Aug 19, 2020
Direct ELISA for investigating the binding of peroxidase-labeled anti-chicken IgY conjugate with avian 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 peroxidase-labeled anti-chicken IgY conjugate with avian immunoglobulins. protocols.io https://dx.doi.org/10.17504/protocols.io.bjxykppw
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: 40664
Abstract
The peroxidase-labeled anti-chicken IgY conjugate cross-reacts with many IgY present in the egg of many and diverse avian species.
References
1. Justiz Vaillant AA, Ramirez N, Cadiz A, Ferrer B, Akpaka P, et al. (2013) Separation and Reactivity of Avian Immunoglobulin Y. J Chromat Separation Techniq 4: 173. doi:10.4172/2157-7064.1000173
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
This ELISA is used to study the interaction of anti-chicken IgY-HRP conjugate with diverse avian immunoglobulins.
The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified avian immunoglobulins or 50 µl of water soluble fraction from egg yolks of avian species 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-anti-chicken 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 should be calculated as the mean of the optical density of negative controls x 2 SD.