Aug 11, 2020
Competitive ELISA to study the inhibition of the SpA- binding to Ab-3 by Ab-2.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Competitive ELISA to study the inhibition of the SpA- binding to Ab-3 by Ab-2.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjkbkksn
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: 40291
Abstract
This immunoassay tested the ability of anti-anti-SpA (Ab-2) to inhibit or interfere with the binding of anti-anti-anti-SpA (Ab-3) to the original antigen- staphylococcal protein A (SpA). It is a confirmation test of the functional capacity of Ab-2. In this ELISA, both Ab-2 and the antigen SpA compete for binding to Ab-3. When Ab-2 is present, it blocks the binding of HRP-labeled SpA to Ab-3, resulting in inhibition of reaction color development.
Guidelines
Use appropriate positive and negative controls, as well as blanks.
Materials
MATERIALS
Substrate (TMB)Millipore SigmaCatalog #EZMADP-60K(kit)
Stop Solution (0.3M HCl)Millipore SigmaCatalog #EZMADP-60K(kit)
96-Well Microtiter™ Microplates, Polystyrene, 280µL, Nonsterile, V-bottomThermo FisherCatalog #2605
Pierce™ Recombinant Protein A, Peroxidase ConjugatedThermo FisherCatalog #32400
Safety warnings
Do not use any reagent where damages to the packaging has occurred.
Prepare materials and ELISA buffer solutions and reagents.
Pipette 53 μl of purified Ab-3 (100 μg/ml) mixed with 2.8 ml of coating buffer into each well.
Incubate the microplate at 37°C for 4 hrs.
Aspirate the contents of the wells.
Fill each well with an appropriately diluted washing solution and aspirate.
Wash the microplate 3 times.
Pipette 53 μl of serial dilutions of pooled Ab-2 (100 μg/ml) in triplicates.
Incubate the microplate at RT for 1 hr.
Rewash the microplate filling each well with 100 μl of washing buffer.
Pipette 53 μl of commercially available peroxidase-labeled SpA conjugate (Sigma-Aldrich) diluted 1:5000 to each well.
Reincubate the microplate at RT for 1 hr.
Repeat step 5.
Pipette 53 μl of TMB (Sigma-Aldrich) to each well.
Incubate in the dark at RT for 14 min.
Measure absorbance at 450 nm in a microplate reader and analyze the results