13 replies to this topic

[bethk]

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.

[baifighter]

It's your mistake to add tween-20 in your blocking buffer ! Try calf serum instead of BSA to block the non-specific sites.

[Fujiwara]

bethk, on Sep 23 2003, 10:14 AM, said:

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.

Dear bethk,

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?)

[enjoylife]

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.

[tambourina]

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.

Any comments/suggestions/recommendations?

Thanks!

[mpanchal]

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.

[amkush]

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]

Edited by amkush, 06 January 2005 - 06:02 PM.

[beth]

mpanchal, on Aug 6 2004, 08:19 AM, said:

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.

Hi, I'm intrigued by your post. We are blocking in BSA and having issues with our ELISA. Why shouldn't you use BSA and why is casein better?

Thanks!

[klinmed]

beth, on Oct 9 2009, 09:15 PM, said:

mpanchal, on Aug 6 2004, 08:19 AM, said:

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.

Hi, I'm intrigued by your post. We are blocking in BSA and having issues with our ELISA. Why shouldn't you use BSA and why is casein better?

Thanks!

We routinely use BSA blocking for immunometric assays.
Our lab runs routine tumor marker immunoassays (PSA, CA125, CEA, thyroglobulin etc) in the clinical laboratory setting. We run thousands of human sera a year in accredited assays generated "in house".

Plates are coated with primary antibody, washed in tris/NaCl buffer + 0.05% tween and blocked overnight in 0.1 % BSA/sorbitol. This is followed by aspiration, air drying and vacuum packing. All steps are done on a plate coating robot.

Our assay buffers contain 0.1% BSA, 0.05 % tween and heat-aggregated irrelevant mouse immunoglobulin (to minimize assay interference from heterophilic antibodies).

We have NEVER had a problem blocking assays for human serum proteins with BSA.

Hope this helps

[Small is BIG]

I am new in this field. We are using 1%BSA (from Sigma) in PBS (pH 7.4).


We routinely use BSA blocking for immunometric assays.
Our lab runs routine tumor marker immunoassays (PSA, CA125, CEA, thyroglobulin etc) in the clinical laboratory setting. We run thousands of human sera a year in accredited assays generated "in house".

Plates are coated with primary antibody, washed in tris/NaCl buffer + 0.05% tween and blocked overnight in 0.1 % BSA/sorbitol. This is followed by aspiration, air drying and vacuum packing. All steps are done on a plate coating robot.

Our assay buffers contain 0.1% BSA, 0.05 % tween and heat-aggregated irrelevant mouse immunoglobulin (to minimize assay interference from heterophilic antibodies).

We have NEVER had a problem blocking assays for human serum proteins with BSA.

Hope this helps
[/quote]

[HBImolbiol]

The blocking buffer that works for your assay is very difficult to predict and there is no all in one solution despite any previous experiences.

The best thing to do when developing a new ELISA is to screen 5 to 10 different blocking formulations such as BSA, milk, serum, casein or some of the proprietary commercial formulations and you can try supplementing with detergent such as NP-40 (Igepal) or Tween 20.  Avoid azide-containing solutions if you are developing with peroxidase conjugated detection antibodies.

If you are getting high background with human serum samples, calculate your theoretical antigen concentration in the sample and ask yourself if the quantity you are applying to your plate is reasonable.  That is, you should generally only require ng/ml quantities for specific detection.  I recommend doing checkerboard titrations of your capture, detection and sample in separate experiments to identify conditions that yield specific detection.

[lizh]

Have u tried blocking for long periods of time? The blocking step that I did for human serum assay was overnight, and it reduced our background.

[AnetteM]

Interestimng thread. I am fairly new to ELISA as well. I am doing a sandwich ELISA with human proteins and have been told that I have to wash and dilute in PBS buffer and not include tween. I get much better binding when I do this. The bacground is though a bit hign but when I include a separate blocking step (incubation 1 h with 0.5 fish gelatin) the binding is abogarated.

Why should I eclude tween? And is there anything I can do to reduce the backgound but still have the affinity?

[v_talibov]

>non-specific binding in a Sandwich ELISA to assay for protein in human serum
If it possible and analyte doesnt show revere hook effect you may dilute it.
Also sometimes removing of high abundant  proteins from samples before ELISA helps.