Oriented Immobilization of Single-Domain Antibodies Using SpyTag

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Technical Note Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Oriented Immobilization of Single-Domain Antibodies Using SpyTag/SpyCatcher Yields Improved Limits of Detection George P. Anderson,† Jinny L. Liu,† Lisa C. Shriver-Lake,† Dan Zabetakis,† Victor A. Sugiharto,‡,⊥ Hua-Wei Chen,‡,⊥ Cheng-Rei Lee,‡,⊥ Gabriel N. Defang,‡ Shuenn-Jue L. Wu,‡ Neeraja Venkateswaran,§ and Ellen R. Goldman*,†

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Center for Biomolecular Science and Engineering, U.S. Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, D.C. 20375, United States ‡ Viral and Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, Maryland 20910, United States ⊥ Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, Maryland 20817, United States § Tetracore, Inc., 9901 Belward Campus Drive, Suite 300, Rockville, Maryland 20850, United States S Supporting Information *

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.

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While sdAb offer several attractive properties relative to conventional antibodies, one limitation is that being small, their covalent attachment to a surface in a random fashion can impair their binding function;13 and it stands to reason that being much smaller than an IgG, their binding ability might be impaired to a greater extent as the binding interface represents a significantly larger portion of the total surface area.14 This limitation can become a critical consideration when conducting assays in complex matrices, such as plasma or serum, which can dramatically reduce assay sensitivity.15−17 Therefore, to achieve highly sensitive detection in complex matrices, creating the most effective assay possible is essential.

number of single-domain antibodies (sdAb), also called VHH or nanobodies, have been developed for integration into immunoassays for detection and diagnostic applications.1−3 Derived from the heavy-chain only antibodies found in sharks and camelids,4,5 sdAb are the smallest naturally occurring antigen binding domains at 12−15 kDa. 6,7 Notwithstanding their small size, sdAb display a high level of specificity and affinity for their antigens; often possessing affinities (KD) in the nanomolar or sub-nanomolar range.8,9 One advantage over conventional antibodies is that sdAb can refold to bind antigen after chemical or heat denaturation, enabling them to retain the ability to bind antigen after exposure to elevated temperatures.10 In addition, sdAb can easily be tailored for specific applications through protein engineering.11,12 © XXXX American Chemical Society

Received: May 3, 2019 Accepted: July 3, 2019

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DOI: 10.1021/acs.analchem.9b02096 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry To overcome this limitation, various methods have been used to orient the sdAb onto the capture surface.13,14,18 These include a number of noncovalent methods, such as the incorporation of a biotinylation tag or expression as a fusion with a biotin binding protein such as rhizavidin.19−22 Other strategies have focused on genetic fusions of sdAb such as with a bacterial S-layer,23 sortase-mediated ligation,24 or addition of an orienting tail that has the potential to influence orientation during covalent attachment by adding lysine or cysteine residues at the C-terminus of the sdAb.25−27 Although these methods can work well, they have limitations. Using the combination of a biotin tag and a biotin binding protein for orientation is generally not compatible for systems that utilize the same avidin−biotin interactions to generate the signal. Another limitation of any noncovalent immobilization is that even when the affinities of that interaction are quite high, in multiplexed situations such as that with MagPlex microspheres (color coded microspheres that can be used inasmuch as a 50plex assay), over an extended storage time, the antibodies of the mixed microspheres could switch places, possibly leading to either false positives or negatives. Sortase ligation, a covalent approach, while highly specific, often has low ligation efficiency and requires extended reaction times unless further remedies are implemented.28 On the other hand, the use of lysine or cysteine containing tails does not guarantee that immobilization will be through the tail, thus, the orientation may not be complete. The SpyCatcher/SpyTag pair is derived from the Streptococcus pyogenes fibronectin-binding protein FbaB, which contains a spontaneous isopeptide bond between lysine and aspartic acid. Splitting FbaB into two domains, combined with engineering the resulting fragments, yielded a 13 amino acid peptide (SpyTag) that binds to and forms a robust covalent bond with its partner SpyCatcher.29 We exploited this system to provide oriented immobilization of sdAb captures for use with Luminex xMAP immunoassays. We produced several sdAb and multimeric sdAb constructs as fusions with the SpyTag peptide. MagPlex microspheres were first functionalized with the SpyCatcher protein and then allowed to cross-link with the sdAb-SpyTag constructs. Initial tests showed the ability of oriented monomer, dimer, and trimer sdAb captures to improve detection versus unoriented sdAb in an assay for staphylococcal enterotoxin B (SEB) spiked into buffer. Next we examined the ability to improve detection of the nonstructural protein 1 (NS1) antigen from dengue virus (DENV) in serum using a dimer sdAb oriented through SpyCatcher/SpyTag. The oriented dimer capture provided significantly lower limits of detection of NS1 from each of the four DENV serotypes spiked into human serum versus an unoriented dimer. Finally, we demonstrated the potential to generate improved detection of the DENV NS1 antigen in clinical samples when using a sdAb dimer capture reagent oriented through SpyCatcher/SpyTag (Figure 1).

