Hello,
I am designing to produce antibodies against 15aa peptides, these antibodies will be used for detection of a pathogen in plant.
The problem is when i blast these 15aa sequences there are a lot of organisms which have strings of 5 or 6aa long same identities. I am afraid that these organisms might cross-react with the antibody or is an epitope vs the 15aa peptide not going to bind to a 5-6aa sequence?
Do i just base my choice on the peptides that show similarities with organisms the least likely to be present in my host?
Hi Vicken, the easiest way for this project is to make polyclonal antibodies against the 15aa peptide.
When you get the antiserum, pass it over a column with immobilized 15aa peptide.
Do stringent washes and elute the remaining Abs with 0.1M glycine pH 2.5.
With this you will have affinity purified Abs that only bind to the 15aa sequence.
Most of the linear epitopes consist of less than 15 aa. Therefore, most likely affinity purification will not be enough. Subtractive affinity purification with unwanted cross-reactants might be an option.
I have more than a decade hands-on experience in designing 10-15aa peptides for antigen-affinity purified antibodies and I added a couple of thousand good-selling products to the market. Despite all the theories, there is no fixed rule about the best designs. In fact, Michael is correct that the antigen affinity purification is not a guarantee to high specificity, but it is a prerequisite. All peptide-generated antibodies need antigen-affinity purification, but then still need to be verified for cross-reactivity in the assay of your requirement, even when the immunizing peptide shows little or no overlap with other proteins in a BLAST query. See https://f1000research.com/articles/3-232/v2 for further info.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies