Hi!
I am trying to develope a sandwich ELISA to detect a molecule in chylomicrons produced by Caco-2 cells.
I normally coat the ELISA plate with anti-ApoB antibody, block with serum albumin, then add either the basolateral media or the chylomicrons isolated in ultracentrifugation and last, the antibody against this other molecule I am looking for (plus the antibody carrying the HRP).
Every time I tried there´s lot of background. Those wells where the chylomicrons shouldn´t have my molecule, show also "positive values" and many times the values make no sense at all, increasing and decreasing with higher dilutions!
Other techniques to detect the molecule of interest worked, but the ELISA doesn´t work!
Should I take any special consideration if I try to do such ELISA with lipoproteins? do they degrade while incubating in the ELISA plate?
Thank you in advance!
you use an antibody at the bottom of the plate to capture the chylomicron. Then a second antibody to detect the molecule of interest. And finally another antibody coupled to HRPO to trace the second antibody. Is not it?
In this case the background noise comes from the conjugated antibody which binds to the antibody at the bottom of the plate.
It is essential that the second antibody used is directly coupled to HRPO to avoid this very natural background.
If it doesn't interfere with your protein/assay, you may wish to use a detergent or lipase to break down the chylomicrons and solubilize or clarify the assay components.
You have several problems in this case:
1. Chylomicrons are large particles, which generally tend to high background signals.
2. The lipids in the chylomicrons might interact with the polystyrene plates.
3. You may not be able to use surfactants, such as Tween 20, which might disintegrate your chylomicrons. Tween 20 is necessary for most washing steps in ELISA.
Do you perform your incubations with buffers containing BSA? If this is not sufficient, stronger blocking agents, such as casein or defatted milk might be used, if these compounds do not interfere with your assay.
I use HSA as blocking agent since BSA could interfere with my assay, and I am affraid also casein or milk would do it too!!... but still I get background.
It is "funny" because sometimes the more diluted the media is, the higher is the signal, and viceversa. Also in those samples that are supposed to don´t contain the molecule that is the target for the 2nd antibody... the signal is higher than in the ones that should contain it...
I realized after so many failed attempts that what I want to do, might be quite challenging, but I would really love to make it work!!
Did you add HSA to all of your reagent and washing solutions? A single blocking step will not make it. How did you perform the washing?
I add HSA during the blocking step and also in the solution containing the antibodies, but not in the washing solution!
The washing steps I did them using PBS
Since you are already partially purifying your chylomicrons via ultracentrifugation is it possible to then extract your protein of interest and directly develop an antigen capture specific that protein. You would need the proper antibodies of course. If you are only interested in detecting the protein and do not require chylomicron structure then I would test various detergents. Perhaps start with Tween-20 at 0.5% in all your buffers and move on from there. We had some luck with Triton X-100 for background removal on certain ELISAs, but you do need to titrate it carefully as it will wipe out antibody/antigen binding at higher concentrations.
In many cases, PBS is not sufficient for ELISA washing steps. Without surfactant, you may end up with extremely high background signals. Tween 20 seems to be mandatory for many assays. If Tween 20 is not suitable, you need to find a surfactant, which is compatible with chylomicrons.
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