Hi folks!
I'm new to the forum and also to immunology and was wondering if I could get some advice about antibody production. I need to produce mAbs to a recombinant protein. There is only a single amino acid difference between the recombinant and natural protein, and I need to produce a mAb that can discriminate between the two.
Does anybody have any tips on immunogen design that might enable me to do this? I do not know if there are any differences in post translational modifications between the two, nor do I have any information about the cell line used to produce the protein.
Thanks in advance for any help or ideas!
Andrea.
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Chelo
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Posted 12 December 2010 - 06:31 AM
Hi Andrea,
if you want to produce antibodies to distinguish both proteins then you cannot immunize with the entire protein. Instead you will have to choose a peptide (12-15 aa long)encompassing the distinct amino acid. You should immunize mice with the peptide version of your choice and then, in the cloning step, you can do differential ELISA (using the peptide you used for immunization and its variant) to find hybridomas producing the desire antibody (i.e. having signal in the ELISA where the first peptide was plated and not in the second).
You check whether your protein is subjected to post-translational modifications at the sequence of your peptide, you can visit http://scansite.mit.edu/ and introduce your peptide sequence.
I hope this helps.
if you want to produce antibodies to distinguish both proteins then you cannot immunize with the entire protein. Instead you will have to choose a peptide (12-15 aa long)encompassing the distinct amino acid. You should immunize mice with the peptide version of your choice and then, in the cloning step, you can do differential ELISA (using the peptide you used for immunization and its variant) to find hybridomas producing the desire antibody (i.e. having signal in the ELISA where the first peptide was plated and not in the second).
You check whether your protein is subjected to post-translational modifications at the sequence of your peptide, you can visit http://scansite.mit.edu/ and introduce your peptide sequence.
I hope this helps.
