Aug 13, 2020
Binding properties of immunoglobulin-binding protein 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 properties of immunoglobulin-binding protein by double immunodiffusion (Ouchterlony) technique. . protocols.io https://dx.doi.org/10.17504/protocols.io.bjqdkms6
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: 40421
The binding of SpLA, SpL and SpG with animal sera, avian IgY, avian egg whites and purified IgG are investigated by double immunodiffusion.
Briefly, 1% agarose gels are prepared and wells cut into the gel using a template.
Initially, aliquots of 25 µl each of SpLA, SpL or SpG at 1 µg/µl were applied to the centre well.
The peripheral wells are filled with 25 µl each of IgY (30 µg/µl), avian egg white diluted 1:2 in PBS pH 7.4, or animal serum.
The gels are incubated at RT for 48−72 hours.
After that the gels are examined for precipitin lines.
Human serum and human IgG are included as positive controls.
The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.
The experiments were repeated using concentrations of each bacterial Ig receptor and animal serum or purified immunoglobulin ranging from 1−51 µg/µl.