Mar 31, 2025
Enzyme-linked immunosorbent assay (ELISA)
- 1Veterinary Medicine University
Protocol Citation: Karyna Tarasova 2025. Enzyme-linked immunosorbent assay (ELISA). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx7eydl8j/v1
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: March 31, 2025
Last Modified: March 31, 2025
Protocol Integer ID: 125756
Abstract
To analyze the expression of TNF- α, supernatants were collected at the end of each designated culture period, centrifuged and stored until subsequent enzyme-linked immunosorbent assay (ELISA). Total protein concentrations were determined by Bradford assay. The concentrations of TNF-α were measured using ELISA Quantitative Sandwich kits according to the manufacturer's instructions.
Materials
Bradford assay (5000006, Bio-Rad Laboratories, California, USA)
ELISA Quantitative Sandwich kits (MBS2701341, MyBiosource, San Diego, USA)
Safety warnings
The ELISA Quantitative Sandwich kit (MBS2701341) has high sensitivity and excellent specificity for detection of TNFa.
Before start
The ELISA Quantitative Sandwich kit (MBS2701341) was acquired from MyBiosource (San Diego, USA).
Sample preparation
Sample preparation
Sample preparation and total protein concentration determination.
Cell culture supernatant was collected.
The supernatant was centrifuged 2000g x 10min x 4oC. The samples were kept on ice.
Total protein concentrations were determined by Bradford assay (5000006, Bio-Rad Laboratories, California, USA) by measuring the absorvance (595 nm) with a plate reader (Varioskan LUX, Thermo Scientific, USA).
Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA)
The standard curve was prepared by serial diluting the stock standard solution in the diluent according to the manufacturer's instructions (MBS2701341, MyBiosource, San Diego, USA).
The standards, blank and samples were added into the pre-coated plate and incubate for 1h at 37oC.
The liquid of each well was removed without wash. 100μL of Detection Reagent A working solution was added to each well and incubated for 1 hour at 37oC.
The wells were washed with 1× Wash Solution three times. The remaining liquid from all wells was completely removed by snapping the plate onto absorbent paper.
100μL of Detection Reagent B working solution was added to each well and incubated for 30 minutes at 37oC.
The wells were washed with 1× Wash Solution three times. The remaining liquid from all wells was completely removed by snapping the plate onto absorbent paper.
90μL of Substrate Solution was added to each well and incubated for 20 minutes at 37oC.
Without removing or washing, 50μL of Stop Solution was added to each well.
The absorvance (450 nm) was measured immediately with a plate reader (Varioskan LUX, Thermo Scientific, USA). TNF-α levels were normalized to total protein concentrations.