Aug 15, 2020
Universal sandwich ELISA for investigating the binding of avian and mammalian immunoglobulins to Streptococcal protein-G (SpG) using a peroxidase-labeled Protein LAG conjugate (SpLAG-HRP).
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Universal sandwich ELISA for investigating the binding of avian and mammalian immunoglobulins to Streptococcal protein-G (SpG) using a peroxidase-labeled Protein LAG conjugate (SpLAG-HRP).. protocols.io https://dx.doi.org/10.17504/protocols.io.bjsfknbn
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: 40487
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Materials
MATERIALS
Anti-Chicken IgY, HRP Conjugate, 300ulPromegaCatalog #G1351
Streptococcal protein G by Sigma Aldrich
This ELISA is used to study the interaction of streptococcal protein-G (SpG) with different avian and mammalian immunoglobulins.
The 96 well microtitre plate is coated overnight at 4°C with 2 µg/µl per well of SpG 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 avian egg yolk, egg white (1:8 dilutions) or 50 µl of sera or 50 µl of mammalian IgG (1mg/ml) is added to the well and incubated for 1.30h at room temperature and the microplate is then rewashed 4X with PBS-Tween.
Then 50 µl of peroxidase-labeled-Protein-LAG (SpLAG-HRP) conjugate diluted 1:15000 in PBS-non-fat milk is added to each well and incubated for 1.30h at RT. The plate is washed 4X with PBS-Tween.
Pipette 50 μl of TMB (Sigma-Aldrich) to each well.
The reaction is stopped with 50 µl of 3M H2SO4 solution.
The plate is visually assessed for the development of colour and read in a microplate reader at 450 nm.
A cut-off point should be calculated as the mean of the optical density of negative controls x 3. The higher the OD value the higher will be the affinity of SpG to immunoglobulins.