Aug 19, 2020
Direct ELISA for investigating the binding of chemically-made Protein-LAG-anti-IgY-peroxidase to both avian and mammalian immunoglobulins.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Direct ELISA for investigating the binding of chemically-made Protein-LAG-anti-IgY-peroxidase to both avian and mammalian immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjxzkpp6
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 19, 2020
Last Modified: August 19, 2020
Protocol Integer ID: 40665
Abstract
Peroxidase-labeled-protein-LAG-anti-IgY conjugate is chemically made. It has unique binding properties. It binds to both avian and mammalian immunoglobulins. It can be used as a reagent in ELISA, Western blotting and dot blot for immunodetection of immunoglobulins and antigens. It may be used to make the immunodiagnosis of infectious diseases involving laboratory, wild, zoo, and farm animals [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
Anti-Chicken IgY, HRP Conjugate, 300ulPromegaCatalog #G1351
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-anti-IgY-HRP (SpLAG-anti-IgY-HRP) with diverse immunoglobulins.
The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified immunoglobulins, 50 µl of any animal sera, or 50 µl of water soluble fraction from egg yolks 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-anti-IgY 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.