Non-specific binding in ELISA assays - Reducing background with human serum (Sep/23/2003 )
Does anyone know how I can eliminate non-specific binding in a Sandwich ELISA to assay for protein in human serum? I tried traditional blocking methods (BSA, PBS, Tween-20) but they didn't help to reduce background.
It's your mistake to add tween-20 in your blocking buffer ! Try calf serum instead of BSA to block the non-specific sites.
All the traditional blocking methods are good since if you work at a optimum concentration of these reagents. To eliminate the background you could increase the concentration of the BSA (up to 5%) or Tween-20 (up to 3-5%). For sandwich ELISA you could try the blocking buffer with 5% of sucrose, 1% BSA and 0.01% of sodium azide. It work well for me...
Calf serum, casein, powder milk, and other can be used also to block the plates...
Try to increase the time with the blocking solution.
On the other hand, the background could be related with the specificity of your coating (is it a monoclonal or policlonal antibody? Which concentration?)
Maybe you can check your solution in those way.Firstly,to adjust concentraction of antigen and antibody you used.Secondly,best to choose the monoclone antibody as enzyme labelled one.Besides, as Fujiwara said,you may change your blocking methods.
By the way,Tween-20 maynot have any bad effects on your test,and avoid adding sodium azide in your buffer if you use HRP labelled antibody.
I am also doing sandwich ELISA and encountering high background. However I am using polyclonal antibody.
Currently I am using 1% BSA, 0.05% Tween 20 and EDTA. I have even tried 1% FBS v/v - decreased background by a mere 0.3.
I have difficulty establishing the dynamic range now. Could it be due to the polyclonal antibody? My blank can read 0.8 while the lesser serial dilutions could register negative readings.
because you are assaying protein of human serum you must not use BSA for blocking. for any serum protein you must use 1% or 0.1% casein.
ya i too have come across similar problem- in a Sandwich ELISA to assay for protein in human serum.
my captrure is monoclonal--blocking with TBS-Tween 20--sample(serum)--primary Ab(polysera-not purified)--detection Ab conjugated to HRP. except the coationg all reactions are at RT.
what i am trying to understand is the efficiency of tween-20 in blocking a well with hydrophilic and hydrophobic sites [maxisorp plates]
You can use top-block as alternative to BSA
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.
Are you sure it is background and not heterophilic abs?
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.