Hi Kaija,

probably nobody has successfully tried yet.

After adding sulfuric acid, you may expect that all proteins on the plate have been very much denatured.

Before adding sulfuric acid, you should get your antibodies off with glycine buffer at pH 2.5 or so. The biotin-streptavidin interaction is practically unbreakable (it's probably the strongest non-covalent interaction in nature).

If you want to isolate these antibodies, I'd suggest you to coat a NZ or PVDF membrane with streptavidin, then immobilize (i.e. add :) your biotinylated peptide, add your IgG containing solution (probably diluted serum) and have the bound IgG analyzed. If you make your setup large enough, you might be able to isolate quantities of these antibodies (elute as suggested above with glycin) .

Or better make an affinity column (the linker might be a bit tricky, ass it needs to be in the same position as the biotin was), this might work better.

What is your plan with the recovered antibodies?

Wo

What first struck my mind was affinity columns. In theory, you could coat a column with your streptavidin, wash, coat with your biotinylated peptides (or you can proceed with this interaction in vitro and coat the column with the bound biotinstrepaticidin complexes), wash again and then add your samples. You can then elute by either altering the pH, competing peptide or adding a chaotropic agent, e.g. urea, guanidine hydrochloride. I haven't done it but if you can spare some time, I think that 's sth you could do.

I agree with Wolfgang, the sulfuric acid has completely denatured the antibodies using standard ELISA methods. You could try stopping the HRP reaction with other reagents, maybe a less concentrated acid?

Basically, there is no need to really stop the reaction with sulfuric acid. Just measure the OD of the blue dye at appropriate wavelength.

Don't know about the pK of the TMB product, but possibly 1M Glycine at pH 2 or 2.5 will do the same job.

Even if, theoretically, you can detach the Ab(s) you will end up having a mixture of x-peptid IgG and secondary IgG that have to be separated to analytical grade. Not worth the time and effort. As mentined before, H2SO4 destroys everything. I will instead try to optimize the sensitivity of the detection limit so that you can save more IgG. For instance, using a fluorimetric rather than colorimetric system. The sensitivity is higher. Of course you will use a fluorochrome-labeled secondary IgG. Hope it helps.

What you need to do is couple you two peptides to an affinity matrix, pull out the specific IgGs from serum by chromatography and if need you still need the ELISA data, assay your purified IgGs by ELISA. The other way round is pointless!

It's will work quite good, also the elution of antibodies from western blot strips. Normally a pH at 4 ... 4.5 is sufficient. You have to know, that there are only a few picograms. So you need the antibodies from severals wells.

To avoid the denaturation by stopping the enzyme reaction at the end of the ELISA:

1. measure the unstopped plate at a distinct time. (measure at 650 nm: http://www.interchim.com/interchim/bio/produits_uptima/tech_sheet/FT-UP66478(TMBsolution).pdf), wash and elute.

2. use ß-Gal instead of HRP, [substrate for ß-Gal: : o-nitrophenyl-ß-D- galactoside (ONPG)]. The reaction takes longer (up to 60 min and more) but there will be no denaturation.

Hi Wolfgang- the recovered antibodies are hopefully to be used on a neuronal cell culture to see if there are any pathological effects. So if I was to bind the antibodies (in the serum) to the plate (in multiple wells, as Stephan Kiessig suggested), and eluted them off with urea or glycine solution, I'm hoping I could alter the solution back to a reasonably neutral pH for the functional assay.

I'm additionally trying to separate IgG1 and IgG2 from these collected antibodies, to see their effect. Any help to that technique would be appreciated, and I've phrased that question here:

https://www.researchgate.net/post/Elution_of_human_antibodies_from_ELISA_for_functional_assay#share

Cheers!