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EK000067-EK0366: Rat GM-CSF ELISA Kit
Range 15.6pg/ml-1000pg/ml
Sensitivity < 1 pg/ml
Specificity: No detectable cross-reactivity with any other cytokine.
Application: For quantitative detection of rat GM-CSF in sera, plasma, body fluids, tissue lysates or cell culture supernates.
Expiration: Four months at 4°C and eight months at -20°C.
Background
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also symbolized CSF2. Human GM-CSF is a glycoprotein that is essential for the in vitro proliferation and differentiation of precursor cells into mature granulocytes and macrophages. The human cDNA clones contain a single open-reading frame encoding a protein of 144 amino acids with a predicted molecular mass of 16,293 daltons and show 69% nucleotide homology and 54% amino acid homology to mouse GM-CSF. The gene for human GM-CSF appears to exist as a single-copy gene.1 Human GM-CSF is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. The GM-CSF gene is localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia.2 A complementary DNA for the T lymphocyte-derived lymphokine, GM-CSF has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulates peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375.3 The standard product used in this kit is recombinant mouse GM-CSF, consisting of 125 amino acids with the molecular mass of 14.8KDa.
Principle
The ELISA Kit is based on standard sandwich enzyme-linked immune-sorbent assay technology. The target specific antibodies are precoated onto 96-well plates. The target from the sample is bound to the microwell. The biotinylated target specific detection antibodies are added to the microwells and followed by washing with the PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with the PBS or TBS buffer. TMB, the HRP (horseradish peroxidase) substrate, is used to visualize color change resulting from the HRP enzymatic reaction. TMB is catalyzed by HRP to produce the blue color. The color changes into yellow after the acidic stop solution is added. The density of the yellow color is proportional to the target amount from the sample captured in the microwells.
Reference
1. Cantrell, M. A.; Anderson, D.; Cerretti, D. P.; Price, V.; McKereghan, K.; Tushinski, R. J.; Mochizuki, D. Y.; Larsen, A.; Grabstein, K.; Gillis, S.; Cosman, D. Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor. Proc. Nat. Acad. Sci. 82: 6250-6254, 1985.
2. Huebner, K.; Isobe, M.; Croce, C. M.; Golde, D. W.; Kaufman, S. E.; Gasson, J. C. The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly. Science 230: 1282-1285, 1985.
3. Grabstein, K. H.; Urdal, D. L.; Tushinski, R. J.; Mochizuki, D. Y.; Price, V. L.; Cantrell, M. A.; Gillis, S.; Conlon, P. J. Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor. Science 232: 506-508, 1986.
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