sdAb Cell-Free Screening by In Vitro Compartmentalization
Single domain antibody plays important roles in immunotherapeutics development. To obtain sdAbs with sub-nanomolar affinities, in vitro approaches are necessary. Here, Creative Biolabs describes the novel cell-free screening of single domain antibody (sdAb) by in vitro compartmentalization (IVC).
Introduction of IVC
Immunotherapy refers to the treatment of diseases by activating or suppressing the immune system. In recent years, immunotherapy presents great potentials against various forms of cancer and has become of great interest to clinicians, biological researchers, and pharmaceutical companies. In this case, the antibodies’ identification with desired specificity and affinity is critical for novel immunotherapeutics development. Compared with conventional antibody, single domain antibodies (sdAbs) present great advantages, including small size, high stability, and great solubility. Based on the mature phage display technology, the sdAbs can be obtained easily. However, the in vitro approaches are necessary if sub-nanomolar affinities are required. Here, Creative Biolabs describes the cell-free screening of sdAb by in vitro compartmentalization.
Fig.1 The emulsion-based selection of antibody fragments is a Darwinian process. (Sepp, et al., 2012)
Cell-free Screening of sdAb by IVC
For IVC selection, the sequence components, namely, Arc operators, single chain antigen recognizing construct (scArc) encoding sequence, translation start codon ATG, T7 promoter, and terminator motifs, are added by subcloning of the sdAb library into pIE2a2A vector. Then, large gene libraries comprised of linear DNA fragments can be generated by PCR, and bacteria transformation is avoided. During selection, the PCR-based library is added to E. coli coupled transcription-translation extract and dispersed rapidly. When the number of genes equals to the number of uniform droplets, the most protein-DNA complexes are formed. Almost 90% of the library can be segregated at one gene per droplet while the gene-to-droplet ratio is 1:10. In this case, the genotype-phenotype linkage is created.
Finally, the sdAb-DNA complexes can be recovered and fractionated according to their affinity and dissociation rate for the target. According to a series of PCR amplification and selection rounds, the sdAbs with desired affinity can be obtained.
Main Steps of sdAb Cell-free Screening by IVC
- Library assembly and characterization
- Preparation of sdAb display library in emulsion
- Biopanning
- Assessment of library fitness
- Cloning and screening of library selection output
It is worth mentioning that the optimal concentration of the capturing antigen allows for the best clones enrichment with the highest affinity in the library. Besides, ten rounds of selection improve library fitness largely and more stringent selection conditions can also play an important lifting role.
Reference
- Sepp, A.; Griffiths A. Cell-free selection of domain antibodies by in vitro compartmentalization. Single Domain Antibodies. Humana Press, Totowa, NJ. 2012: 183-198.
Case Study
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