Abstract
Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. 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. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.
Highlights
Pure and abundant before it can be used for biopanning
The recombinant ubiquitin protein and its complementary E. coli lysate were prepared for the biopanning simulations while the rMERS-NP and its complementary E. coli lysate were produced for ‘Yin-Yang’ biopanning
The overexpression of recombinant ubiquitin (rUbi) and rMERS-NP were visible in their respective crude preparations as a thicker band
Summary
Pure and abundant before it can be used for biopanning This presents a major limitation when attempting to develop antibodies against challenging target antigens. We propose a proof of concept panning process coined ‘Yin-Yang’ panning using antigen preparations devoid of purification for biopanning. The crude extract consisting of the antigen of interest in its native form alongside the endogenous E. coli proteins are used to achieve a balance between positive and negative selection. The proposed approach was able to isolate monoclonal scFv clones against the MERS-NP utilizing a crude preparation. The antibodies were verified and identified for specificity against MERS-NP This approach highlights the possibility of carrying out antibody phage display biopanning using crude antigens to identify antigen specific monoclonal antibodies
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