Questions regarding ELISA

Question

I have recently peformed an ELISA (Enzyme-linked immunosorbent assay), but I still have some questions, let me first outline what I did:

• We had a number (20) of tubes containing fake 'bodily fluids' , namely saliva. One of these tubes contained the Epstein-Barr virus, which on its turn causes Pfeiffer's disease, aka the 'kissing disease' (so we basically conducted an experiment to see how fast the virus would spread within a class if everybody started kissing each other, if you don't mind the crudity).

• Everybody mixed their saliva with the saliva of 3 other people, so at the end everybody had a tube with the saliva of 4 different people in it.

• Since we worked in pairs of 2, every duo got a microtiter plate with 12 wells in it: 3 wells for a positive control, 3 wells for a negative control, 3 wells for the fluids of 1 of the duo and 3 wells for the fluids of the other one. You pippete in (is that correct?) the fluids accordingly.

• After a while you rinse and clean the wells with wash buffer.

• Then you pippete in the primary antibody in each well.

• After a while you rinse and clean the wells with wash buffer.

• Following this you pippete in the secondary antibody (conjugated with the enzyme HRP).

• After a while you rinse and clean the wells with wash buffer.

• Finally, you add the enzyme substrate (TMB) to each well. This causes a blue color if when in contact with HRP. The positive controls must come out blue, the negative must come out colorless, and the other wells can go either way, depending if you have the virus.

I hope this is not to crudely explained, again. These are my questions concerning the experiment:

1. What do the components of the acronym 'ELISA' actually stand for? Does immunosorbent refer to the binding of the antigens to the wells? My English isn't too good, and these acronyms and abbreviations often seem foreign to me (nevermind that they are foreign since I'm not English, you get the point I hope).

2. When you pippete in the saliva in the wells, what actually sticks to the wells? All of the antigens in your saliva? Because it's hard to believe that they make special microtiter plates for every single antigen seperately. I assume it is only the antigens, since the primary antibodies attach to the antigens only, so there is no need for the microtiter plate itself to bind to antibodies.

3. How would we get the primary and secondary antibody? This is my assumption: The primary antibody we get is by injecting some antigens of the Epstein-Barr Virus into a human and then taking a serum. The secondary antibody could be gotten by injecting a random human primary antibody into ANY animal, irrelevant which anyimal, and then taking a serum. After that you conjugate an enzyme to the secondary antibody. Is this correct?

Answer 1

ELISA is Enzyme-Linked ImmunoSorbent Assay (capitalisation to point out source of acronym).

You can read about the various ways to do an ELISA here.

In your case this was probably an indirect ELISA. All ELISA techniques make use of the fact that proteins stick to plastic. In your case the protein components of the saliva, including the virus (the viral coat is proteinaceous) will stick to the wells. After rinsing out the sample the wells are treated with another protein (usually bovine serum albumin (BSA)) to completely block all available protein binding sites. At this stage these wells will no longer bind protein non-specifically, but will bind anti-EBV Ig because of the presence of the EBV particles bound to the plastic. This bound anti-EBV can then be detected with a secondary antibody. The protein complexes will build as follows:

plastic/EBV/rabbit anti-EBV/goat anti-rabbit IgG-HRP conjugate

It would also be possible to do this experiment as a sandwich ELISA. In this format the wells would have been pretreated with anti-EBV serum (or purified anti-EBV IgG) then blocked with BSA. At this stage these wells will no longer bind protein non-specifically, but will bind EBV because of the presence of the EBV-specific antibody. Once samples have been incubated in these wells (so that any EBV can bind to the antibody coating the wells) another antibody (raised in a different animal) is added, and this will bind to the EBV that has been captured by the original antibody that coats the wells. This latter antibody is then detected with a conjugate. The protein complexes will build as follows:

plastic/mouse anti-EBV/EBV/rabbit anti-EBV/goat anti-rabbit IgG-HRP conjugate

Your description of how to get the antibodies is basically correct, except that the primary antibody will probably have been raised in rabbits or mice, and certainly not in humans.

Added later: I didn't read the first question carefully enough. An ELISA is an assay because it measures something. It is based on a binding reaction mediated by an antibody-antigen interaction (immunosorbent). It is enzyme-linked because the secondary antibody is present as an antibody-enzyme conjugate. It is the amount of this conjugate that remains bound at the final step which is what is measured - the assay is set up so that the amount of this enzyme is a measure of the amount of the original molecule that is being assayed.