Phage Display: Not getting antibodies after 3 rounds of panning - (Mar/05/2007 )
Hi all
I am trying to select antibodies by phage display against a human protein (ca 23 kDa, glycosylated) expressed in Pichia Pastoris. I checked the protein before starting selections on SDS-PAGE and by Western. On SDS-PAGE the protein showed the expected bands with no apparent impurities. On Western, I could also see the expected bands, although the intensity of staining was quite weak compared to a control of commercial recombinant protein (could that be a problem of the used polyclonal antibody against the protein?). Furthermore, the proteins (sample and control) showed a single band of a bigger size in *reducing* conditions than in non-reducing conditions (3 bands there), although the protein is known to be dimerized in physiologic conditions.
I checked protein concentration with a commercial ELISA and that was ok.
I then coated the protein at 50µg/ml in PBS overnight on maxisorp tubes and started panning using a synthetic antibody phage display library. After 3 rounds of panning the titers of the eluted phages (1st round 2x10e5; 2nd round 1.8x10e5; 3rd round 4.8x10e6) did not rise as high as the titers of a control selection using Glutathion-S-Transferase (21kDa) (1st round 2x10e5; 2nd round 1x10e7; 3rd round 2.9x10e9) at same coating conditions.
I nevertheless screened 96 colonies for expression of binding scFvs in ELISA and got no positive clones for my target protein (positive control for coating of target protein and commercial antibody worked – although somewhat faint) while the control selection yielded about 50% positive clones.
This protein is really giving me pain, I also tried selection before using 10-7M biotinylated protein and using streptaviding beads. It did not work.
I am thinking that maybe the protein itself is somehow not a good target for phage display. Maybe due to aggregation? How would I see that?
I will try doing a fourth round, do you guys think that could help?
Any ideas?
Thanks in advance
marinder
I am trying to select antibodies by phage display against a human protein (ca 23 kDa, glycosylated) expressed in Pichia Pastoris. I checked the protein before starting selections on SDS-PAGE and by Western. On SDS-PAGE the protein showed the expected bands with no apparent impurities. On Western, I could also see the expected bands, although the intensity of staining was quite weak compared to a control of commercial recombinant protein (could that be a problem of the used polyclonal antibody against the protein?). Furthermore, the proteins (sample and control) showed a single band of a bigger size in *reducing* conditions than in non-reducing conditions (3 bands there), although the protein is known to be dimerized in physiologic conditions.
I checked protein concentration with a commercial ELISA and that was ok.
I then coated the protein at 50µg/ml in PBS overnight on maxisorp tubes and started panning using a synthetic antibody phage display library. After 3 rounds of panning the titers of the eluted phages (1st round 2x10e5; 2nd round 1.8x10e5; 3rd round 4.8x10e6) did not rise as high as the titers of a control selection using Glutathion-S-Transferase (21kDa) (1st round 2x10e5; 2nd round 1x10e7; 3rd round 2.9x10e9) at same coating conditions.
I nevertheless screened 96 colonies for expression of binding scFvs in ELISA and got no positive clones for my target protein (positive control for coating of target protein and commercial antibody worked – although somewhat faint) while the control selection yielded about 50% positive clones.
This protein is really giving me pain, I also tried selection before using 10-7M biotinylated protein and using streptaviding beads. It did not work.
I am thinking that maybe the protein itself is somehow not a good target for phage display. Maybe due to aggregation? How would I see that?
I will try doing a fourth round, do you guys think that could help?
Any ideas?
Thanks in advance
marinder
your phage by panning have not enrich. in phage display the protein purification is very important, purity ofyour protein is low ,maybe influence result.
your antibody library can contain all of the antibody, maybe your library have not needing antibody.
Hi marinder,
can i just ask what kind of antibody library you're using?
regards,
Miha
can i just ask what kind of antibody library you're using?
regards,
Miha
Hi Miha
I am using the ETH-2 Gold library. It is a library created by artificially diversifying a germline VH and VL fragment by degenerate PCR, and displays single chain antibodies (scFv) on the phage tip.
regards, marinder
[/quote]
your phage by panning have not enrich. in phage display the protein purification is very important, purity ofyour protein is low ,maybe influence result.
your antibody library can contain all of the antibody, maybe your library have not needing antibody.
