Aug 19, 2020
ELISA for quantification of IL-6 in human serum or plasma.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. ELISA for quantification of IL-6 in human serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjy7kpzn
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 19, 2020
Last Modified: August 19, 2020
Protocol Integer ID: 40703
Abstract
Interleukin-10 is a pleiotropic cytokine playing a critical role as a regulator of myeloid and lymphoid cell function. Due to the ability of IL-10 to blocking cytokine synthesis and many accessory cell functions of antigen-presenting cells such as macrophages this cytokine is a potent suppressor of the effector functions of macrophages, T-cells and NK cells. IL-10 also participates in regulating proliferation and differentiation of B-lymphocytes, mast cells and thymocytes [1].
Reference
1. Ouyang W, O'Garra A. IL-10 Family Cytokines IL-10 and IL-22: from Basic Science to Clinical Translation.Immunity. 2019;50(4):871-891. doi:10.1016/j.immuni.2019.03.020
Materials
MATERIALS
IL-10, Interleukin-10, humanBio Basic Inc.Catalog #RC212-21.SIZE.2ug
An anti-human IL-6 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
Add 50 µl of human serum. Human IL-6 present in the serum sample binds to antibodies adsorbed to the microwells.
The microplate is blocked with 3% non-fat milk-PBS buffer and later wasth o remove unbound proteins.
Fifty (50) µl of biotin-conjugated anti-human IL-6 antibody is added.
The microplate is rewashed with PBS-Tween buffer.
One hundred µl of streptavidin-HRP is added and binds to the biotin-conjugated anti-human IL-6 antibody.
The plate is washed following incubation to remove the unbound Streptavidin-HRP.
Add 50 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
Incubate the microwells in the dark for 15 min.
A colored product is formed in proportion to the quantity of human IL-6 present in the sample or standard.
The reaction is terminated by addition of 3M H2SO4 and absorbance is measured at 450 nm.
A standard curve is made from 7 human IL-6 standard dilutions and the human IL-6 sample concentration determined.
For better results place the microplate on a shaker in every incubation.