Figure 1. Schematic representation comparing the random orientation of sdAb achieved by standard chemical cross-linking vs the highly oriented sdAb-SpyTag fusion capture surface obtained by first coating microspheres with SpyCatcher.

previously described.35 Unless otherwise specified, other reagents were from Sigma-Millipore, VWR, or Thermo Fisher. DENV NS1 ELISA. The detection of DENV NS1 antigen in human serum samples was performed using the DENV Detect NS1 ELISA Kit (InBios, Seattle, WA) according to the manufacturer’s instruction with minor modifications. Instead of adding 50 μL of human serum to each well, as per product insert, 10 μL or 1 μL of samples were used to conserve the clinical samples. Clinical Samples. A total of 24 acute serum samples (including four for each of the four DENV serotypes, four nonDENV febrile illnesses, and four undifferentiated clinical samples) were tested. Sample collection expressed as days post-onset of fever for the samples tested are listed in Supporting Information, Table 1. These serum samples are deidentified, previously collected samples from DoD-IRB approved prospective studies in DENV endemic areas. They were characterized by established reference testing methods and are allowed to be used for this study under NMRC IRB Determination of Human Subject Research Project # PJT-1706. Protein Constructs. The components ACVE, SpyTag (ST), and SpyCatcher (SC) were prepared in the BglBrick format and utilized BglBrick cloning to produce fusions.31,35 The gene for the DD7-DD7-ST construct was synthesized by Eurofins Genomics with flanking Nco I and Not I restriction sites and cloned into pET22b. Protein sequences of the individual BglBricks as well as the DD7-DD7-ST are provided in the Supporting Information. In all cases, protein production and purification was as described previously. 36 After purification, protein concentration was determined by optical density at 280 nm; samples were divided into aliquots and quick frozen in dry ice. Protein samples were stored at −80 °C until use. MagPlex Sandwich Fluoroimmunoassays. To evaluate MagPlex sandwich immunoassays, each sdAb or SpyCatcher was immobilized onto a separate set of microspheres using the standard protocol, with 30 μL of each set of microspheres coated and diluted to a final volume of 300 μL following immobilization. The sdAb were immobilized in PBS pH 7.2, while the SpyCatcher was immobilized at a final concentration of 1 mg/mL by dilution of protein with 10 mM sodium acetate (pH 4.5), representing ∼85% final volume. Immobilization of SpyCatcher to the microspheres at lower pH, which gave ∼30% improvement in signal, was determined empirically by the preparation of SpyCatcher bead sets at a range of pHs, data not shown. Once the SpyCatcher bead sets were prepared, they

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EXPERIMENTAL PROCEDURES Materials. The SEB-specific sdAb ACVE30,31 and A3,32 as well as the sdAb that recognize the DENV NS1 antigen (DD5, DD1, and DD7)33 have been described previously. All cloning enzymes were from New England Biolabs. SEB was from Toxin Technologies. Recombinant NS1 from the four DENV serotypes was from the Native Antigen Company. Normal Human Serum (NHS) was from Sigma-Millipore. Expression plasmid, pET22b-BglII, used for the BglBrick cloning34 was B

