Print This PageSingle-Chain Antibody Library - FAQs
Q. Are there any restrictions as to who can receive the single-chain antibody library kit?
A. A kit will only be provided to entities, organizations, government agencies, and research universities that will use the kit only for research projects or teaching which is funded solely by the U.S. Government.
Q. How much does a kit cost?
A. There is no cost to qualified institutions, except for shipping charges.
Q. What will be provided in the single-chain library kit?
A. The library kit consists of the following:
- scFv 109 library in yeast strain EBY100
- pPNL6 - a starting plasmid vector for library construction
- EBY100 - yeast strain the library is in
- pPNL9 - secretion vector used for final production of scFv
- YVH10 - yeast strain used for secretion of scFv
- scFv Control in EBY100 - positive control for scFv expression and antigen binding
- Control biotinylated Antigen
Q. Some proteins we use seem to stick to all the yeast. Why?
A. Before starting any selection we routinely check the protein of interest for binding to a single scFv clone that is not induced (the control sent with the library), the library not induced and the library in the induced state. You should see no labeling in any case. If there is labeling it could be binding to the yeast surface or the scFv in a nonspecific fashion. We have the most reproducible results and experience with peptides and secreted proteins. However, we have also obtained antigen specific scFv to a variety of cytoplasmic proteins. We recommend trying different selection buffers to decrease or eliminate the nonspecific binding. For example, 0.1% tween/PBS, No EDTA, etc...
Q. I have gone through five rounds of selection and all I got is secondary reagents binders or nothing. What's wrong?
A. It could be a large number of possibilities. The most common are enumerated below.
- Did you check that the sorter was set up properly? Did the flow cytometer operator show you what was sorted by rerunning that sample or doing a test sort of labeled beads? Did you set up a dilution plate from the sorted cells? Was the number what you expected? We generally see about a 40-70% plating efficiency from the number of cells sorted.
- Do you know if your antigen is biotinylated? How? We use the Pierce HABA methodology to determine moles of biotin per mole of antigen and strive for a 1-2 moles of biotin /mole per of antigen.
- Try increasing the concentration of antigen up to 1µM.
- There may be no binders to your protein.
- Note, we have never not obtained a streptavidin binder if we go through five rounds of selection.
- You may want to try a test selection using biotinylated EGF. The quality of the aliquot of the library you received was checked by doing a multiplex screen using biotinylated EGF and f3 other peptide antigens. Binders were obtained for all. We have repeated this quality control at least twice, so the EGF will not be provided.
Q. Can I just use flow cytometric sorting or just use magnetic beads to get my antigen-specific clones?
A. Yes, however we believe combining the two is the most powerful way to screen the complete diversity of the library. In our paper, Hen egg lysozyme (HEL) was isolated using the Macs system only.
Q. I would like to do a test selection on the library with an antigen I know will work. What do you suggest?
A. HEL was used as test antigen by Dane Wittrup's group at MIT and three high affinity scFv clones were identified. HEL is cheap and is easily biotinylated. We do not recommend using the 378p peptide included as a control, as we did not include enough antigen for an entire selection. We have used a number of types antigens that are published in the Nature Biotech paper, any of these would work well.
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