[/quote]
hi biovector
You are right, antigen purity is important. however on sds-page i have not seen any contaminants in the sample. Mass spec also tells me that my antigen is pure.
I start to think as well that there might not be any binder against my protein in the library at all.
does anyone know whether glycosylation or disulfide-bridges in the antigen can affect panning success?
best, marinder
I am trying to select antibodies by phage display against a human protein (ca 23 kDa, glycosylated) expressed in Pichia Pastoris. I checked the protein before starting selections on SDS-PAGE and by Western. On SDS-PAGE the protein showed the expected bands with no apparent impurities. On Western, I could also see the expected bands, although the intensity of staining was quite weak compared to a control of commercial recombinant protein (could that be a problem of the used polyclonal antibody against the protein?). Furthermore, the proteins (sample and control) showed a single band of a bigger size in *reducing* conditions than in non-reducing conditions (3 bands there), although the protein is known to be dimerized in physiologic conditions.
I checked protein concentration with a commercial ELISA and that was ok.
I then coated the protein at 50µg/ml in PBS overnight on maxisorp tubes and started panning using a synthetic antibody phage display library. After 3 rounds of panning the titers of the eluted phages (1st round 2x10e5; 2nd round 1.8x10e5; 3rd round 4.8x10e6) did not rise as high as the titers of a control selection using Glutathion-S-Transferase (21kDa) (1st round 2x10e5; 2nd round 1x10e7; 3rd round 2.9x10e9) at same coating conditions.
I nevertheless screened 96 colonies for expression of binding scFvs in ELISA and got no positive clones for my target protein (positive control for coating of target protein and commercial antibody worked – although somewhat faint) while the control selection yielded about 50% positive clones.
This protein is really giving me pain, I also tried selection before using 10-7M biotinylated protein and using streptaviding beads. It did not work.
I am thinking that maybe the protein itself is somehow not a good target for phage display. Maybe due to aggregation? How would I see that?
I will try doing a fourth round, do you guys think that could help?
Any ideas?
Thanks in advance
marinder
Hi Marinder
Well I've also spent more than two months with the same problem while trying to select antibodies from an immune phage antibody library. The serum ELISA gave me good signal against the immunogen suggesting the immune system was specifically responding. But when panning enrichment was observed after each round but polyclonal ELISA gave no signal as well as monoclonal ELISA.
After spending a month on my desk I received a fresh antigen to pan on and it worked. What I'm trying to say is that it seems like your antigens is the problem in this case. I'll suggest you stop panning and start from expression again otherwise you'll pan for months without any success.
The other factor may be your phage library concentration >10^12/ml, check that as well.
Good Luck!!
Well I've also spent more than two months with the same problem while trying to select antibodies from an immune phage antibody library. The serum ELISA gave me good signal against the immunogen suggesting the immune system was specifically responding. But when panning enrichment was observed after each round but polyclonal ELISA gave no signal as well as monoclonal ELISA.
After spending a month on my desk I received a fresh antigen to pan on and it worked. What I'm trying to say is that it seems like your antigens is the problem in this case. I'll suggest you stop panning and start from expression again otherwise you'll pan for months without any success.
The other factor may be your phage library concentration >10^12/ml, check that as well.
Good Luck!!
Hi Chickgene
Thanks for your response and your suggestions. I also think the antigen is the problem.
Phage library concentration is high enough (about 5x10^12tu/ml).
I am now trying to express my protein of interest in mammalian cells. Only problem is that they make really not much!! Until I have enough antigen from this source, I will try deglycosylating the protein expressed from pichia pastoris and try panning again. Maybe the sugars added by P.pastoris hinder the panning. The Mass Spec people at our institute had quite some problem with digesting the protein, probably also because of glycosylation.
Best, Marinder