Problem:
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:
Question 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).
Question 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.
Question 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? Please describe your answer.