Hello, I have a curious situation. I have been biopanning against antigen. My phagemid vector has an amber stop codon which allows for expression of soluble antibody into bacterial supernatant which I use for ELISA usually. I also monitor enrichment by polyclonal phage ELISA. When polyclonal phage ELISAs show strong signals I pick individual colonies which I then induce antibody expression in and then use the supernatant for an ELISA. This time even though the polyclonal phage elisas have shown high signals the monoclonal ELISAs with soluble antibody are not giving me any strong signals. I confirmed that antibody is being produced by the cells by WB. I switched to screening individual clones by monoclonal phage ELISA and got high signals for a lot of my clones. (the positive clones have many different sequences). Why would an ELISA not work with soluble antibody and only work with phage for so many different clones? This has happened to me twice now with different experiments.
This is a quite common finding. I have seen a similar situation dozens of times. Only a fraction (highly variable) of clones able to produce positive phages produce active soluble antibody fragments. Some people claim that the conformation of PIII-fused protein is different. Although this is a possibility, in my opinion its more likely that aggregation-prone fragments tend to aggregate when secreted to periplasm, while they are protected from aggregation when attached to phage particles quickly exported to supernatant. I think so because I have observed higher success rate when phage-positive clones are not tested in production of soluble fragments, but their antibody genes are transferred to a mammalian expression system or to optimized baterial secretion vectors (for instance using chaperones). Often these antibody fragments are active both phage-displayed and expressed in these alternative expression system, but not in classical soluble fragments' production experiments like the one you describe. I would suggest to move to one of these other expression systems starting with your positive clones.
This is a quite common finding. I have seen a similar situation dozens of times. Only a fraction (highly variable) of clones able to produce positive phages produce active soluble antibody fragments. Some people claim that the conformation of PIII-fused protein is different. Although this is a possibility, in my opinion its more likely that aggregation-prone fragments tend to aggregate when secreted to periplasm, while they are protected from aggregation when attached to phage particles quickly exported to supernatant. I think so because I have observed higher success rate when phage-positive clones are not tested in production of soluble fragments, but their antibody genes are transferred to a mammalian expression system or to optimized baterial secretion vectors (for instance using chaperones). Often these antibody fragments are active both phage-displayed and expressed in these alternative expression system, but not in classical soluble fragments' production experiments like the one you describe. I would suggest to move to one of these other expression systems starting with your positive clones.
Thanks for the help! I have never had this problem before so I was surprised to see it was all my clones.
Hi all.
I am not on phagemid antibody production and evaluation but ... thinking about your problem and the response of Gertrudis Rojas I was wondering If diluting it further wouldn't improve your results.
My thought is based on the fact that you may be achieving high amounts of antibody production and then some hook effect (unbalanced amounts of antibody to antigen in the reaction) or competition among them.
Just another thought for you to think about. Of course, Gertrudis Rojas probably has the best response about it.
All the best.
The above is a possibility, of course, but usually the problem of negative antiody fragments remains the same irrespective of the dilution. Expression levels with phagemid vectors tend to be not so high.
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