kit (CSB-E17153 h; CUSABIO), and Human IGF-1 Quantikine ELISA kit (DG00; R&D Systems). Protocols of ELISA for these ligands are summarized in Table S1 .
Human egf quantikine elisa kit
Manufactured by R&D Systems
Sourced in United States, China
The Human EGF Quantikine ELISA Kit is a quantitative sandwich enzyme immunoassay designed to measure human epidermal growth factor (EGF) levels in cell culture supernates, serum, and plasma.
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Lab products found in correlation
Human epiregulin elisa kit, by Cusabio (2 mentions)
Human amphiregulin elisa kit, by USCN (2 mentions)
Human neureglin 1 elisa kit, by Cusabio (2 mentions)
Human igf 1 quantikine elisa kit, by R&D Systems (2 mentions)
Human hgf quantikine elisa kit, by R&D Systems (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Human mmp 9 quantikine elisa kit, by R&D Systems (1 mentions)
Total mmp 2 quantikine elisa kit, by R&D Systems (1 mentions)
Human kgf fgf 7 quantikine elisa kit, by R&D Systems (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
Tgf β1 human elisa kit, by Thermo Fisher Scientific (1 mentions)
Vegf human elisa kit, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Au5800, by Beckman Coulter (1 mentions)
Immage 800, by Beckman Coulter (1 mentions)
Amicon ultra 15 3k, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Human amphiregulin quantikine elisa kit, by R&D Systems (1 mentions)
Human tarc quantikine elisa kit, by R&D Systems (1 mentions)
14 protocols using human egf quantikine elisa kit
1
Quantifying Signaling Ligands in Solid Tumors
2
Quantifying Human EGF in Urine
3
Quantification of Secreted Mediators
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The medium of each culture was collected immediately after 6 h or 24 h exposure to ES and subjected to ELISA assays to quantify the secretion of different mediators. The Human KGF/FGF-7 Quantikine ELISA Kit (DKG00, R & D systems), Human EGF Quantikine ELISA Kit (DEG00, R & D systems), Human MMP-9 Quantikine ELISA Kit (DMP900, R & D systems) and total MMP-2 Quantikine ELISA Kit (MMP200, R & D systems) were used. The absorbance was measured at 450 nm using a Microplate Reader Model 680 (Bio-Rad, Philadelphia, PA, United States). The measurements were performed in triplicate for each condition. The minimum detectable concentrations were under 0.7 pg/ml for EGF, 15 pg/ml for FGF-7, under 30 pg/ml for MMP2, and under 160 pg/ml for MMP9, as reported by the manufacturer. Each experiment was repeated three times (n = 3), and the means ± SD were calculated and presented. To minimize the intra- and inter-assay variations, the collected supernatants from the different experiments (n = 3) were run in the same plate of either FGF-7, MMP-9, MMP-2, or EGF. Each supernatant was run in triplicate, and the value was rejected if a triplicate was over 10% different (intra-assay CV) from the other. Using the supernatants of the various experiments in the same plate gave us an inter-assay CV of less than 10%.
4
Protein Release Assays in PLMA Hydrogels
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The protein release assays were performed in PLMA hydrogels at 10, 15, and 20% (w/v) without encapsulated spheroids. The samples (n = 6) were placed into falcons with PBS (5 mL, Thermo Fischer Scientific, USA) and incubated with constant agitation (60 rpm) in a water bath at 37 °C. Over 14 d, an aliquot (1 mL) was taken at each time‐point and fresh PBS (1 mL) was added. The collected aliquots were stored at −20 °C. For total protein quantification, Micro BCA Protein Assay Kit (Thermo Fisher Scientific, USA) was used. ELISA assays were performed to quantify the release of vascular endothelial growth factor (VEGF Human ELISA Kit, Invitrogen, ThermoFisher Scientific, USA), transforming growth factor β1 (TGF‐β1 Human ELISA Kit, Invitrogen, ThermoFisher Scientific, USA), and epidermal growth factor (Human EGF Quantikine ELISA Kit, R&D systems, Minneapolis, USA).
