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Peptide dotblot and ELISA - How are synthetic peptides efficent as antigens in immune assays? (Jun/24/2009 )

Hi all,
I wonder if someone can tell me how to improve retaining of synthetic peptides on PVDF or nitrocelulose membranes for dotblots? I found a paper reporting dotblots failed with peptides (~20aa long) but direct coating ELISA seemed to work better. I would apperciate much if you can point me towards a good review on this issue. Thanks.

-bachai-

Friend of mine was trying to do that with 5-7 AA peptides. Western-blot and dot-blot failed, but she managed to develop quite reliable ELISA for quantitative determination. She's using competitive ELISA with peptide conjugated to poly-lysine (with EDC) for coating.

-K.B.-

K.B. on Jun 25 2009, 05:44 PM said:

Friend of mine was trying to do that with 5-7 AA peptides. Western-blot and dot-blot failed, but she managed to develop quite reliable ELISA for quantitative determination. She's using competitive ELISA with peptide conjugated to poly-lysine (with EDC) for coating.


Thankyou very much K.B. Your friend's peptides (5-7aa) were obviously too short for an immune assay so she had to couple them with a poly-lysin as carier molecule for ELISA. Can you tell me what EDC is? Did your friend published the method somewhere, or can she share the trick with me? Many thanks.

-bachai-

EDC is a coupling agent, N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide, CAS Number 1892-57-5.

You can find brief description of preparation of immunization conjugate, antigen conjugate for ELISA and ELISA in: doi:10.1016/j.idairyj.2008.10.011 (contact me if you don't have an access).

-K.B.-

Thanks K. B. The link indeed can not be opened from my computer.

-bachai-

That's strange... DOI links should work fine. Check your inbox - I've sent you this paper.

-K.B.-

K.B. on Jul 3 2009, 04:22 AM said:

That's strange... DOI links should work fine. Check your inbox - I've sent you this paper.

Thanks K.B. I've got it.
I've tried a 19-mer peptide which was both OK in direct coating ELISA (PolySorp plastics, carbonat buffer), and competition ELISA. The peptide concentrations in range 0.1-2mM gave strong and specific signal with Abs, yet did not appear to reach the saturation.

-bachai-