Aug 15, 2020
Binding properties of Human IgG to immunoglobulin-binding proteins tested 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 Human IgG to immunoglobulin-binding proteins tested by double immunodiffusion (Ouchterlony) technique.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjskkncw
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Created: August 15, 2020
Last Modified: August 15, 2020
Protocol Integer ID: 40492
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Abstract
1. IBP-HRP conjugates can be prepared in the laboratory by the periodate method.
2. The Human IgG used can be commercially available of prepared in the laboratory from human sera by affinity chromatography (using an SpG-agarose column).
3. SpG-agarose column are widely commercially prepared.
4. PURE-1A antibody purification kit will be discontinued. However there are others product for this purpose as Montage Antibody Purification Kit with PROSEP-G media.
Materials
MATERIALS
Affinity Purification Using HiTrap™ Columns
Montage Antibody Purification Kit with PROSEP-G media
PURE-1A (antibody purification kit)-- Sigma Aldrich
The binding of Human IgG to peroxidase-conjugated Immunoglobulin-binding protein (IBP-HRP) including SpA-HRP, SpG-HRP, SpL-HRP, SpLA-HRP, SpAG-HRP, SpLG-HRP, SpLAG-HRP and turtle serum (Sigma-Aldrich) as a negative control 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 of purified human IgG (Sigma-Aldrich) at a concentration of one (1) µg/µl are applied to the centre well. Human IgG could be prepared homemade by purifying human serum by SpA-affinity chromatography (PURE-1A, Sigma-Aldrich).
The peripheral wells are filled with 25 µl each of bacterial protein conjugates including SpA-HRP, SpG-HRP, SpL-HRP, SpLA-HRP, SpAG-HRP, SpLG-HRP, SpLAG-HRP diluted 1:5.
The gels are incubated at RT for 48−72 hours incubated in a humidity chamber.
After that the gels are examined for precipitin lines.
Turtle serum is included as negative controls and all IBP-HRP conjugates suppose to react with human IgG.
The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.