Aug 12, 2020
ELISA for anti-SpA antibodies in hyper-immune egg whites.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. ELISA for anti-SpA antibodies in hyper-immune egg whites.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjnrkmd6
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 12, 2020
Last Modified: August 12, 2020
Protocol Integer ID: 40369
Materials
MATERIALS
96-Well Microtiter™ Microplates, Polystyrene, 330µL, Nonsterile, Flat-bottomThermo FisherCatalog #9205
3 3′5 5′-Tetramethylbenzidine Liquid Substrate Supersensitive for ELISASigma AldrichCatalog #T4444-100ML
Staphylococcal Protein-ASigma Aldrich
Anti-Protein-A
The 96 well polystyrene microplate (U-shaped bottom) is coated with 500 ng of SpA (Sigma-Aldrich) in coating buffer for 4 h at 37oC.
The microplate is washed four times with PBS-Tween-20 and blocked with 3% non-fat milk in PBS, 25 μl/well, 1h, RT.
The microplate is washed four times again.
Samples of egg whites are added in 1:10 dilutions.
After incubation for 1h at RT the microplate is washed four times.
Then, 50 μl of peroxidase-labelled protein-A diluted 1:3000 (Sigma-Aldrich) is added.
The microplate is incubated for 1h at RT.
The microplate is washed four times.
Tetramethylbenzidine (TMB) solution (50 μl) is added to each well.
Perform a further incubation of 15 min in the dark
The reaction is stopped with 3M H2SO4.
Read the microplate in a microplate reader at 450 nm.
Calculate the cut-off point as appropriate.