peptides fixation - (May/26/2011 )

Hello everyone again,

I use small peptides (17 Kda) as antigens in indirect ELISA. I coat my plates in Carbonate/bicarbonate Buffer (Sigma) and store for 48 hours at 4ºC.
I use 20 ug per plate. Sometimes I think my peptide hasn't attached enough in plate (Costar plates High Binding - Corning.
I have read about fixation of peptides with glutaraldehyde and others.
Does anyone have experience with this kind of fixation. What would be the glutaraldehyde concentration to fix?

Thank you in advance.

We have done many experiments with peptides coated on Costar High binding plates and they generally stick OK. We usually coat 1 ug/ml in carbonate buffer but you can try as high as 5 ug/ml if you feel that the peptides are detaching from the plate. If you can you can also try a competitive type of ELISA. That way you don't have to coat the peptides. If you get good inhibition then you can start worrying about the attachment of the peptide. There are a couple of vendors selling covalent coupling plates for these purposes e.g. Nunc and Pierce amino reactive plates, however they are pretty expensive.

Sticking is often not a problem, it's the conformation on the plate that's the problem, and if the epitope is squashed like a fly on the windshield, then the detection reagent will only bind with low efficiency. Costar 4HBX and Nunc Maxisorp are designed to orientate antibodies with the antigen binding site up, so using these for antigen down assays is hit and miss depending on the charge of the peptide and where the epitope for the antibody lies on the peptide. Costar 2HB or Nunc polysorp may work better for antigen down.

As Biomiha says, the in-solution competitive format, with immobilized antibody and labelled peptide may work better. If antigen down is important for you, then a biotinylated peptide and strepavidin surface may work. Simply pre-form the biotinylated peptide, and streptavidin (10 ug/mL each) in PBS for 30 mins at RT, and then coat the complex after a 10 x dilution in PBS on a 4HBX or maxisorp plate.
I am not entireley sure what the glut approach will do for you, what would you be trying to cross link to what?

The route you want to take depends on the availability and # of reagents that you have at your disposal. Let us know what you have and we can advise.