Aug 21, 2020
ELISA for quantification of IL-22 in human serum.
- 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-22 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj3rkqm6
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: August 21, 2020
Last Modified: August 21, 2020
Protocol Integer ID: 40785
Abstract
Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces.
Different cells in both innate and acquired immunities produce IL-22, but the primary sources are T cells. Th22 cell is a new line of CD4+ T cells, which differentiated from naive T cells in the presence of various pro-inflammatory cytokines including IL-6. IL-22 inhibits IL-4 production. It also has essential functions in mucosal surface protection and tissue repairy. [1]
Reference
1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.
An anti-human IL-22 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C.
Add 50 µl of human serum. Human IL-22 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 wash to remove unbound proteins.
Fifty (50) µl of biotin-conjugated anti-human IL-22 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-human IL-22 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 15 min.
A colored product is formed in proportion to the quantity of human IL-22 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 IL-22 standard dilutions and the human IL-22 sample concentration is determined.
For better results place the microplate on a microplate shaker in every incubation.