DOI: 10.1021/acs.analchem.9b02096 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

increased apparent affinity through avidity.39,40 When immobilized, the flexible linkers between individual binding domains may provide for a greater range of motion with improved binding ability. The use of multimer sdAb constructs toward a low molecular weight toxin might also enable the capture of multiple toxins per sdAb construct, as SEB at 28 kDa is relatively small41 and steric hindrance should be minimal. We employed ACVE, a stabilized anti-SEB sdAb, as the basis for our capture reagent constructs. The A3 anti-SEB sdAb, which has a melting temperature above 80 °C, was used as the reporter in our detection assay.32 Two sets of ACVE constructs (having either one, two, or three ACVE in the construct) were prepared with and without a C-terminal SpyTag. In general, all the constructs were well expressed; yields are listed in Table 1.

were allowed to react with various sdAb-SpyTag fusions (50 μg) overnight, then any unbound sdAb-SpyTag was removed by washing with PBST (PBS+0.05% Tween) prior to storage. No noticeable degradation of the SpyCatcher beads or the sdAb-SpyTag in performance was observed due to storage at 4 °C during the course of our experiments (i.e., several months). Similar assay protocols were followed for the detection of both SEB and NS1.33 To perform the assay, relevant bead sets were resuspended by shaking and mixed together, using 0.5 μL of each MagPlex bead set for each sample to be tested (≥50 MagPlex beads/set). In many of the NS1 assays, we included four different control beads, similar to Radix Biosolution’s Assay CheX beads,37 that allow one to better assess the results. Using a magnet, the microspheres were washed twice with PBST and resuspended into the desired volume, that is, 5−10 μL/sample. To generate standard curves, antigen was spiked in the first row of a 96-well, polystyrene, round-bottom, microtiter plate containing either PBSTB (PBST + 1 mg/mL BSA) or a 50:50 (1:1) mix of normal human serum and LowCross Buffer (Candor, Wangen, Germany); then a serial dilution was made down the rows of the plate. The mixture of MagPlex microspheres was added to each well and the plate placed in the dark at 4 °C for 30 min. Next, the plate was washed three times with PBST using a 96 F magnet (BioTek, Winooski, VT). The beads were then incubated with 50 μL/ well of biotinylated (Bt)-sdAb (2 μg/mL diluted in PBSTB). After 30 min, the beads were washed three times and incubated for 15 min with 5 μg/mL Streptavidin-RPhycoerythrin conjugate (SAPE) diluted into PBSTB (50 μL/well). After a final two washes, an 85 μL aliquot of PBST was added to each well, and the binding was measured on the MAGPIX instrument (Luminex, Austin, TX). The median value obtained by the evaluation of ≥50 microspheres for each set was plotted. The standard error of the mean (SEM) was typically less than ±10% the mean of three replicates. When performing amplified assays, after the first SAPE incubation, the beads were washed three times, and 50 μL/well of Bt-Goat anti-streptavidin diluted to 1 μg/mL in PBSTB was added and incubated for 15 min. The beads were washed as before, and again, 50 μL/well of 5 μg/mL SAPE was added and incubated for 15 min. The beads were then washed a final time and measured as above. The same assay was performed on archived patient serum samples, except that the microspheres were added to the LowCross Buffer prior to dilution of the patient serum samples. To further analyze the data and to segregate DENV NS1 positive samples from negative samples, the ratio of the signal to background was determined by dividing the signal from the sample by the signal obtained from unspiked 50% NHS. To make a reliable determination of the presence of DENV NS1, a ratio of at least 2 was selected, as this ratio represents a value that provides nonoverlapping error bars when both the blank (zero) and samples with detectable amounts of DENV NS1 are plotted with error bars that represent three times the SEM.

Table 1 construct name

construct description

yield (mg/L)

ACVE ACVE-ACVE ACVE-ACVE-ACVE ACVE-ST ACVE-ACVE-ST ACVE-ACVE-ACVE-ST

sdAb monomer linked sdAb dimer linked sdAb trimer sdAb with SpyTag sdAb dimer with SpyTag sdAb trimer with SpyTag

35.6 16.6 10.2 8.4 3.6 1.1

We compared the immobilization of sdAb using standard EDC chemistry with sdAb anchored via SpyCatcher/SpyTag, as shown in Figure 2 and Supporting Information, Figure 1.

