Hi ,

Make sure that: the time of incubation in each step is the same, also incubation time of substrate is the same, the ELISA plate is same brand in each experiment , the tips is attach fully to the multichannel pipets used and the buffer used to prepare your reagents is good.

Try to replace BSA instead of Casein.

Do you use the saturation step for wells at the beginning of the experiment with the coating wells?

Which buffer is used in Coating step (Carbonate or PBS)?

Which enzyme is in your conjugate HRPerox or Alkaline Phosphatase?

Thanks

Than you Dr Gaber for your input

I don’t use saturation step , just STDs , what is it ?

coating buffer is PBS with some Proclin + the coatin Ab. The enzyme is HRPerox.

What do you think of repeated low Absorbance?

of course you must repeat it,

With respect to the saturation step:

After coating step, there are some unoccupied binding sites on wells, thus we must block these sites by a saturation step using a solution of BSA 1.5 % in PBS buffer. This solution considers a good blocker of non-specific binding on well surfaces.

Or you can use your STDs in PBS containing Tween 20 to prevent the non-specific binding .

Good luck