Soluble proteins are desirable for protein purification whilst insoluble proteins must be solubilized, purified and re-folded. Initially, solubility of the antigen is governed by the secretion of antigen into the cytoplasm (soluble form), periplasm (soluble form) and as inclusion body (insoluble form due to protein aggregation). Nevertheless, several complications can be anticipated during antigen expression that hinders the availability of a target antigen for biopanning. This is evident with the reports of antibody development for dengue virus 10, herpes simplex virus 11, cytomegalovirus 11, Zika virus 12, Ebola virus 7, 13, Plasmodium species 14, Mycobacterium tuberculosis 15, and nematodes responsible for filariasis 16. The main assurance is that these proteins are mainly prepared with good purity and yield. The target antigens used for biopanning includes recombinant proteins, peptides, tissues and whole cells 8, 9. The affinity enrichment process involves several steps: immobilization of target antigen, binding of phage to target antigen, elimination of unbound phage and elution of phage 7. To identify a target specific antibody, an in vitro selection process underlined by the affinity enrichment concept known as biopanning is commonly used 2. A detailed review on antibody phage display is provided by Ponsel, et al. It has since been widely employed for the in vitro development of monoclonal antibodies, trading places with the conventional hybridoma technology which was still mainly animal host dependent 2, 3. Phage display technology is regarded as an important tool used for the discovery of novel ligands against various targets of interest 1. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display. Three unique monoclonal antibodies were isolated in the process. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. A naïve human scFv library with kappa light chain repertoire with a library size of 10 9 was developed. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. This includes production complexity, downstream purification, quality and yield. Target antigen preparation is a main bottleneck associated with the panning process. Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |