discordance between western blot and ELISA - (Oct/24/2007 )

Hi,

I'm searching for the presence of some antibodies in autoimmune patients' sera for an specific antigen. I`ve been succesfull at detecting them by western blot, however, i still cannot see them by an ELISA assay.
I wonder if anyone can help me with the reason of this discordance and how can I solve it.
I'll be very thankful

---

usually WB are performed with antibodies directed against the reduced form of the antigen. So, maybe you have good antibodies against the reduced protein but not against the native form?

---

hi,
the given information is not sufficient to understand and answer the problem.
1-How did u perform the ELISA?
-did u coat Ag of interest on to the wells? or it is a capture assay?
-did u dilute ur serum? or used undiluted serum for the detection?
2-How did u perform the WB?
-how did u prepare ur samples? did u enrich the Ab fraction in the serum? or loaded the serum directly?
-what kind of antibodies did u use?

payeli

---

Hi,

Thank you very much for your interest.

For my ELISA, I coat the plate directly with my antigen, diluted in carbonate buffer. Then I block with BSA, and I use diluted serum of the patients (1:30). Finally I use a secondary antibody coupled with HRP for the detection.

For the WB, I boil my antigen in sample buffer. After the blot, I block the membrane with PBS-milk. Again, I use diluted serum (1:50) , and I use a secondary antibody coupled with AP for the detection step.

I hope this information may be useful

---

did you have a control, where you do not coat the antigen, but block, add serum and secondary antibody?

---

hi,
nice explanation. perform the experiment as follwed...

Exp:Boil your Ag which you will use for your ELISA plate coating. then go ahead with the ELISA protocol.

i hypothesize that, in the native Ag (unboiled) the epitope which should be recognized by your serum antibodies might be inaccessible. the reason is you detect denatured Ag with the serum but not the native one in ur ELISA!!!!!

if you can see the color in ELISA that would confirm my assumption.

Let me know what happend?

gud luk
sravan payeli

---

Hello,
I also had the same problem. I tried to detect AMPK and phosphorylated AMPK using both Western Blot and (in cell) ELISA.
The ELISA thing did not work eventhough the cells were fixed with formaldehyde and permeabilized by Triton-X100.
Does anyone have any experience with in-cell ELISA?
Thanks

---

your antibody might recognize the denatured form of the protein.
Try with an other antibody

---

I aggree with Missele and Payeli above and as Payeli suggested above you could boil your antigen prior to your ELISA.

The question is what relevance is this to the disease process? If the autoantibodies recognise the denatured form of the antigen, is this likely to be found in the patients? Could the antigen be denatured, mis-folded or truncated or degraded to produce short polypeptides which are recognised by the autoantibodies in the patients? Otherwise I think people will question the clincal relevance of your work.

You might need to test under what conditions the antigen from control or diseased patients is recognised. So try a sandwich ELISA using another antibody from mouse/rabbit for the antigen which works in ELISA as a capture antibody then some kind of clinical sample from autoimmune patients and controls for the antigen (denatured by boiling and adding reducing agents and non-denatured) and autoantibody from diseased patient serum then detection antibody and substrate.

Best wishes,
Ceri

---

What's the antigen? is it commercially available or do you prepare it yourself. As Ceri said, if you patient sera only recognises the denatured form of the antigen you'd need to think of the biological relevance of that. As you can really change your antibodies, I'd check the stability and "nativity" of your antigen, how similar it really is to what the antibodies will encounter in vivo.

---