Sep 04, 2020
ELISA for quantification of granulocyte macrophage-colony stimulating factor (GM-CSF) in tissue culture supernatant, human serum or plasma.
- Angel A Justiz-Vaillant1,
- Belkis Ferrer-Cosme2
- 1University of the West Indies St. Augustine;
- 2"Saturnino Lora Torres" Provincial Teaching Clinical Surgical Hospital. Cuba
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant, Belkis Ferrer-Cosme 2020. ELISA for quantification of granulocyte macrophage-colony stimulating factor (GM-CSF) in tissue culture supernatant, human serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bktykwpw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: September 04, 2020
Last Modified: September 04, 2020
Protocol Integer ID: 41560
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Abstract
Granulocyte macrophage-colony stimulating factor (GM-CSF) is a monomeric glycoprotein. It is a cytokine secreted by macrophages, T cells, natural killer cells, mast cells, endothelial cells and fibroblasts. It acts as a growth factor. [1]
Reference
1. Egea L, Hirata Y, Kagnoff MF. GM-CSF: a role in immune and inflammatory reactions in the intestine. Expert Rev Gastroenterol Hepatol. 2010 Dec;4(6):723-31. doi: 10.1586/egh.10.73. PMID: 21108592; PMCID: PMC3291482.
An anti-human granulocyte macrophage-colony stimulating factor (GM-CSF) coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
Add 50 µl of human serum or plasma into the wells. GM-CSF present in the serum sample binds to antibodies adsorbed into the microwells.
The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
Fifty (50) µl of biotin-conjugated anti-GM-CSF antibody is added. The optimal dilution must be investigated.
The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-GM-CSF antibody.
The plate is washed following incubation to remove the unbound Streptavidin-HRP conjugate.
Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
Incubate the microwells in the dark for 15 min.
A colored product is formed in proportion to the quantity of GM-CSF present in the sample or standard.
The reaction is terminated by addition of 100 µl 3M H2SO4 and the absorbance is measured at 450 nm.
A standard curve is made from 7 human GM-CSF standard dilutions and the human GM-CSF sample concentration is determined.
For better results place the microplate on a microplate shaker in every incubation.