Aug 13, 2020
Competitive enzyme-linked immunosorbent assay for investigating SpL binding to mammalian and avian immunoglobulins
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Competitive enzyme-linked immunosorbent assay for investigating SpL binding to mammalian and avian immunoglobulins. protocols.io https://dx.doi.org/10.17504/protocols.io.bjqckmsw
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 13, 2020
Last Modified: August 13, 2020
Protocol Integer ID: 40420
Abstract
This ELISA was based on the theory that antibodies present in different samples would compete with human IgG for binding to SpL, resulting in inhibition of human IgG-SpL interactions [1].
Reference:
1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.West Indian Med J. 2013;62(1):12-20.
This ELISA is based on the theory that antibodies present in different samples would compete with human IgG for binding to SpL, resulting in inhibition of human IgG-SpL interactions.
The samples tested are commercially prepared pooled sera from skunk, coyote, raccoon, duck, and also commercially prepared purified immunoglobulins from cat and chicken (SigmaAldrich Co, St Louis, Missouri).
The microplate is coated with 50 µl of commercial human IgG (1 µg/well overnight at 4°C).
Serial doubling dilutions (1:4 to 1:1024) of 30 µl of each sample are made in a separate microplate to which 30 µl of the conjugate SpL-HRP diluted 1:1000 in non-fat milk is added.
The microplate is incubated for one hour at RT and then 50 µl of each sample is transferred to the human IgG coated microplate and incubated for one hour.
The microplate is then washed four times with PBS-Tween 20 buffer (SigmaAldrich Co, St Louis, Missouri), and 50 µl of the substrate OPD (3 mg/ml) is added to each well and incubated at RT for 15 minutes.
The reaction is stopped with 3M H2SO4 and the microplate is visually assessed and read at 492 nm.
The percentage of the binding inhibition (I%) of the SpL-human IgG interactions by different samples was calculated.