Oriented Immobilization of Single-Domain Antibodies Using SpyTag/SpyCatcher Yields Improved Limits of Detection

Abstract

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Spy chemistry‐enabled protein directional immobilization and protein purification

“…The 13 aa SpyTag with a short linker is chemically synthesized on the agarose, while the POI fused with SpyCatcher and intein Mtu ΔI-CM (H73V/T430S) is captured by the SpyTag-modified resin, followed by the self-cleavage of the intein to release the POI. PDB, Protein Data Bank [Color figure can be viewed at wileyonlinelibrary.com] component in conjunction with glutathione S-transferase (GST) for immobilization of proteins on magnetic microspheres and nanoparticles for detection (Anderson et al, 2019;Ma et al, 2018).…”

Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly

“…It has been shown that surface-coated SpyCatcher1 can be used as a capture reagent for in vitro assays 17,27 ( Figure 4A). We wanted to establish such an oriented immobilization with the faster-reacting SpyTag2 and SpyCatcher3, which should provide an advantage at the low protein concentrations used for immobilization in ELISA assays.…”

“…Nevertheless, there is a small number of studies in which periplasmic expression of SpyTagged proteins were described 16,17,18 . Interestingly, during our initial investigation, we also discovered one antibody which could be produced in our standard E. coli expression strain, albeit with low yields.…”

“…Proof of concept for such an approach was demonstrated for one antibody by Geyer and colleagues 16 , and two publications 17,18 describe periplasmic bacterial expression of nanobodies with a SpyTag.…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…With respect to conventional IgGs (150 kDa), the tiny dimension of nanobody confers the exclusive capacity to bind to cryptic epitopes of viruses [2], a characteristic that at the present could be particularly useful to produce reagents suitable for coronavirus studies. Over the years, it emerged that VHHs are effective crystallography chaperones and molecular tools for protein structural characterization [3][4][5], convenient carriers for radioisotopes with extremely short half-life to use for in vivo PET/ SPECT imaging [6][7][8][9][10], valid immunoreagents for the controlled and oriented functionalization of nanoparticles, nanogels and biosensors [11][12][13][14] and that they can be even internalized by mammalian cells when provided with a suitable leader peptide [15]. More recently, their minimal dimension has been particularly appreciated by scientists looking for binders suitable to optimize the performance of super resolution microscopy [16][17][18] and to create tandem chimeric antigen receptors (CARs) that show higher target specificity due to the possibility of binding simultaneously multiple antigens [19].…”