Aug 13, 2020
Enzyme linked immunosorbent assay for investigating the immunoglobulin-binding bacterial protein (IBBP) to avian egg yolk antibodies.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Enzyme linked immunosorbent assay for investigating the immunoglobulin-binding bacterial protein (IBBP) to avian egg yolk antibodies.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjnzkmf6
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: 40377
This ELISA is used to study the interaction of proteins A, L and LA with different avian IgY preparations.
The 96 well microtitre plate is coated overnight at 4°C with 1 µl/mg per well of unlabelled SpL, SpA or SpLA in carbonate-bicarbonate buffer pH 9.6.
Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
50 µl of different IgY preparations (1 mg/ml) are added and incubated for 1h at room temperature and washed.
Then 50 µl of rabbit anti-chicken IgG-HRP conjugate diluted 1:15000 in PBS-non-fat milk is added to each well and incubated for 1h at RT. The plate is washed 4X with PBS-Tween.
50 µl of 4 mg/ml o-phenylenediamine solution (OPD) is added and the plates are incubated 15 minutes at RT.
The reaction is stopped with 50 µl of 3M H2SO4 solution.
The plate is visually assessed for the development of colour.
The plate is read in a microplate reader at 492 nm.
A cut-off point is calculated.