Aug 15, 2020
Binding of avian immunoglobulins to peroxidase-labeled anti-chicken IgY conjugate by double immunodiffusion (Ouchterlony) technique.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Binding of avian immunoglobulins to peroxidase-labeled anti-chicken IgY conjugate by double immunodiffusion (Ouchterlony) technique.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjsiknce
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
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Created: August 15, 2020
Last Modified: August 15, 2020
Protocol Integer ID: 40490
Abstract
In this experiment anti-chicken IgY developed in rabbit cross-reacts with a large panel of avian species including ostrich, duck, pigeon, and quail.
Materials
MATERIALS
Anti-Chicken IgY, HRP Conjugate, 300ulPromegaCatalog #G1351
The binding of avian immunoglobulins to a secondary antibody rabbit anti-chicken IgY antibody was investigated by the Ouchterlony technique.
Briefly, 1% agarose gels are prepared and wells cut into the gel using a template.
Initially, aliquots of 25 µl each of rabbit anti-IgY antibody at 1 µg/µl are applied to the centre well.
The peripheral wells are filled with 25 µl each of IgY (30 µg/µl) of various avian immunoglobulins Y (IgY) including duck, pigeon, chicken, bantam hen, ostrich, pheasant,guinea hen, goose, quail, cattle egret and other species. This IgY were isolated from the egg by the method of Polson, 1990.
The gels are incubated at RT for 48−72 hours on a humidity chamber.
After that the gels are examined for precipitin lines.
Chicken IgY is included as a positive controls.
The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.