Peptide dotblot and ELISA - How are synthetic peptides efficent as antigens in immune assays? (Jun/24/2009 )
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.
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. on Jun 25 2009, 05:44 PM said:
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.
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).
Thanks K. B. The link indeed can not be opened from my computer.
That's strange... DOI links should work fine. Check your inbox - I've sent you this paper.
K.B. on Jul 3 2009, 04:22 AM said:
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.