Hi,
Could you help me for solving the problem for the following ELISA ( Check board ELISA) ?
Although the serial double fold dilution of the Ag ( purified protein) and the serial double fold dilution of sera(non heat treated) I could not see any decrease in the OD with the decrease of Ag concentration .
What could be the causes for that ?
Thanks for your support
I made another ELISA with different Ab concentration but I think ,
I might not be the only problem. so could you have a look through this result to help me for getting the possible cause for making the OD did not change with Ag variation .
best,
zeinab
You get a sensible signal response to antibody dilution.
The negative control is negative, as it should be.
The only thing that doesn't look right is that the antigen dilution has not much effect on the signal. Even more puzzling, is that the more dilute antigen gives a larger signal than the more concentrated antigen. Check to make sure the antigen concentrations weren't reversed on the plate.
Thanks for your replay , it is not reversed plate what could be the cause for that
do I have any problem with coating ( carbonate bi carbonate buffer ph 9.6 as Na2CO3 5.3 g, NaHCO3 4.2 g , NAN3 1.0g , h2o 900ml .
do I have any problem in blocking as used general block of ( mammalian protein based blocking buffer with BSA ).
do I have a problem in washing my washing buffer is PBST20 with out azide .
The fact that you get no signal in the blank wells, but you do get a signal in the wells treated with antigen, suggests (1) that the antigen is binding, (2) that blocking is working properly, and (3) that the antibody is recognizing the antigen. The only explanation I can think of is that there is too much antigen, even at 1:26,500 dilution. The lower absorbance values at the highest antigen concentrations may be the result of insufficient washing to get rid of unbound antigen, causing some of the antibody to be lost due to binding to free antigen.