Why capture ELISA failed - (Nov/29/2007 )
I am doing ELISA with the purified antigen and two purified monoclonal IgG antibodies.
Firstly, it is good in my indirect ELISA with the antigen and the monoclonal antibodies, including the biotinylated ones. The specific bindings were shown for signal/noise ratio (s/n) was at least greater than 30. I have done a series of titrations and found out the optimal conditions for the Ag/Ab reactions.
However, when I coated either of the two monoclonal antibodies (150 / 100 / 50 ng/well) to the plate (Nunc MaxiSorp) for my capture ELISA, it produced almost no signals (s/n = less than 4) regardless of the antigen dilutions changing from 1 pg to 100 ng/well. The detection mAb I used is biotin-labelled and differs from the one coated onto the plate (strep-HRP + TMB system). Furthermore, I then tried the polyclonal Ab (from goat) as the detection Ab in the capture ELISA, but it still didn't work well.
No problem with IgG coating----The Ab-coating plate was detected with anti-mouse IgG-HRP and result was positive, indicating that the monoclonal Ab was coated onto the plate.
Also, no problem with buffer-----I found no significant differences between two coating buffer I tried: carbinate (pH9.5) and PBS (pH7.4).
Anybody there can give me an idea what was going wrong? Is it possible that the coated IgG had been "injured" with its binding arms to grasp the antigen? or....
Firstly, it is good in my indirect ELISA with the antigen and the monoclonal antibodies, including the biotinylated ones. The specific bindings were shown for signal/noise ratio (s/n) was at least greater than 30. I have done a series of titrations and found out the optimal conditions for the Ag/Ab reactions.
However, when I coated either of the two monoclonal antibodies (150 / 100 / 50 ng/well) to the plate (Nunc MaxiSorp) for my capture ELISA, it produced almost no signals (s/n = less than 4) regardless of the antigen dilutions changing from 1 pg to 100 ng/well. The detection mAb I used is biotin-labelled and differs from the one coated onto the plate (strep-HRP + TMB system). Furthermore, I then tried the polyclonal Ab (from goat) as the detection Ab in the capture ELISA, but it still didn't work well.
No problem with IgG coating----The Ab-coating plate was detected with anti-mouse IgG-HRP and result was positive, indicating that the monoclonal Ab was coated onto the plate.
Also, no problem with buffer-----I found no significant differences between two coating buffer I tried: carbinate (pH9.5) and PBS (pH7.4).
Anybody there can give me an idea what was going wrong? Is it possible that the coated IgG had been "injured" with its binding arms to grasp the antigen? or....
the coated antibody is bound with random orientation. not all will be available to bind the antigen.
thanks mdfenko.
yes, i agree. but i could not find significant signal changes by adding the antigen dilitions from 1 pg to 100 ng/well to the mAb-precoated plate.
hi,
efficient coating does not say much about the ability of ur antibody, if the Fab is destroyed, still u can see the signal with the anti mouse igG HRP.
careful before u conclude something
gud luk
sravan.
No problem with IgG coating----The Ab-coating plate was detected with anti-mouse IgG-HRP and result was positive, indicating that the monoclonal Ab was coated onto the plate.
efficient coating does not say much about the ability of ur antibody, if the Fab is destroyed, still u can see the signal with the anti mouse igG HRP.
careful before u conclude something
gud luk
sravan.
No problem with IgG coating----The Ab-coating plate was detected with anti-mouse IgG-HRP and result was positive, indicating that the monoclonal Ab was coated onto the plate.
sravan, thanks a lot for your comment.
do you mean the Fab fragments could be demaged after being coated to the plate wells?
hi,
yes.... cud be some how the Fab is damaged coz of ur buffer or coz of any storage condition...
there can be several reasons for that....
OR the antibody is fine BUT the epitope on the antigen is destroyed????
make sure that step, then proceed further..
gud luk
sravan.