Hi,
Dear all,
I want to measure human serum IL-1beta and TNF-alpha by using precoated sandwich ELISA kits from BD. I am getting serum absorbance values lower than the zero standard/blanks. I am following the kit protocol exactly but still this problem is persisting. The absorbance of serum if not containing any measurable amount should conform to the blank values. What could be the possible reason for that? Any comments or suggestions are highly appreciated.
Do you have protein in the buffer used for diluting your standards? This is a common problem when no or low protein diluent is used for the standard. Suggest you add 1 - 5 % albumin, milk or serum to you dilution buffer.
I did lot of ELISA in my life, but I do not remember ever having this problem. However I can suggest: a) try a wide range of sample dilutions; b) pay attention to the temperature of the buffer (presence of crystals etc.); c) as already suggested, try with different blocking agents (albumin, milk powder, normal serum of the same species of the conjugate).
Thanks for nice comments and suggestions. The standard/sample diluent contains the animal serum. I have tried different dilutions of the sample but still the absorbance of dilutions is lower than blank.
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