Antigen Selection and Design for Antibody Generation

[moleculardude]

How can I select and design a peptide for antibody generation?

Does anyone have experience with a coupling strategy?

thanks in advance!

Moleculardude

[BAS]

I have the same question... specifically when we know the antigen is membrane associated, how can we make sure that the designed peptide will surely be exposed to be recognized by antibody invivo....

[BAS]

Ok.. Yesterday, I found this site when I was looking for answers regarding peptide design for membrane bound protein antigen. I was excited to see that we have common questions and thought some body out there must have an answer...
so where is (s)he?

I have generated several peptide antibodies (both poly and mono) over last few years. I design peptide based on antigenic sites predicted by softwares (like Antigenic) in a given amino acid sequence. A length of 20 amino acid is enough to generate antibody. However, the titer may not be as good as a polyclonal raised with full length protein. My problem at this point is I have to generate an antibody against a membrane bound protein. The software predicted antigenic sites are all located in the transmembrane domain. thus, I am sure that the antibody (even if it has a resonable titer against the peptide) will not see the site in vivo and thereby will fail to recognize the antigen.
Is there any body you know who can help me out in this?
Thanks
BAS

[immunoassay]

Originally Posted by moleculardude

Does anyone have experience with a coupling strategy?
Moleculardude

There is a few usually method for coupling peptide to carrier, e.g. EDC, NHS. I have linked a few of haptens to BSA，OVA，KLH, maybe we can discuss the linking futher by email. my email: [Only registered users see links. ]

[iihay]

Hi,

There is no easy answer to this one as it is very sequence specific. In a lot of cases you may not be able to do anything about it. In some cases you can use other poor looking regions and get some sucess. Keep in mind that all antigen prediction software are flawed to a degree and the best looking regions may actually fail to give an antibody to the protein anyway.

I hesitate to post this as I don't want to be seen to promote my company but it is best to let somebody have a look at this for you specifically and we can do that for you foc. I have therefore sent a private message to keep it off the main forum, hope this is ok!

Iain

[Antibodydude]

Hi All,

Alternative to using peptide will be to use DNA expression vector for immunization. This is especially good approach for membrane bound antigens, and there is no need to make peptide or protein. You can PCR out your sequence and insert into an an expression vector. There are several good reference on this as well as companies doing this. Let me know if you need more information.

[subhankar2505206]

Hi Guys
I am looking to design an antibody. I also designed the peptide sequence based on several prediction methods. However, when I submitted the sequence, I got a reply back from the company making the antibody that since my residue has a central cysteine, they cannot synthesize the antibody. However, on my antigenecity plots the peptide sequence containing this cysteine comes out as the best. On the hydrophobicity plots too, this region is the least hydrophobic. Now the question I have is: Is the presence of a single cysteine enough to make the peptide antigenic? I actually came across a paper wherein a peptide containing an internal cysteine was actually being tried as a vaccine. Can you please give me your views on this?
Thanks and hoping to hear from some of you.
sincerely
Subh