Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays

Abstract

Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development.

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes

“…One of the clones had higher affinity as Fab than as scFv-CL fragment, while the other two lost either some or all of their affinities. As suggested by Chan et al (2011), structural changes might occur at variable domains upon conversion of the scFv to Fab, which may lead to change or loss of specificity. Also, in the case of the anti-STR and anti-PSA antibodies, the scFvs were analyzed as fusions with alkaline phosphatase, which is known to form dimers.…”

“…By using synthetic repertoires, the frameworks with the most favorable biophysical characteristics can be chosen as the library templates supporting the development of the retrieved antibodies into desirable final products (Tiller et al, 2013). However, scFv clones derived from synthetic libraries are sometimes unable to fold correctly or do not retain their affinity after conversion to a Fab or IgG format (Chan et al, 2011). Therefore, efficient and fast cloning methods are needed for the conversion of multiple clones from scFv to the more conventional formats to detect the best candidates for further development.…”

Phage and Yeast Display

“…Specifically, the presence of the constant heavy 1 and constant light domains may influence conformational characteristics of the V H and V L fragments. Compared with the scFv format, certain reports have described the increased capacity of selected Fabs to retain antigen specificity when converted to a full-length IgG antibody (34). In addition to favorable biochemical properties, in vivo studies demonstrated the biological potential of Fab antibody fragments.…”

Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis

“…Briefl y, a MaxiSorp immunotube (Nunc) was coated with 200 g/ml of Mtb -derived mycolic acid dissolved in n-hexane, after which the hexane was allowed to evaporate overnight at 4°C. Screening of the library was performed on antigen coated immunotubes essentially as described previously for protein antigens and modifi ed for lipid antigens ( 19 ). Briefl y, Fab-phage were incubated with coated immunotubes for 2 h at room temperature followed by washing with PBS/0.5% Tween-20 to remove nonspecifi c bound phage; specifi cally bound phage were then eluted by digestion with trypsin solution for 30 min at 37°C.…”