Intracellular Antibody Capture (IAC) Methods for Single Domain Antibody (sdAb)
Intracellular single domain antibodies (sdAbs) refer to the recombinant proteins consisting of one variable region domain fragment. With the ability to bind intracellular molecules specifically and further interfere with their particular functions within various cellular compartments, intracellular sdAbs have been served as important tools for biological diagnosis and therapeutics. Here, Creative Biolabs describes the novel intracellular antibody capture (IAC) methods for sdAb generation.
Introduction of Intracellular sdAb
In modern biological research, monoclonal antibodies are important reagents for academic research and medical fields. However, their usage is largely restricted in vitro or for extracellular molecules on viable cells. In this case, the intracellular antibodies are necessary to modify and interfere protein functions inside viable cells. However, because the reducing environment precludes protein folding, most antibodies and fragments obtained using genera methods (including hybridomas and phage display) do not work inside cells. IAC technology was developed to solve this problem. Besides, the studies have shown that single domain antibodies, rather than other antibody fragments, are the most effective intracellular antibody fragments.
Fig.1 Schematic representation of IAC method.
Intracellular Antibody Capture (IAC) Method
Intracellular sdAbs can be utilized as powerful laboratory reagents in bioscience studies and progenitors of drug-like molecules in medical applications. The IAC method described here is based on yeast two-hybrid (Y2H) selections to obtain intracellular antibodies against any desired protein antigens. In Y2H, the tested proteins are expressed in yeast nuclei fused either to a DNA binding domain (bait) or to a transcriptional activation domain (prey). The reporter gene is activated and affects a particular phenotype of the yeast when the bait and prey interact. Here, the target proteins or protein domains are expressed as baits and intracellular sdAbs as preys. The intracellular sdAbs contain three complementary determining regions (CDRs), which are flexible peptide loops to bind antigens specifically. In conclusion, the IAC method uses a series of optimized single domain antibody libraries to obtain sdAbs against target proteins via in vivo isolation. Then the affinity maturation is performed to develop sub-nM affinity antibody fragments.
Fig.2 Schematic outline of intracellular antibody capture technology (IACT), suicide (or silencing) intracellular technology (SIT), and antigen-antibody interaction-dependent apoptosis (AIDA). (Weidle, et al., 2013)
Features of IAC Methods
- Avoid the preparation of native antigens in vitro, especially for the antigens which are difficult to prepare as recombinant proteins.
- The ability to isolate intracellular antibodies with high specificity and affinity via direct genetic selection.
- Critical isolation of intracellular sdAbs binding native antigens.
- Low false positive since two different bait systems are used in screening.
- Large scale screening after CDR randomization from the first round selected intracellular sdAbs.
Reference
- Weidle, U.; et al. The translational potential for target validation and therapy using intracellular antibodies in oncology. Cancer Genomics & Proteomics. 2013, 10(6): 239-250.
Case Study
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