Need help with ELISA! I am a newbie! - (Feb/26/2008 )
Hi everyone!
I was told to perform ELISA (but no one in my lab has ever done it, and our technician is gone!). I am trying to detect the presence of an antigen (oviductin) in the medium that was used to culture a cell line (immortalized oviductal cell line).
I did a bit of research on the web, and I found there are different types of ELISA: in-direct, sandwich, competitive....
I wonder which ELISA should I use if my goal is to detect the presence of an antigen?
Given that I have the following in my lab:
- rabbit anti-human oviductin antibody against whole molecule (primary; polyclonal antibody)
- rabbit anti-human oviductin antibody against core protein (primary; polyclonal antibody)
- HRP-conjugated goat anti-rabbit IgG (secondary antibody)
So what reagent do I need to use in order to give the signal at the end?
Actually I also have - Biotinylated goat anti-rabbit IGg... can this be used in ELISA? 
I don't know if I have given enough information... 
Thank you so much for your time!!
I would recommend you sandwich ELISA. Much more sensitive than direct ELISA.
The bad point is that you can't do it with your antibodies. You should maybe biotinylate one.
So you would coat a plate with around 50 ng of a non biotinylated antibody (the best would be to biotinylate the polyclonal, but you have to try which one is best biotinylated), wash, block (try with BSA or non fat dry milk), wash, incubate with sample, wash, incubate with biotinylated antibody (you can't use a not biotinylated and then an anti-rabbit, because it would recognize the antibody used for the coating), wash, incubate with streptavidin-HRP, wash, add 100 µL of substrate TMB, let the blue color develop, and add H2SO4 2N (50 µL) to stop the reaction and read.
If you can't biotinylate your antibody, try direct ELISA by coating around 50-100 ng of your sample, and then block, incubate with antibody (you have to try all your antibodies), biotinylated anti-rabbit, streptavidin-HRP, TMB.
Here I would recommend you to use biotinylated anti-rabbit rather than streptavidin-HRP because you enhance the signal with biotin.
The bad point is that you can't do it with your antibodies. You should maybe biotinylate one.
So you would coat a plate with around 50 ng of a non biotinylated antibody (the best would be to biotinylate the polyclonal, but you have to try which one is best biotinylated), wash, block (try with BSA or non fat dry milk), wash, incubate with sample, wash, incubate with biotinylated antibody (you can't use a not biotinylated and then an anti-rabbit, because it would recognize the antibody used for the coating), wash, incubate with streptavidin-HRP, wash, add 100 µL of substrate TMB, let the blue color develop, and add H2SO4 2N (50 µL) to stop the reaction and read.
If you can't biotinylate your antibody, try direct ELISA by coating around 50-100 ng of your sample, and then block, incubate with antibody (you have to try all your antibodies), biotinylated anti-rabbit, streptavidin-HRP, TMB.
Here I would recommend you to use biotinylated anti-rabbit rather than streptavidin-HRP because you enhance the signal with biotin.
Hi Missele! Thanks for your reply!
I am completely new to this technique.. so I just wonder how do you 'coat'? Do I just simply add my sample to the mircotiter plate and incubate for some time? And then decant the sample after incubation?
Another question: for the second method that you mentioned.. I still need to add H2SO4 2N (50µL) after 100 µL of TMB to stop the reaction right? And then I read it at 450nm?
Thank you again! I'll perform the technique next week... hopefully it will work!
Coat is to incubate the plate with the "capture Ab" in the sandwich ELISA or with the sample in the direct ELISA. After the incubation, your Ab or sample are sticked to the bottom of the wells. I usually incubate it diluted in blocking buffer overnight at 4ºC and, if possible, inside some kind of "humid chamber" partially filled with water (in my case a tupperware container
) to prevent excesive evaporation. Then you remove your capture Ab or sample and wash. Another question: for the second method that you mentioned.. I still need to add H2SO4 2N (50µL) after 100 µL of TMB to stop the reaction right? And then I read it at 450nm?
Thank you again! I'll perform the technique next week... hopefully it will work!
Yes you just incubate the antibody and it will stick to the plastic as said Pumuki.
My favourite coating buffer is PBS. But you might also try carbonate buffer.
use maxisorb plates.
I'm used to coat overnight at 4°C, in the fridge. I cover the plate with a sticker and I don't need a humid chamber.
yes, for the second method you also need to add H2SO4, and then you read at 450nm
Thanks for the reply again!
For the standard curve, i saw other people were using Bradford or BCA protein assay, is it possible if I use Lowry assay instead? Because that's the one I have learned before, so it would be easier for me since I already have the kit/reagent + know how to do it.
And I saw other posts, most of the people mentioned they have a purified protein sample. Mine is not purified, its basically cultured medium from a cell line, is it ok? And I need to further dilute it with coating buffer?
Missele mentioned PBS as the coating buffer, so I just simply dilute my sample with PBS? That simple?