Figure 2. Comparison of immobilization methodology on the ratio of the signal divided by background following capture of SEB at various concentrations. The ACVE-ACVE dimer oriented on the microsphere via a SpyTag (ST) tail fused to the C-terminus and bound by a chemically attached SpyCatcher (ACVE-ACVE-ST-SC) provided the best results.

The oriented immobilization achieved through reacting the sdAb constructs containing a C-terminal SpyTag with the SpyCatcher functionalized MagPlex beads provided improved detection relative to EDC cross-linking via lysine residues, with the dimer outperforming either the monomer or the trimer construct. Avidity and increased range of motion of the binding elements likely account for the improvement of the dimer versus monomer. However, the oriented trimer performed worse than the dimer, while there is not a simple explanation for why it should perform worse, not improving could indicate the third domain may sterically hinder the first

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RESULTS AND DISCUSSION Our first implementation using SpyCatcher/SpyTag for orienting sdAb capture reagents included a comparison of monomer, dimer, and trimer sdAb constructs for the detection of SEB. The SEB toxin, a highly stable 28 kDa protein, is of interest both due to its role in foodborne illnesses and potential for use as a biothreat agent.38 Multimers of sdAb are advantageous for use in detection assays as they can provide C

DOI: 10.1021/acs.analchem.9b02096 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry and second domains, thus, resulting in no additional signal gain.42 The results on detecting SEB using the oriented dimer prompted us to use the same strategy toward improving a sdAb-based assay for DENV NS1 antigen. The NS1 antigen serves as a biomarker for the acute phase of DENV infection. DENV is the causative agent of dengue fever, a mosquitoborne viral infection endemic in many tropical and subtropical countries, and exists as four antigenically distinct virus serotypes (DENV-1, -2, -3, and -4).43 The ability to achieve sensitive detection of NS1 has potential both for diagnostic assays as well as for surveillance of mosquito populations in DENV prone environments.44,45 Multiplex MagPlex immunoassays have previously been demonstrated for the detection of IgM and IgG to antigens of arboviruses, including DENV.46 Previously we reported the isolation, engineering, and use of sdAb for the sensitive detection of NS1 antigen from DENV spiked into 50% NHS.33 Through the use of both captures and reporters with a lysine containing tail, we achieved sensitive and specific detection for all four serotypes. However, when we applied the assay to clinical samples, we did not achieve adequate sensitivity as compared to a commercial ELISA for NS1 detection and were not able to detect NS1 from any of the DENV-1 or DENV-2 positive samples (Supporting Information, Table 1). We hypothesized that a strategy to improve detection would be a combination of a commercial buffer that promotes improved signal over nonspecific binding in serum samples along with engineering the sdAb in an effort to achieve a more sensitive assay. In tests comparing the signal generated from DENV NS1 spiked into NHS diluted 50% with PBSTB versus diluted 50% with LowCross buffer, we observed an improvement primarily at low NS1 concentrations by approximately a factor of 2, which is significant, as this is the region where improvement was required. Signal generated by high concentrations of NS1 was unaffected, suggesting that the LowCross buffer decreases nonspecific binding of the NS1 to other serum components improving its accessibility for the sdAb (Supporting Information, Figure 2). Antibody engineering involved constructing a dimer of DD7, our NS1 capture sdAb, containing a C-terminal SpyTag that could be immobilized in an oriented fashion on SpyCatcher coated MagPlex beads. The SpyTag-containing dimer of DD7 is termed DD7-DD7-ST. We performed amplified xMAP assays for the detection of NS1 antigen from the four DENV serotypes that compared our original capture with the lysine tail (DD7-GS3K), with DD7-DD7-ST immobilized through conventional EDC chemistry and DD7-DD7-ST immobilized via the SpyCatcher. Use of SpyCatcher/SpyTag to produce an oriented dimer capture led to an increase in signal at all concentrations of NS1 and led to a dramatic improvement in our limits of detection of NSI spiked into 50% NHS. Assays were performed with two reporter sdAb constructs, DD5GS3K and DD1-GS3K; representative results are shown in Figure 3 and Supporting Information, Figure 3. A limited panel of clinical samples comprising one representative from each DENV serotype was assessed using the oriented DD7-DD7-ST capture and the DD7-GS3K capture, both paired with the DD1-GS3K reporter. Results are shown in Figure 4 and Supporting Information, Table 2. Use of the oriented dimer leads to robust detection of NS1 antigen from the DENV-1 serotype that was not detected utilizing the original DD7-GS3K capture. Similarly, detection

