ELISA and surface - (Aug/27/2009 )
Hi everyone here, i am a new member here and I have quit poor experience in immunoassay (ELISA). I am involving in determination of food proteins as antigens on the stainless steel plate surfaces but the problem what I have, the secondary Ab binds directly to the surface and give me a positive result. Any one has suggestion to prevent the attachment between Ab and the surface, I used several blocking agents and also PBST solution in variable Tween20 concentrations but, without any a good result.
Many thanks for your help
To restate: You are running an elisa to detect food protein(s). These proteins are the antigen(s) in your elisa. Your secondary antibody non-specifically attaches to the surface of the microtiter plate. You have used several blocking agents and wash between your sample, reagent additions.
1. Are you sure the ab is binding to the plate and not the primary antibody?
2. Your blocking agent should not have any surfactant
3. Are you blocking for sufficient period of time and is the concentration of the blocking agent sufficient?
4. Another possibility are you surce your primary ab is not binding your secondary ab?
5. Finally is there anything in your sample which could bridge the two antibodies?
sgt4boston on Aug 27 2009, 06:00 PM said:
1. Are you sure the ab is binding to the plate and not the primary antibody?
2. Your blocking agent should not have any surfactant
3. Are you blocking for sufficient period of time and is the concentration of the blocking agent sufficient?
4. Another possibility are you surce your primary ab is not binding your secondary ab?
5. Finally is there anything in your sample which could bridge the two antibodies?
Dear, sgt4boston
I am grateful for your reply but, i am afraid the explanation for my problem wasn’t a clear enough. I used stainless steel plate (flat) instead of plastic micro titration plate because I want to determine the proteins which adsorb (attach )on stainless steel surface.
Before doing my experiment (ELISA) I used Blank ( only secondary Ab and then TMB substrate) to make sure the secondary Ab doesn’t bind to stainless steel surface. Unfortunately, the result was positive (false positive ) that means the secondary Ab attached directly to the surface without existing proteins (antigens) and primary Ab . so, i want any advise or suggestion to solve this problem and again many thanks for your help.
I'm totally unfamiliar with proteins and metalbinding but isn't it possible that the tmb substrate itself reacts because the metal functions as a katalyst? TMB is know to be very sensitive and can easily give high backgrounds.
Look onto this link for information about substrates.
http://www.piercenet.com/Products/Browse.c...7C-1FA2CA04F788