I am a new guy come working with antibody. Recently I've got a great trouble and hope you guys can help
Say I have a peptide LVRPEVDVMCTAFHDNEETFLK. Because of the hydrophilicity, the sequence DNEETFLK in the C terminus are used to prepare an antibody to capture the original peptide. We pay to a biotech company who is expert in this. The company use the DNEETFLK-KLH conjugate as immunogen, and DNEETFLKC (with an extra C in C termins) as the antigen when they screen the antibody from animal serum. For what I've know, the extra C is for conjugation with the solid substrate during immunoaffinity chromatography and shouldn't be an issue I am going to mention.
Okay so I have the antibody, antigen (DNEETFLKC) and the LVRPEVDVMCTAFHDNEETFLK peptide. I did a direct ELISA with DNEETFLKC. It works perfectly (at 2ng /well level) . I did a competitive with DNEETFLKC, it works too. Now I did a direct ELISA with LVRPEVDVMCTAFHDNEETFLK, no, I don't see any signal. I coated the plate with DNEETFLKC and use LVRPEVDVMCTAFHDNEETFLK as the competitor. The longer peptide (serial dilution from 3 ug to 1 ng /well) didn't bind to the antibody at all.
At first we though there should have some trace residues causing the trouble from the synthetic LVRPEVDVMCTAFHDNEETFLK (done by other company).
I've spiked the DNEETFLKC to the LVRPEVDVMCTAFHDNEETFLK and the competitive ELISA works. So this eliminated the residue factor.
Then I suspect it is related to the orientation of the antibody-antigen pair. The epitopic sequence here is in the C terminus but not N or in the middle of LVRPEVDVMCTAFHDNEETFLK . However, the antibody recognizes the DNEETFLK from the N terminus during the antibody production.
My question is: will this be the troubling causing factor? that I will need an antibody recognizing from C terminus of DNEETFLK if I need it to recognize LVRPEVDVMCTAFHDNEETFLK?
Thanks again for your patient to read the long post. I will send an enquiry to the antibody production company too but I also appreciate any comments from you guys!