The Quantikine Human Flt-3/Flk-2 Ligand Immunoassay is a 4.0 or 4.5 hour solid phase ELISA designed to measure soluble human Flt-3 Ligand in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant human Flt-3 Ligand and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate recombinant Flt-3 Ligand accurately. Results obtained measuring natural human Flt-3 Ligand showed dose-response curves that were parallel to the standard curves obtained using the recombinant Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human Flt-3 Ligand.
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision
The recovery of Flt-3 Ligand spiked to three different levels throughout the range of the assay in various matrices was evaluated.
Average % Recovery
Cell Culture Media (n=5)
Citrate Plasma (n=5)
EDTA Plasma (n=5)
Heparin Plasma (n=5)
To assess the linearity of the assay, five samples were spiked with high concentrations of Flt-3 Ligand in various matrices and diluted with the appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: Flt-3 Ligand
Flt-3 Ligand, also known as FL, is an alpha-helical cytokine that promotes the differentiation of multiple hematopoietic cell lineages. Mature human Flt-3 Ligand consists of a 158 amino acid (aa) extracellular domain (ECD) with a cytokine-like domain and a juxtamembrane tether region, a 21 aa transmembrane segment, and a 30 aa cytoplasmic tail. Within the ECD, human Flt-3 Ligand shares 71% and 65% aa sequence identity with mouse and rat Flt-3 Ligand, respectively. Human and mouse Flt-3 Ligand show cross-species activity. Flt-3 Ligand is expressed as a noncovalently-linked dimer by T cells and bone marrow and thymic fibroblasts. Each 36 kDa chain carries approximately 12 kDa of N- and O-linked carbohydrates. Alternate splicing and proteolytic cleavage of the transmembrane form can generate a soluble 30 kDa fragment that includes the cytokine domain. Alternate splicing of human Flt-3 Ligand also generates membrane-associated isoforms that contain either a truncated cytoplasmic tail or an 85 aa substitution following the cytokine domain. Both transmembrane and soluble Flt-3 Ligand signal through the tyrosine kinase receptor Flt-3/Flk-2. Flt-3 Ligand induces the expansion of monocytes and immature dendritic cells as well as early B cell lineage differentiation. It synergizes with IL-3, GM-CSF, and SCF to promote the mobilization and myeloid differentiation of hematopoietic stem cells. It cooperates with IL-2, -6, -7, and -15 to induce NK cell development and with IL-3, -7, and -11 to induce terminal B cell maturation. Animal studies also show Flt-3 Ligand to reduce the severity of experimentally induced allergic inflammation.
Refer to the product for complete assay procedure.
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
Prepare all reagents, standard dilutions, and samples as directed in the product insert.
Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
100 µL Assay Diluent
Add 100 µL of Assay Diluent to each well.
50 µL Standard, Control, or Sample
Add 50 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.
200 µL Conjugate
Add 200 µL of Conjugate to each well. Cover with a new plate sealer.
For Serum & Plasma Samples: Incubate at room temperature for 2 hours. For Cell Culture Supernate Samples: Incubate at room temperature for 1.5 hours.
Aspirate and wash 4 times.
200 µL Substrate Solution
Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.
100 µL Stop Solution
Add 100 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.
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but also provides information about sample types, species, and experimental conditions.
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