Aug 23, 2020
ELISA for quantification of tumor necrosis factor alpha (TNF-α) in 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 tumor necrosis factor alpha (TNF-α) in human serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj69krh6
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: Working
We use this protocol and it's working
Created: August 23, 2020
Last Modified: August 23, 2020
Protocol Integer ID: 40897
Abstract
Tumor necrosis factor (TNF) is a cell signaling cytokine involved in systemic inflammation. It is one of the proteins that make up the acute phase reaction [1].
Reference
1. Pasquereau S, Kumar A, Herbein G. Targeting TNF and TNF Receptor Pathway in HIV-1 Infection: from Immune Activation to Viral Reservoirs.Viruses. 2017;9(4):64. Published 2017 Mar 30. doi:10.3390/v9040064
An anti-human TNF-α 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. Human TNF-α present in the serum or plasma 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-TNF-α 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-TNF-α antibody. The optimal dilution of this conjugate must be investigated.
The plate is washed following incubation to remove the unbound Streptavidin-HRP.
Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
Incubate the microwells in the dark for 20 min.
A colored product is formed in proportion to the quantity of TNF-α 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 TNF-α standard dilutions and the human TNF-α sample concentration is determined.
For better results place the microplate on a microplate shaker in every incubation.