Non-specific binding in ELISA assays - Reducing background with human serum (Sep/23/2003 )
Proteins, bsa, fbs, fish, etc work well for blocking unbound sites on the plastic. Detergents only for washing. Sugars or dextrins to protect the ab or ag coated on the wells (storage).
If it is heterophilic rx then investigate removal of Fc portion of ab or Other possibilities include preincubation of sample with blocking reagents such as non-specific IgG of class similar to abs in the sytem. or commercial heterophilic blocking reagents.
Good thinking, sgtboston.
Preincubation of sample with an isotype control is an excellent idea.
However, removal of Fc portion (using Fab only) may not work, because the Fc portion is required for the enzyme/streptavidin conjugation or secondary antibody (anti-IgG) binding.
How about pre dilution of sample prior to assay? Try at least 1:10 or 1:100 this should reduce nsb (of course you still need the sensitivity).
To go around this add polymer to your assay which will boost your signa.
You will have to balance sample dilution, polymer, and wash (surfactant) to maximize signal minimize background (nsb).
Try diluting your detection antibody in 1% whole calf serum.
This did the trick for me when I was testing human serum from autoimmune patients.
Flickers
I am trying to detect anti-bodies against Tat protein (HIV-1) from human serum.
I am coating 100ng of the protein in carbonate buffer, blocking with 2% BSA followed by 10% sheep serum.
The wash contains 0.2% Tween20, 50mM Tris and 100mM NaCl. The human serum is diluted (1:100) in sheep serum.
I still see a high back ground of 0.4 even in "no antigen" control. however, the no antobody control is fine (0.04).
how do I reduce this back ground? One more point is that the normal serum too is behaving similarly.
what grade whole calf serum do you use? (of where did you order it?)
As I said back in July to another email, If you have heterophilic abs the surfactants, bsa, etc etc are NOT going to help. You have to add similar isotypic ab or commercial blockers to the assay. Try material from scantibodies, omega, and bioreclamation.
If not heterophilic abs then your bsa, surfactants, non-human proteins, casein, etc etc may work.
Thanks!
i am not an expert on ELISA so i have to admit that the existence of heterophilic ab's was new to me, i looked it up and it might indeed be the reason for the high background.
is there a way to check if my ab's are indeed heterophilic or is it trial and error?
i also looked up the scantibodies site and the commercial blockers are quite expensive, (only add it to the ab's or all samples?), what do you use when working with heterophilic ab's?
is it the same effect as blocking with normal (whole) serum from the same host as the second ab?
and if both ab's are from the same host (sheep anti bovine casein) then i have to use a completely different normal serum, like mouse or rat?
in which step do i add the normal serum?
If you are still struggling can you remind (us) of the nature of your assay. Id be interested to know the species of your antibodies and is the secondary anti IgG specific? in ref to earlier quote I am not a chemist but i am pretty sure I have used HRP conjugated Fab2 antibodies with no problems from Dako and Sigma. Sgt4 boston hit it on the head in terms of where to use the correct reagents, detergents are for washing not blocking normally. Proteins for blocking plates and in conjugate step to reduce background (normally 1%BSA) and sugars for drying plates down for storage.
sparky
and if both ab's are from the same host (sheep anti bovine casein) then i have to use a completely different normal serum, like mouse or rat?
in which step do i add the normal serum?
i don't use anti IgG. it is a sandwich for casein, i coat with affinity purified sheep anti bovine caseine, add sample, add sheep anti bovine caseine HRP (for block i tried bsa and gelatine and) (conjugate is diluted in blockbuffer)
i wonder if the heterophilic antibodies can also be found in animal serum or only human serum?
BSA is a major component of the serum.
if you used a polyclonal primary, may be you have anti-BSA Ab.
I would recommand you to use something very far from serum protein.
may be the coldfish gelatin (which is usually used in immunohistochemistry, but avoid to let it too long at RT because it stincks!!!)
Maxime