Figure 3. Enhancement of the Dengue NS1 assay via use of DD7DD7-ST oriented capture sdAb on SpyCatcher coated microspheres vs the sdAb construct DD7-GS3K that is partially oriented via the multilysine C-terminal tail. Similar enhancement was achieved when the capture sdAb was paired with either of two reporters: Bt-DD5GS3K, which has high specificity to DENV NS1, or Bt-DD1-GS3K which binds a different epitope and cross reacts with West Nile virus NS1. Samples were run in duplicate, and two separate SpyCatcher bead sets were utilized. The black line indicates a ratio of two.

of NS1 from DENV-3 increases from a weak to a strong positive. Unexpectedly, no detection of NS1 from DENV-2 was observed even utilizing the oriented dimer capture. Similar results were observed when pairing the captures with the DD5GS3K reporter (Supporting Information, Table 3). Currently, it is not clear why the assay as formulated fails to detect NS1 of the DENV-2 serotype in the clinical sample(s) tested here. The data obtained from our control bead sets did not point to any obvious reason. Thus, one possibility is that the epitope being recognized by DD7 is not available in those patient serum samples. Our initial thought was that it was being captured but not recognized by the biotinylated sdAb, however when tested using a different sdAb that recognizes a different epitope no improvement in detection of the DENV-2 patient samples was realized. Another possibility is that these serum samples also possess a level of antidomain antibodies that interferes with this assay, but that remains to be confirmed.16 D

DOI: 10.1021/acs.analchem.9b02096 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

DTRA RD Chemical and Biological Technologies Department, Project Number HDTRA1518114 (CB3636). The work at the Naval Medical Research Center was supported by Work Unit Number 6000.RAD1.L.A1224. G.D. is a U.S. military service member and S.W. is a U.S. government employee. The work of these individuals was prepared as part of official government duties. Title 17 USC. §105 provides that “Copyright protection under this title is not available for any work for the U.S. Government.” Title 17 USC. §101 defines a U.S. government work as a work prepared by a military service member or employee of the U.S. government as part of that person’s official duties. The views expressed are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of the Army, Department of Defense, or the U.S. government. V.S., H.C. and C.L. are employed by the Henry M. Jackson Foundation for the Advancement of Military Medicine and are funded to do this work by the U.S. Government.

Figure 4. Detection of NS1 from the four DENV serotypes. Comparison of DD7-GS3K capture (red) and DD7-DD7-ST capture oriented using two different SpyCatcher bead sets (blue and green). In all cases, the DD1-GS3K was used as a reporter. Undifferentiated clinical sample (UCS) and a sample positive for malaria (Mal) and chikungunya virus (CHIKV) were included as controls. Samples were run in duplicate. Samples with a signal to background ratio higher than two (black line) were labeled positive.

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CONCLUSIONS Several studies have found sdAb to be inherently thermostable, demonstrating antigen binding after exposure to elevated temperatures, which suggests they are well suited for long-term field applications in low resource regions where cold chain is often scarce. Recognition elements based on sdAb should offer the specificity of conventional antibodies with the potential for use and storage at elevated temperatures. Here, we take advantage of their ability to be genetically engineered, to prepare dimer sdAb constructs with a C-terminal SpyTag that makes for facile orientation of the sdAb onto microspheres precoated with SpyCatcher. This technique provided a dramatic improvement in signal intensity, which leads to a many-fold improvement in limits of detection and may facilitate the adaptation of sdAb into a variety of diagnostic platforms.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b02096. Protein sequences and supplemental figures and tables (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 202-404-6052. ORCID

Ellen R. Goldman: 0000-0001-9533-7455 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work at the U.S. Naval Research Laboratory was supported by the Joint Science and Technology Office and E

DOI: 10.1021/acs.analchem.9b02096 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

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DOI: 10.1021/acs.analchem.9b02096 Anal. Chem. XXXX, XXX, XXX−XXX