Aug 11, 2020
Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-Salmonella antibody in layer hen's egg yolks.
- Angel Justiz-Vaillant1
- 1University of The West Indies. St. Augustine. Trinidad and Tobago.
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel Justiz-Vaillant 2020. Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-Salmonella antibody in layer hen's egg yolks.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjj3kkqn
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 11, 2020
Last Modified: August 11, 2020
Protocol Integer ID: 40283
Abstract
Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-Salmonella antibody in layer hens was a reproducible and feseable test used to meassure IgY development after vaccination.
Guidelines
After adding the substrate, keep the ELISA plate in a very dark place to get reproducible results.
Materials
MATERIALS
Anti-Chicken IgY, HRP Conjugate, 300ulPromegaCatalog #G1351
LPSSigma-aldrichCatalog #L3129
eBioscience™ TMB Solution (1X)Thermo FisherCatalog #00-4201-56
Nunc™ 96-Well Polystyrene Round Bottom Microwell Plates, U 96 well plate, Non-Treated, clear, with lid, SterileThermo FisherCatalog #268200
ELISA Coating Buffer (5X)BioLegendCatalog #421701
U-shaped bottom's ninety-six well polystyrene microplate purchased at Sigma-Aldrich, St. Louis USA was incubated with (2 µg/well) of the LPS (Sigma –Aldrich) from Salmonella Typhimurium in coating buffer (overnight at 4 ºC.)
The microtiter plates was washed four times, with 10 % PBS-Tween-20.
The microplate was blocked with 3% non-fat milk in PBS (25 µl/well).
The microplate was incubated 1 hr at RT .
The microplate was washed four times.
Then a 50 µl aliquot of the egg yolk (Ig)Y solutions in a concentration of 1.25 mg/ml was added in triplicate. The IgY concentration was assessed by ELISA and sample were titrated with sample buffer until it got the expected IgY concentration.
After incubating for one hour at RT, the microplate was washed four times.
Fifty (50 µl) of the anti-IgY-HRP conjugate (Sigma-Aldrich) diluted to 1:30000 with conjugate diluent was added into each well.
The microplate was incubated for 1 hr at RT.
Then, the microplate was washed four times.
Fifty (50 µl) of tetramethylbenzidine (TMB, Sigma-Aldrich) was added into each well.
The microplate was further incubated for 15 minutes in the dark.
Fifty (50 µl) 3M HCl was added to the microplate for stopping the reaction.
After that, reaction color development was measured with a microplate reader (Synergy™ Neo Hybrid Multi-Mode Microplate Reader).
The cut-off point was an OD of 0.51, and it was calculated from the XOD of the negative control times 3. This ELISA tested triplicates of a total of 90 IgY preparations.