In this 96 well plate the wells with the darker wells have a greater concentration of a specific protein than lighter wells. Any well with no color can be considered to have none of the specific protein.
Biologists who work with smaller organisms, like bacteria and cell cultures, or larger organisms, like mice and rats, need a way to really know if a specific enzyme or any molecule for that matter is actually there. For instance if we are looking for a protein that may be related to cancer we not only need to know if our organism has this protein, but also a way to quantify how much of the protein is there. Sometimes in biology the enzymes that lead to cancer are required for normal cell activity, but when expressed at greater amounts than normal they can cause serious problems.The ELISA assay is simple test that can fairly accurately predict the both the existence of our protein of interest, but also can measure the quantity of this protein.
ELISA is an acronym for enzyme linked immunosorbent assay, the technique utilizes our understanding of antibodies to bind our protein of interest as an antigen. You can read a bit more about antibodies from my other articles on the immune system, but in this assay they are stuck to a surface. This well plate has 96 wells each coated with an antibody that will bind only a specific antigen. For each ELISA each different protein we want to measure requires a specific plate with a specific antibody.
Lets look at an example where we are quantifying the amount of testosterone in blood plasma. Our blood plasma samples would be added in small amounts to the wells of our ELISA plate and given sufficient time for the antibodies to bind the testosterone hormone. Additionally we may add a solution containing a “detecting antibody”. After allowing the binding to take place our plate can be washed several times to remove all of the extra contents that did not bind. The washing process will flush out everything except our testosterone bound to the antibody on the plate and the detecting antibody also bound to the testosterone molecule. We call this a detecting antibody because it is important in allowing us to measure the quantity of testosterone. The detecting antibody is a primary antibody because it binds the testosterone hormone directly, but after the washing phase we will next add a secondary antibody. The secondary antibody labelled with some fluorphore or chromophore and is secondary because it binds the primary antibody rather than the testosterone hormone. The fluorophore or chromophore on the secondary antibody is what allows the detection of testosterone. By using a spectrophotometer or fluorometer a light of a specific wavelength will be absorbed and the absorbency can be measured. I will explain a bit more about these devices on a separate post, but now our testosterone hormone can be measure in terms of absorbency. Additionally to our well plate we would add several different known concentrations of pure testosterone to several wells as a control to ensure the plate is binding testosterone, but also to compare absorbance values when quantifying our protein concentration from our samples.
ELISA is an important technique in research as we are using new compounds to directly affect the concentrations of specific enzymes or other molecules, but also a key component of clinical biology. ELISA can be used to detect the presences of HIV antibodies of a suspected infected individual. It is used to detect endotoxins of bacterium in certain foods to guarantee the food has not been contaminated with deadly E. coli. Overall ELISA is a remarkably simple, but highly universal technique many biologists rely on everyday.