Measurement of Macrophage-Produced Cytokines Using ELISA

2024-09-13

Measurement of Macrophage-Produced Cytokines Using ELISA

Goal: To understand cytokine production by macrophages. 

Macrophages can indicate different inflammatory responses, depending on the type of stimulation. 

The cytokines I measured (e.g., TNF-α, IL-6, IL-10) are crucial mediators in immune responses and are secreted by macrophages during inflammation.

Throughout the procedure, washing of the wells was necessary to remove non-specific material, ensuring that only specific interactions were detected. Serial dilutions of TNF-α standard were prepared to generate a standard curve, which was used to estimate the concentration of TNF-α in the unknown samples.

To quantify TNF-α levels, the sandwich ELISA technique was used. This method relies on antibodies specific to TNF-α: a capturing antibody immobilizes the antigen on the well surface, while a detection antibody binds to a different epitope on the same antigen. After a series of washing and incubation steps, a substrate is added that reacts with an enzyme linked to the detection antibody, producing a color change. 

Intensity of the color ∝ the amount of TNF-α in the sample

The absorbance is measured using a spectrophotometer.

The steps involving the 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate need to be done in the dark because TMB is light-sensitive. Exposure to light can cause premature oxidation of the TMB, leading to non-specific color development and inaccurate results.

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