Human GM-CSF ELISA Assay Kit

$390.00

The Eagle Biosciences Human Granulocyte macrophage colony stimulating factor (GM-CSF) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Granulocyte macrophage colony stimulating factor (GM-CSF) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Human Granulocyte macrophage colony stimulating factor (GM-CSF) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: MSF31-K01 Categories: , ,

Human GM-CSF ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 1000 pg/ml
Incubation Time: 3 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µL

Additional Information

Assay Principle

The Eagle Biosciences Human Granulocyte macrophage colony stimulating factor (GM-CSF) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for GM-CSF has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any GM-CSF present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for GM-CSF is added to the wells and binds to the combination of capture antibody- GM-CSF in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of GM-CSF present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven GM-CSF standard dilutions and GM-CSF sample concentration determined.

  1. Prepare all reagents and working standards as directed in the previous sections.
  2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
  3. Add 100µL of Standard, control, or sample, per well. Cover with the adhesive strip provided.
  4. Incubate for 1.5 hours at 37C.
  5. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350µL) using a squirt bottle, manifold dispenser or auto-washer. 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.
  6. Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37C.
  7. Repeat the aspiration/wash.
  8. Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37C. Avoid placing the plate in direct light.
  9. Repeat the aspiration/wash.
  10. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37C. Avoid placing the plate in direct light.
  11. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  12. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. (optionally 630nm as the reference wave length;610-650nm is acceptable)

Typical Standard Curve

human-gm-csf-elisa-kit


Assay Background

Granulocyte macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine with multiple effects on hematopoietic cells (1-4). It mobilizes CD34+ progenitor cells into the periphery and stimulates their proliferation, survival and differentiation into neutrophils, monocytes/ macrophages, eosinophils, and myeloid dendritic cells (4-8). On these terminally differentiated myeloid cells, GM-CSF is also needed for inducing their effector functions (7-10). In addition, GM-CSF has been shown to stimulate the proliferation and differentiation of the erythroid and megakaryoctye progenitor cells (4).

GM-CSF is produced by a number of different cell types, including keratinocytes, mature and immature NK cells, type II alveolar cells), endothelial cells, monocytes, bone-marrow mesenchymal stem cells, CD4+ and CD8+ T cells, megakaryocytes, B cells, eosinophils, chondrocytes and fibroblasts.

Human GM-CSF cDNA encodes a 144 amino acid (aa) residue precursor protein with a 17 aa putative signal peptide and a 127 aa mature protien (11 – 13). Natural GM-CSF is a monomer that contains both N- and O-linked.

Manual

Product Manual