3 replies to this topic

[Winnie123]

Hi all,

        I am setting up a sandwich ELISA assay with human serum. My standard is purified His-tagged protein. I am supposed to check the level of my protein of interest in human serum. I found that my blank has higher absorbance value than my human serum. I am suspecting thaat there may be interfering antobodies in the serum so I diluted with blocking buffer by two folds. I still get the same result. Does this mean that the human serum does not contain my protein of interest or the antibodies cannot detect the protein? How can I strengthen my experimental design? Thanks.

 

Regards,

Win

[Missle]

Could you try a western blot of the serum - just to confirm that the protein is or is not present?  That could help with the ELISA troubleshooting...

[Ben Lomond]

So you presumably have a capture and detection reagent specific for your protein of interest and you are building a curve in a matrix (diluent) that isn't human serum and perhaps less complex....so the serum has a blocking property and blocks non-specific binding sites for the assay reagents so you get higher background in the curve matrix (buffer or something that doesn't have the blocking property).  Let us know what reagents you are using for the assay and what you are trying to detect and we'll try to advise.

[tkf]

May be your protein of interest is present at low concentrations in human serum and your ELISA is not sensitive enough. In addition to what B Lomond said above, I would recommend adding some mouse serum to your standard diluent/buffer so that it mimics human serum and you get similar backgrounds. After that you can start optimizing the concentrations of your Ab, detection reagents etc to make your assay more sensitive.