I have recently peformed an ELISA (Enzyme-linked immunosorbentassay), but I still have some questions, let me first outline whatI 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 'kissingdisease' (so we basically conducted an experiment to see how fastthe virus would spread within a class if everybody started kissingeach 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 differentpeople in it.
Since we worked in pairs of 2, every duo got a microtiter platewith 12 wells in it: 3 wells for a positive control, 3 wells for anegative control, 3 wells for the fluids of 1 of the duo and 3wells for the fluids of the other one. You pippete in (is thatcorrect?) the fluids accordingly.
After a while you rinse and clean the wells with washbuffer.
Then you pippete in the primary antibody in each well.
After a while you rinse and clean the wells with washbuffer.
Following this you pippete in the secondary antibody (conjugatedwith the enzyme HRP).
After a while you rinse and clean the wells with washbuffer.
Finally, you add the enzyme substrate (TMB) to each well. Thiscauses a blue color if when in contact with HRP. The positivecontrols must come out blue, the negative must come out colorless,and the other wells can go either way, depending if you have thevirus.
I hope this is not to crudely explained, again. These are myquestions concerning the experiment:
What do the components of the acronym 'ELISA' actually standfor? Does immunosorbent refer to the binding of the antigens to thewells? My English isn't too good, and these acronyms andabbreviations often seem foreign to me (nevermind that they areforeign since I'm not English, you get the point I hope).
When you pippete in the saliva in the wells, what actuallysticks to the wells? All of the antigens in your saliva? Becauseit's hard to believe that they make special microtiter plates forevery single antigen seperately. I assume it is only the antigens,since the primary antibodies attach to the antigens only, so thereis no need for the microtiter plate itself to bind toantibodies.
How would we get the primary and secondary antibody? This is myassumption: The primary antibody we get is by injecting someantigens of the Epstein-Barr Virus into a human and then taking aserum. The secondary antibody could be gotten by injecting a randomhuman primary antibody into ANY animal, irrelevant which anyimal,and then taking a serum. After that you conjugate an enzyme to thesecondary antibody. Is this correct?
I have recently peformed an ELISA (Enzyme-linked immunosorbentassay), but I still have some questions, let me first outline whatI 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 'kissingdisease' (so we basically conducted an experiment to see how fastthe virus would spread within a class if everybody started kissingeach 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 differentpeople in it.
Since we worked in pairs of 2, every duo got a microtiter platewith 12 wells in it: 3 wells for a positive control, 3 wells for anegative control, 3 wells for the fluids of 1 of the duo and 3wells for the fluids of the other one. You pippete in (is thatcorrect?) the fluids accordingly.
After a while you rinse and clean the wells with washbuffer.
Then you pippete in the primary antibody in each well.
After a while you rinse and clean the wells with washbuffer.
Following this you pippete in the secondary antibody (conjugatedwith the enzyme HRP).
After a while you rinse and clean the wells with washbuffer.
Finally, you add the enzyme substrate (TMB) to each well. Thiscauses a blue color if when in contact with HRP. The positivecontrols must come out blue, the negative must come out colorless,and the other wells can go either way, depending if you have thevirus.
I hope this is not to crudely explained, again. These are myquestions concerning the experiment:
What do the components of the acronym 'ELISA' actually standfor? Does immunosorbent refer to the binding of the antigens to thewells? My English isn't too good, and these acronyms andabbreviations often seem foreign to me (nevermind that they areforeign since I'm not English, you get the point I hope).
When you pippete in the saliva in the wells, what actuallysticks to the wells? All of the antigens in your saliva? Becauseit's hard to believe that they make special microtiter plates forevery single antigen seperately. I assume it is only the antigens,since the primary antibodies attach to the antigens only, so thereis no need for the microtiter plate itself to bind toantibodies.
How would we get the primary and secondary antibody? This is myassumption: The primary antibody we get is by injecting someantigens of the Epstein-Barr Virus into a human and then taking aserum. The secondary antibody could be gotten by injecting a randomhuman primary antibody into ANY animal, irrelevant which anyimal,and then taking a serum. After that you conjugate an enzyme to thesecondary antibody. Is this correct?
