Aug 14, 2020
Sandwich ELISA for investigating the binding of Protein-LG (SpLG) to avian immunoglobulins using anti-IgY-peroxidase as conjugate.
- Angel A Justiz-Vaillant1,
- Monica F. Smikle2
- 1University of the West Indies St. Augustine;
- 2University of the West Indies. Mona Campus
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant, Monica F. Smikle 2020. Sandwich ELISA for investigating the binding of Protein-LG (SpLG) to avian immunoglobulins using anti-IgY-peroxidase as conjugate.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjq4kmyw
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 14, 2020
Last Modified: August 14, 2020
Protocol Integer ID: 40444
Materials
MATERIALS
Anti-Chicken IgY, HRP Conjugate, 300ulPromegaCatalog #G1351
Nunc™ 96-Well Polystyrene Round Bottom Microwell Plates, V 96 well plate, Non-Treated, clear, without lid, SterileThermo FisherCatalog #260210
Protein-L from P. Magnus
Streptococcal protein G by Sigma Aldrich
This ELISA is used to study the interaction of protein-LG (SpLG) with diverse avian immunoglobulins.
The 96 well microtitre plate is coated overnight at 4°C with 2 µg/µl per well of recombinant SpLG or a mixture of SpL with 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 or egg white (1 mg/ml) is added and incubated for 1.30h at RT and the microplate is then rewashed 4X with PBS-Tween.
Then 50 µl of peroxidase-labeled-anti-IgY conjugate diluted 1:15000 in PBS-non-fat milk is added to each well and incubated for 1.30h at RT. After that the plate is washed 4X with PBS-Tween.
Pipette 50 μl of 3,3',5,5' - tetramethylbenzidine (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 can be calculated as the mean of the optical density of negative controls x 3. The higher the OD value the higher will be the binding affinity of SpLG to avian immunoglobulins.