5
Quantifying EGF Levels in Pancreatic Cells
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Mia-paca-2 and Panc-1 cells were plated in 12‐well plates and Enzyme‐linked immunosorbent assay (ELISA) was performed to detected the EGF levels in the supernatants after cultured for 48 hours using the Human EGF Quantikine ELISA Kit (#DEG00, R&D Systems, Minneapolis, MN, USA) according to manufacturer’s instructions
6
Urinary Biomarkers Quantification Protocol
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Urinary protein, albumin, and N-acetyl-beta-D-glucosaminidase levels were measured with automated biochemistry analyzers. Urinary protein was measured with a Beckman Coulter AU5800 (CA, USA) clinical chemistry analyzer using the pyrogallol red method. Albumin was measured with a Beckman Coulter IMMAGE 800 (CA, USA) clinical chemistry analyzer using a nephelometric immunoassay. N-acetyl-beta-D-glucosaminidase was measured with a Hitachi 7180 (Tokyo, Japan) clinical analyzer using the MNP-G1CNAc substrate method. Urinary EGF was measured after urine samples were cryopreserved for 3 months. Urinary EGF was measured with the Human EGF Quantikine ELISA Kit (SEG00, R&D Systems, USA) respectively. The samples were measured in duplicate. The concentrations of all the above urinary biomarkers were divided by urinary creatinine, osmolality, or (specific gravity-1) × 100 [32 (link)] for normalization of hydration status. Intra-assay variation and inter-assay variation were within 4% and 8%, respectively (evaluated by quality control, QC21, R&D Systems). The levels of urinary albumin in nine patients were excluded from data analyses for exceeding the upper limit of measurements.
7
Quantifying Secreted EGF in CM
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We used CM of CD163+, CD204+, and CD206+ cells as samples and DMEM (Life Technologies Corporation) with 2% FBS (Sigma-Aldrich) as the control for ELISA. We concentrated the CM using the Amicon® Ultra-15 3K (Merck Millipore, Burlington, MA, USA); a total of 15 ml of culture medium was centrifuged at 4000 ×g in 25 °C for 20 min to make 200 μl of sample. The samples were examined using the Human EGF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s guidelines, and EGF concentration was measured by using the Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 450 nm.
8
Measurement of ErbB Pathway Ligands
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We selected the ligands, such as EGF, TGF-alpha, AREG, EREG, NRG, HGF and IGF-1, that had previously been reported to be associated with activation or cross-talk of the downstream signal pathway of the ErbB family in solid tumors. We used ELISA kits to measure serum levels of ligands as follows: Human HGF Quantikine ELISA Kit (DHG00, R & D Systems, Minneapolis, MN, USA), Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, Hubei, China), Human amphiregulin ELISA kit (E90006Hu, Uscn Life Science, Wuhan, Hubei, China), Human EGF Quantikine ELISA kit (DEG00, R & D System, Minneapolis, MN, USA), Human TGF-alpha Quantikine ELISA kit (DTGA00, R & D System, Minneapolis, MN, USA), Human Neureglin-1 ELISA kit (CSB-E17153 h, CUSABIO, Wuhan, Hubei, China) and Human IGF-1 Quantikine ELISA kit (DG00, R & D System, Minneapolis, MN, USA). Protocols for ELISA of these ligands are summarized in Supplementary Material 2 .
9
Quantifying Plasma EGF and AREG
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Plasma EGF and AREG concentrations were measured with the Human EGF Quantikine ELISA Kit (#DEGFR0) and Human Amphiregulin Quantikine ELISA Kit (#DAR00; both from R&D Systems, Minneapolis, MN, USA), respectively, according to the manufacturer’s instructions.
10
EGF Quantification in Cell Culture
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Cells (2 × 104 per well) was plated in a 96‐well plate and Enzyme‐linked immuno sorbent assay (ELISA) was performed to detected the EGF levels in the medium after cultured for 48 hours using the Human EGF Quantikine ELISA Kit (#DEG00, R&D Systems) according to manufacture's protocols.
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