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A biochemical assay to determine protein concentrations

Today’s experiment was a biochemical assay to determine the concentration of a protein released by brain cells following their pro-inflammatory activation with a bacterial component. To do that, I treated the cells in culture with the appropriate compounds and incubated them overnight. Then, I collected whatever the cells released and used these samples in a sandwich ELISA (Enzyme-Linked Immunosorbent Assay) to detect whether or not the protein of interest was one of the things that the cells released, and how much of it they released. This will show how pro-inflammatory the cells became after the treatment.

Timelapse: loading the samples into a plate pre-coated with the capture antibody.

What is a sandwich ELISA and how does it work?

This biochemical assay is used to detect the presence of an antigen (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. The steps of a sandwich ELISA are as follows: First, a plate is prepared to which a known quantity of capture antibody is bound. Then, any nonspecific binding sites on the surface are blocked. The antigen-containing sample is then added to the plate, and captured by the antibody. The plate is washed to remove any unbound antigen. Then, a specific antibody is added, and binds to the antigen (hence the ‘sandwich’: the antigen is stuck between two antibodies). Enzyme-linked secondary antibodies are applied as detection antibodies that also bind specifically to the antibody. The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. The plate is then washed again to remove the unbound antibody-enzyme conjugates. Finally, a chemical is added to be converted by the enzyme into a colour signal. The absorbance of the plate is measured by absorption spectroscopy to determine the presence and quantity of the antigen.

Colour change before and after addition of an acid. The colour intensity is proportional to the concentration of the protein.
Colour change before and after addition of an acid. The colour intensity is proportional to the concentration of the protein.