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

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. Natural GM-CSF is a monomer that contains both N- and O-linked glycosylation. Mature human GM-CSF shares

approximately 55%, 63% and 68% aa sequence homology with mouse, rat and canine. GM-CSF, respectively. Human GM-CSF is not biologically active on mouse cells, but was reported to have some activity on canine cells.

GM-CSF exerts its activity through binding to a high affinity receptor complex consisting of two membrane glycoproteins. The presence of the spliced variants in the heteromeric receptor complex can regulate the functions of the complex.

This assay 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 coloured 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.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human GM-CSF (812)

2. 2 vials human GM-CSF Standard lyophilized, 2000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human GM-CSF monoclonal antibody

4. 2 vials Streptavidin-HRP solution,

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

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