In our experience, a 5AA difference may certainly be plenty enough to be picked by an ELISA with high / very high selectivity.

However: (a) do you mean "5 AA are deleted", or (b) "5 residues are changed to a different AA"? Also: (c) are these "5 changed AA" lumped together, or are they scattered in a longer / how long sequence? In addition: (d) are these differences "exposed", i.e. at the N- or C-terminus of a cleaved peptide, or are they "embedded", i.e. contained within a longer sequence of the protein / gene product you are interested in, and "not exposed" at either the N- or C-teminus of the actual biological product/s present in cells / tissues / biological fluids?

All the above considerations will have significant consequence on the way you can try and produce selective / highly selective antibodies for your ELISA. Especially on the way one can design the (hopefully) immunogenic peptide/s and its/their conjugation. E.g. with respect to a peptide we work with, which is amidated at its C-terminus, we have set up an ELISA which selectively reacts with the amidated form, and not (

For an ELISA to pick up the difference between both peptides, at least one of your antibodies should be able to tell the difference between the two. You could try and develop these antibodies, by immunizing mice with the longer peptide en select antibodies that respond to the lengthy peptide and not the shorter one.

Do you have data (western blot for example) that shows that your ELISA antibodies can differentiate the two isoforms? 

To answer one of your questions: 5 AA difference should be more than enough to generate a differential respons, assuming you are using the right antibodies.

Hello ! I recommend using a biotin tagged peptide on a streptavidin plate for the best results followed by your diluted serum incubation and secondary IgG.

Otherwise try coating the plate with 5ug/mL peptide in sodium bicarbonate buffer pH9 for 1 hr at 37 c

In our experience, a 5AA difference may certainly be plenty enough to be picked by an ELISA with high / very high selectivity.

However: (a) do you mean "5 AA are deleted", or (b) "5 residues are changed to a different AA"? Also: (c) are these "5 changed AA" lumped together, or are they scattered in a longer / how long sequence? In addition: (d) are these differences "exposed", i.e. at the N- or C-terminus of a cleaved peptide, or are they "embedded", i.e. contained within a longer sequence of the protein / gene product you are interested in, and "not exposed" at either the N- or C-teminus of the actual biological product/s present in cells / tissues / biological fluids?

All the above considerations will have significant consequence on the way you can try and produce selective / highly selective antibodies for your ELISA. Especially on the way one can design the (hopefully) immunogenic peptide/s and its/their conjugation. E.g. with respect to a peptide we work with, which is amidated at its C-terminus, we have set up an ELISA which selectively reacts with the amidated form, and not (

Peptides needs to get bound at the solid phase. That’s realized in proteins via the hydrophobic AA. Their hydrophobic tails interact with the polystyrene ring at the plastic surface.

If you are trying to bind peptides, you need to have either a hydrophobic spacer either at the C- or N-terminus. The other way is the use of the free amino group for covalent binding. It this part of the peptide involved in the antigen-antibody reaction, this doesn’t work.

Therefore I suggest to use larger peptides (13 – 25 AA), include a hydrophobic spacer (Ala, Ala, Ala, or Phe, Phe, Phe) Try this on both ends of the peptide. If those modification changing the recognition by the antibody can be checked by pepscan (see: http://www.pepperprint.com/high-content-peptide-microarrays/) = peptide microarrays. A simple method allows you the check this on a very small scale with a high efficacy.

Thanks to you ALL for the answers. You see, I have 3 peptides, 2 that have 4 and 5 mismatches respectively spread over 10 amino acids and the 3 is highly conserved in 2 parasites. The logic is that if the patients serum contain specific antibodies; I should have same response for the conserved peptide and differential responses for the specific peptides. Additions the portions I am working on are in the extracellular domain and extracellular loop of a GPCR. Amino acids differences are changes.

Hi Robert,

those exchanges in the AA sequence needs to get checked in at least two different ways.

1. comparison of reaction vs. solid phase bound peptides (via conservative binding in i.e. carbonate buffer pH 9.6). Single exchanges can change the charge of the peptide and therefore the properties needs for solid phase binding. Here it's helpful to use always the same spacer and the same hydrophobic tail.

2. by inhibition of the solid phase binding (either with the natural antigen at the solid phase or with one of the peptides each per experiment at the solid phase. Than you can check the inhibition of the antigen (peptide) recognition of peptide 1 with peptide 2 ....5. etc. These are the pre-experiments for the detection of the affinities to the different peptides using the method described by Friguet (http://www.ncbi.nlm.nih.gov/pubmed/3981007). It's an old method but you can get easily by ELISA hard affinity data.

If there is an immune response to a given peptide they may be useful for what you intend to do.  That is a big if.  Below is an old paper looking at the immune response to an immunogenic region of HIV GP120.  There are differences and some cross reactivity.  It is hard to predict.  After this paper was written it became clear that the MN strain was the major infectious strain at the time.  So the results were not that earth shaking, but it does show if you choose your peptide carefully you can discern differences.

C-terminal fragments of gp120 and synthetic peptides from five HTLV-III strains: prevalence of antibodies to the HTLV-III-MN isolate in infected individuals.

Devash Y, Matthews TJ, Drummond JE, Javaherian K, Waters DJ, Arthur LO, Blattner WA, Rusche JR.

AIDS Res Hum Retroviruses. 1990 Mar;6(3):307-16.

PMID:

2340200

[PubMed - indexed for MEDLINE]