Aug 13, 2020

Public workspaceUniversal sandwich enzyme linked immunosorbent assay for investigating protein-AG (SpAG) interactions with immunoglobulins using a SpL-HRP conjugate.

DOI
dx.doi.org/10.17504/protocols.io.bjpxkmpn
  • 1University of the West Indies St. Augustine;
  • 2University of West Indies. Mona Campus
  • University of the West Indies
  • angel.vaillant@sta.uwi.edu
Angel A Justiz-Vaillant
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DOI: dx.doi.org/10.17504/protocols.io.bjpxkmpn
Protocol Citation: Angel A Justiz-Vaillant, Norma McFarlane-Anderson 2020. Universal sandwich enzyme linked immunosorbent assay for investigating protein-AG (SpAG) interactions with immunoglobulins using a SpL-HRP conjugate.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjpxkmpn
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: 40407
This ELISA is used to study the interaction of a protein-AG (SpAG) with different immunoglobulin preparations from mammalian species.
The 96 well microtitre plate is coated overnight at 4°C with 2 µg/µl per well of a mixture of protein-A and protein-G 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 animal serum (1 mg/ml) is added and incubated for 1h at room temperature and the microplate is rewashed 4X with PBS-Tween.
Then 50 µl of peroxidase-labeled SpL conjugate diluted 1:3000 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 plate is incubated 15 minutes at RT in the dark.
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 492 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 SpAG to mammalian immunoglobulins.