Human G-CSF Quantikine QuicKit ELISA

Catalog #: QK214 Datasheet / COA / SDS

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QK214

Control Products Available

QC269: Human G-CSF Quantikine QuicKit Control Group 269

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Summary Sample Values Precision Recovery Linearity Data Examples Product Datasheets Preparation and Storage Background Related Research Areas

Human G-CSF Quantikine QuicKit ELISA Summary

Assay Length

80 minutes

Sample Type & Volume Required Per Well

Cell Culture Supernates (25 uL), Serum (25 uL), EDTA Plasma (25 uL), Heparin Plasma (25 uL)

Sensitivity

3.57 pg/mL

Assay Range

39.1 - 2,500 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma)

Specificity

Natural and recombinant human G-CSF.

Cross-reactivity

< 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.

Interference

No significant interference observed with available related molecules.

Sample Values

Serum/Plasma - Ten serum and plasma samples from apparently healthy volunteers were evaluated for the presence of human G-CSF in this assay. No medical histories were available for the donors used in this study. All samples measured less than the lowest human G-CSF standard, 39.1 pg/mL.

Cell Culture Supernates - Human peripheral blood mononuclear cells (PBMCs) (1 x 106 cells/mL) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin sulfate. Cells were left unstimulated or stimulated with 10 ug/mL PHA for 1 or 5 days. Aliquots of the cell culture supernates were removed and assayed for levels of human G-CSF.

Condition	Day 1 (pg/mL)	Day 5 (pg/mL)
Unstimulated	ND	ND
Stimulated	915	1184

ND=Non-detectable

THP-1 cells (2.5 x 105 cells/mL) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, and 50 µM beta -mercaptoethanol. To differentiate the cells into macrophages, 50 ng/mL PMA was added when seeding cells and incubated overnight. The next day, media was removed, and cells were washed once with media before replacing normal media and incubated for 2 days. Cells were then left unstimulated or stimulated with 100 ng/mL or 1000 ng/mL LPS for 1 day. Aliquots of the cell culture supernates were removed and assayed for levels of human G-CSF.

Condition	Day 1 (pg/mL)
Unstimulated	ND
Stimluated with 100 ng/mL LPS	1813
Stimluated with 1000 ng/mL LPS	2721

Product Summary

The Quantikine^® QuicKit™ Human G-CSF Immunoassay is a one step, 80-minute solid phase ELISA designed to measure human G-CSF levels in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human G-CSF and antibodies raised against the recombinant protein. Results obtained using natural human G-CSF showed linear curves that were parallel to the standard curves obtained using the QuicKit™ standards. These results indicate that this kit can be used to determine relative mass values for natural human G-CSF.

Precision

Intra-Assay Precision (Precision within an assay) Two samples of known concentration were tested twenty times on one plate to assess intra-assay precision

Inter-Assay Precision (Precision between assays) Two samples of known concentration were tested in ten separate assays to assess inter-assay precision. Assays were performed by at least three technicians

Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma

	Intra-Assay Precision		Inter-Assay Precision
Sample	1	2	1	2
n	20	20	10	10
Mean (pg/mL)	260	1351	246	1426
Standard Deviation	3.36	26.2	27	70.9
CV%	1.3	1.9	11	5

Recovery

The recovery of human G-CSF spiked to three levels in samples throughout the range of the assay was evaluated.

Sample Type	Average % Recovery	Range %
Cell Culture Supernates (n=4)	101	93-105
EDTA Plasma (n=2)	102	100-106
Heparin Plasma (n=2)	94	90-99
Serum (n=2)	79	77-84

Linearity

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human G-CSF were diluted with calibrator diluent to produce samples with values within the dynamic range of the assay.

Scientific Data

N/A

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Human G-CSF ELISA Standard Curve

N/A

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Human G-CSF QuicKit Recovery Competitor Comparison G-CSF is spiked at three known concentrations throughout the range of the assay and run to measure response of the spiked sample matrix. Culture media recovery is 102% compared to 122% for the top competitor. In spike and recovery experiments, natural samples are spiked with the recombinant target analyte of interest to identify interference caused by sample matrices.

N/A

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Human G-CSF QuicKit Spiked Linearity Competitor Comparison G-CSF is spiked at high concentration in various matrices and diluted with appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay. The linearity in serum is between 92%-112% compared to 121%-153% for the top competitor. The linearity in plasma is between 92%-93% compated to 143%-195% for the top competitor. Linearity is used to assess matrix effects and accurate measurement of sample values to provide confidence in your results.

Product Datasheets

Product Datasheet

COA

SDS

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: G-CSF

Granulocyte-colony stimulating factor (G-CSF) is a 24-25 kDa monomeric glycoprotein that regulates the proliferation, differentiation, and activation of hematopoietic cells in the neutrophilic granulocyte lineage. Mature human G-CSF is a 178 amino acid (aa) O-glycosylated protein that contains two intrachain disulfide bridges. In humans, alternate splicing generates a second minor isoform with a 3 aa deletion. Mouse and human G-CSF share 76% aa sequence identity, and the two proteins show species cross-reactivity. G-CSF is produced by activated monocytes and macrophages, fibroblasts, endothelial cells, astrocytes, neurons, and bone marrow stroma cells. In addition, various tumor cells express G-CSF constitutively.

Human G-CSF receptor (G-CSF R) is a 120 kDa type I transmembrane glycoprotein that belongs to the hematopoietin receptor superfamily. The mature protein consists of a 603 aa extracellular domain (ECD), a 23 aa transmembrane segment, and a 186 aa cytoplasmic domain. The ECD contains an N-terminal Ig-like domain, a cytokine receptor homology domain, and three fibronectin type III domains. Alternate splicing of human G-CSF R generates additional isoforms including a potentially soluble form of the receptor. The ECDs of mouse and human G-CSF R share 63% aa sequence identity. G-CSF R forms a complex with the ligand in a 2:2 ratio. It is expressed on monocytes, neutrophils, megakaryocytes, platelets, myeloid progenitors, trophoblasts and placenta, endothelial cells, and various tumor cell types.

G-CSF is an important regulator for granulopoiesis in vivo, and mutations in G-CSF R are associated with congenital neutropenia. G-CSF can support the growth of multilineage hematopoietic progenitor cells and mobilize them from the bone marrow into the bloodstream. G-CSF enhances the functional capacity of mature neutrophils and supports their survival by limiting the rate of apoptosis. G-CSF also enhances M-CSF induced monocytopoiesis from hematopoietic progenitor cells and stimulates the proliferation of peripheral Th2-inducing dendritic cells (30, 31). It promotes the development of T cell immune tolerance as well as tissue recovery following myocardial infarction and cerebral ischemia.

Long Name:

Granulocyte Colony Stimulating Factor

Entrez Gene IDs:

1440 (Human); 12985 (Mouse)

Alternate Names:

C17orf33; chromosome 17 open reading frame 33; colony stimulating factor 3 (granulocyte); CSF3; CSF3OS; Filgrastim; GCSF; G-CSF; GCSFlenograstim; granulocyte colony-stimulating factor; Lenograstim; MGC45931; Pluripoietin

Related Research Areas

Assay Procedure

These assays utilize an anti-tag coated microplate. Standards, samples, and controls are added to plate, followed by subsequent addition of an antibody cocktail. Following a 1 hour incubation, plates are washed and substrate is added and incubated for 20 minutes. Stop solution is then added and plates are read using a standard microplate reader.

QuicKit ELISA assay procedure