sandwich ELISA not working

[marcbcn]

Hi everyone!

I trying to do a sandwich ELISA but i'm not able to detect my protein of interest. I get the same absorbance in blank wells that in positive control with the recombinant protein. The antibodies work in WB, but noy in ELISA, someone could help to find the problem?

I use the following reagents:

- a mouse monoclonal Ab as a capture Ab
- a rabbit polyclonal Ab as a detection Ab
- anti-Rb- HRP seconadry Ab
- Quantablue kit for detecting peroxidase activity

I am washing with PBS-tween20 (x3)
I have used as a blocking buffer gelatine with Tween20 and also BlockAce (BSA), none of them worked.

Thank you very much in advance!

[danfive]

Originally Posted by marcbcn

Hi everyone!

I trying to do a sandwich ELISA but i'm not able to detect my protein of interest. I get the same absorbance in blank wells that in positive control with the recombinant protein. The antibodies work in WB, but noy in ELISA, someone could help to find the problem?

I use the following reagents:

- a mouse monoclonal Ab as a capture Ab
- a rabbit polyclonal Ab as a detection Ab
- anti-Rb- HRP seconadry Ab
- Quantablue kit for detecting peroxidase activity

I am washing with PBS-tween20 (x3)
I have used as a blocking buffer gelatine with Tween20 and also BlockAce (BSA), none of them worked.

Thank you very much in advance!

Use Carb/Bicarb pH 9.0 buffer for overnight capture antibody binding, done at 4C. Then block with StartingBlock or Superblock (follow manuf. inst. on incubation times) from Pierce. Background should be minimal, then proceed to adjust antibody concentrations, especially be careful with using too much anti-rb-HRP conjugate.

[Elisadeveloper]

If your anti-rabbit secondary reagent (presumably from goat) has not been absorbed against mouse serum proteins, then it will have high cross reactivity with the mouse coating antibody. To eliminate this, use something like Jackson's 711-035-152 or 111-035-144 or another vendor's equivalent and/or include 5% or so mouse IgG in the anti-rabbit antibody diluent. Better still go with a labelled rabbit anti-target antibody if you have one.

If this is not the problem and you are using a mouse absorbed secondary reagent, and you simply are getting no response at all, it could be possible that the antibodies that you are using recognize the denatured western protein, but not the folded recombinant protein in the ELISA well. Are there reports that these antibodies are good for ELISA?