Aug 18, 2020
Copy of Direct ELISA for investigating the binding of chemically-made Protein-LAG to immunoglobulins.
- 1University of the West Indies St. Augustine
Protocol Citation: Angel A Justiz-Vaillant 2020. Copy of Direct ELISA for investigating the binding of chemically-made Protein-LAG to immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjxxkppn
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 18, 2020
Last Modified: August 18, 2020
Protocol Integer ID: 40663
Abstract
Protein LAG (SpLAG) is an immunoglobulin-binding protein that interacts with the Fc and Fab regions of many mammalian immunoglobulins. It is produced by a chemical coupling of individual proteins and them mixing it up to the appropriate protein ratio. SpLAG binds well to some avian immunoglobulin [1].
References
1.Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175
Materials
MATERIALS
Horseradish peroxidase (HRP)Gold BiotechnologyCatalog #P-100
Nunc™ 96-Well Polystyrene Round Bottom Microwell Plates, V 96 well plate, Non-Treated, clear, without lid, SterileThermo FisherCatalog #260210
Staphylococcal Protein-ASigma Aldrich
Protein-L from P. Magnus
Streptococcal protein G by Sigma Aldrich
This ELISA is used to study the interaction of protein-LAG (SpLAG) with diverse immunoglobulins.
The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified immunoglobulins or 50 µl of any animal sera in carbonate-bicarbonate buffer pH 9.6.
Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
Then 50 µl of peroxidase-labeled-protein-LAG conjugate diluted 1:5000 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 should be calculated as the mean of the optical density of negative controls x 2 SD.