Mouse GM-CSF ELISA Assay
The Mouse GM-CSF ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 1000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Mouse Granulocyte macrophage colony-stimulating factor
The Eagle Biosciences Mouse 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.
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- Prepare all reagents and working standards as directed in the previous sections.
- 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.
- Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) 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.
- Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
- Repeat the aspiration/wash.
- 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 37°C. Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
- Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- 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)
Granulocyte macrophage colony-stimulating factor (GM-CSF), a 22 kDa glycosylated protein, was initially characterized as a factor that can support the in vitro colony formation of granulocyte macrophage progenitors. It is also a growth factor for erythroid, megakaryocyte, and eosinophil progenitors. GM-CSF is produced by a number of different cell types (including T cells, B cells, macrophages, mast cells, endothelial cells, fibroblasts, and adipocytes) in response to cytokine or inflammatory stimuli. On mature hematopoietic cells, GM-CSF is a survival factor for and activates the effector functions of granulocytes, monocytes/macrophages, and eosinophils (1, 2). GM-CSF promotes a Th1 biased immune response, angiogenesis, allergic inflammation, and the development of autoimmunity (3 5). It shows clinical effectiveness in ameliorating chemotherapy induced neutropenia, and GM-CSF transfected tumor cells are utilized as cancer vaccines (6, 7). Mature mouse GM-CSF shares 49% -54% amino acid sequence identity with canine, feline, human, and porcine GM-CSF and 69% with rat GM-CSF. GM-CSF exerts its biological effects through a heterodimeric receptor complex composed of GM-CSF Rα/CD116 and the signal transducing common β chain (CD131) which is also a component of the high affinity receptors for IL-3 and IL-5 (8,9). In addition, GM-CSF binds a naturally occurring soluble form of GM-CSF Rα (10). The activity of GM-CSF is species specific between human and mouse. Mouse GM-CSF is only weakly active on rat cells, although rat GM-CSF is fully active on mouse cells (11).
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
1. MartinezMoczygemba, M. and D.P. Huston (2003) J. Allergy Clin. Immunol. B 112:653.
2. Barreda, D.R. et al. (2004) Dev. Comp. Immunol. 28:509.
3. Eksioglu, E.A. et al. (2007) Exp. Hematol. 35:1163.
4. Cao, Y. (2007) J. Clin. Invest. 117:2362.
5. Fleetwood, A.J. et al. (2005) Crit. Rev. Immunol. 25:405.
6. Heuser, M. et al. (2007) Semin. Hematol. 44:148.
7. Hege, K.M. et al. (2006) Int. Rev. Immunol. 25:321.
8. OnettoPothier, N. et al. (1990) Blood 75:59.
9. Hayashida, K. et al. (1990) Proc. Natl. Acad. Sci. 87:9655.
10. Pelley, J.L. et al. (2007) Exp. Hematol. 35:1483.
11. Oaks, M.K. et al. (1995) J. Interferon Cytokine Res. 15:1095.