Very weird results! Any thoughts? - (Mar/28/2008 )

Hi!

I performed direct ELISA few times: Coat antigen overnight (with bicarbonate/carbonate coating buffer) --> wash 6 X 3 min --> block with 1%BSA for 4 hours at RT --> wash 6 X 3 mins --> incubate with primary Ab overnight (diluted in 1%BSA) --> wash 6 X 3 mins --> incubate with secondary Ab (biotinylated) for 1 hour --> wash 6 X 3 mins --> incubate with Streptavidin-HRP conjugate for 30 mins at RT --> wash 6 X 3 mins --> 100ul TMB --> 50ul H2SO4 to stop.

I coated the plate with different dilutions of my antigen. (20ug/ml; 10ug/ml; 2ug/ml)

Results: VERY weird!
- The concentration of my antigen in interest in the 20ug/ml dilution is LOWEST; then 10ug/ml has medium amount of my antigen; 2 ug/ml dilution has the HIGHEST amount of my antigen.

The standard curve looks perfectly normal though (i.e. 0.125 ng/ml has the lowest amount, whereas 2ng/ml has the highest amount of my anitgen).

I performed it several times and I still obtained similar results. I am sure I didn't mistake one well from the other one. The standard curve has high R^2 value as well (e.g. R^2=0.991). So it's probably not because I didn't vortex the solution enough etc.

Any thoughts regarding why I obtained these weird results? Because normally I would expect lower concentration of my antigen in the 20ug/ml dilution, and have higher concentration in the 2ug/ml dilution.

OR..is there something wrong with my protocol?

Any suggestions would be great! I am so confused! Like... it looks like there're some competitive binding going on (according to the data I obtained). Could it possibly be with BSA? Any thoughts?


Thanks a lot!

hi,

use different conc of antigen ranging from 100ng - 2ug n check how your ELISA results look like..

gud luk
regards

Hi!

I performed direct ELISA few times: Coat antigen overnight (with bicarbonate/carbonate coating buffer) --> wash 6 X 3 min --> block with 1%BSA for 4 hours at RT --> wash 6 X 3 mins --> incubate with primary Ab overnight (diluted in 1%BSA) --> wash 6 X 3 mins --> incubate with secondary Ab (biotinylated) for 1 hour --> wash 6 X 3 mins --> incubate with Streptavidin-HRP conjugate for 30 mins at RT --> wash 6 X 3 mins --> 100ul TMB --> 50ul H2SO4 to stop.

I coated the plate with different dilutions of my antigen. (20ug/ml; 10ug/ml; 2ug/ml)

Results: VERY weird!
- The concentration of my antigen in interest in the 20ug/ml dilution is LOWEST; then 10ug/ml has medium amount of my antigen; 2 ug/ml dilution has the HIGHEST amount of my antigen.

Hi,
what are the results of your elisa?
you mentioned 2ug/ml is the highest concentration but it is the otherway round
check it out once
all the best
leelaram

you sure your dilution is right?

How much are you diluting your samples? that is, what is the concentration of our undiluted antigen? You might have an inhibitor/competitor in your antigen preparation.

The suggestion of Dr House to scale down the value of coating your antigen is very adequate Sometimes you can have trouble if you use too much protein for coating abnd that can results of minoring the values of the Elisa. The same can happen in Western Blot if there' too much protein loaded on the gel !