A Low-Cost Biological Agglutination Assay for Medical Diagnostic Applications

Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.


INTRODUCTION
Affordable point-of-testing diagnostic technology applied to resource-limited sites is one of the most promising biotechnologies for improving global health (Daar et al. 2002). The development of new diagnostics and their successful adoption in the field by end users requires consideration of various diverse factors including, but not limited to, scientific challenges, economic restrictions and practical considerations (Giljohann and Mirkin 2009).
High impact technology will allow sensitive and specific detection, it will be low cost and portable to ensure accessibility, and it will have a user-friendly design that does not rely on sophisticated equipment (Urdea et al. 2006).
Immunoassays are a dominant technology in in vitro diagnostics (Borrebaeck 2000).
Antibodies can be generated for the target analyte with very high specificity and relative ease.
Among the various types of immunoassay formats for the development of rapid diagnostic tests, latex agglutination tests (LAT), which use antibody molecules immobilized on latex particles to detect the presence of an analyte, are available for more than 300 diseases and biomolecules (Ortega-Vinuesa and Bastos-Gonzalez 2001). Multivalent immuno-latex particles can recognize their target analyte molecules through specific interactions and form higher order complexes (Ortega-Vinuesa and Bastos-Gonzalez 2001). As the agglutination reaction proceeds, extensive cross-linking between analyte molecules and latex particles leads to the formation of very large complexes that can be visibly detected by the naked eye or monitored spectrophotometrically (Price 2001).
The use of whole cells as a bioanalytical platform for in vitro medical diagnostics offers a number of favorable characteristics such as low cost of production. Whole cells are selfreplicating and can manufacture recognition elements such as antibodies, eliminating the need for expensive purification steps. In addition, as living organisms, whole cells can provide physiologically relevant data on the bioavailability of the analyte (van der Meer and Belkin 2010). Bacteria, especially model organisms such as Escherichia coli, are often used as bioreporters as they are amenable to genetic engineering and easy to culture. Bacterial whole cell biosensors have been genetically engineered to detect medically relevant analytes such as metabolites (Goers et al. 2017), toxic chemicals (Horswell and Dickson 2003) and mercury in urine (Roda et al. 2001), hydroxylated polychlorinated biphenyls in serum (Turner et al. 2007) and nitrogen oxides in both serum and urine samples (Courbet et al. 2015). Whole-cell biosensors usually rely on intracellular detection of the target analyte (Goers et al. 2013), but such designs are limited to the detection of analytes that can diffuse or be actively transported across the bacterial cell membrane. Nevertheless, there is a multitude of medically relevant analytes such as protein biomarkers of disease state (Assicot et al. 1993;Haverkate et al. 1997;Ohman et al. 1996;Thompson et al. 2004) that cannot cross the membrane barrier. A few bacterial whole-cell bioreporter designs have been demonstrated that detect the analyte extracellularly (Looger et al. 2003;Webb et al. 2016), but these are of limited versatility in terms of choice of target because of their mechanism of action.
Merging bacterial surface display technology and antibody engineering can provide a mechanism for the detection of extracellular analytes with whole-cell biosensors in a biological equivalent of the latex agglutination test (LAT). Single chain antibody fragments (scFv) have been displayed on the surface of bacteria using engineered display proteins that localize to the outer membrane of the cell (Daugherty et al. 1998;Francisco et al. 1993;Fuchs et al. 1991). However, scFvs often have poor solubility, misfold and aggregate when expressed in bacterial hosts. As an alternative, single domain antibodies or nanobodies, are antibody fragments from heavy chain-only antibodies produced by the Camelidae family or cartilaginous fish. They have high stability when expressed in bacterial hosts (Muyldermans 2013;Salema and Fernandez 2017) and have been demonstrated to functionally display on the outer membrane of E. coli (Salema et al. 2016a;Salema et al. 2016b;Salema et al. 2013) and other bacterial species (Cavallari 2017;Fleetwood et al. 2013).
Here we demonstrate the development of a biological equivalent of the LAT that utilizes the E. coli surface display of camelid nanobodies as a detection element. As a starting point, we used a nanobody against green fluorescent protein (GFP) with a tandem dimeric GFP (tdGFP) analyte to induce cross-linking as a proof-of-concept. Using this convenient platform and a mathematical model of the agglutination process, we explored the effects of variables such as the number of bacterial particles in the assay and the nanobody expression level on the limit of detection of the assay. The total number of nanobodies in the reaction was found to be the primary factor influencing the limit of detection. With this understanding, we then explored the modularity of the platform by creating a whole-cell biosensor for the detection of human fibrinogen (hFib), a clinically relevant biomarker high levels of which are associated with cardiovascular disease and inflammation (Willis et al. 2004) and low levels of which can be used in the diagnosis of disseminated blood clotting (Lowe et al. 2004). Here, we propose a modular biosensing platform that harnesses the versatility and sensitivity of the LAT, but without the requirement for expensive steps to isolate and purify antibodies.

Strains, plasmids and cell culture conditions
ΔlacX74 recA1 endA1 araD139 Δ(ara-leu)7697 galU galK λ -rpsL(Str R ) was used in this study. The pNVgfp (Salema et al. 2013) and pNVFIB1-2 plasmids (Salema et al. 2016a), which express the anti-GFP nanobody and two variants of an anti-human fibrinogen nanobody surface displayed through a -intimin, respectively, are described elsewhere. The pNVgfp02 -10 constructs were assembled by standard cloning procedures from the pNVgfp construct by replacing the T7 promoter of the original plasmid with a BBa_J2310x constitutive promoter and a designed RBS element.
For nanobody expression from pNVgfp and pNVFIB1-2, a single colony of freshly transformed DH10B cells was inoculated into LB medium supplemented with 34 g/mL chloramphenicol and grown for 2 hours at 37°C with shaking at 250 rpm. For anti-GFP nanobody expression cell cultures were induced with 1mM IPTG and incubated at 30°C for 3 hours and 250 rpm shaking. Cells were kept overnight at 4°C, then harvested by centrifugation, washed twice and re-suspended in sterile phosphate buffered saline (PBS) pH 7.4 to a final OD600 of 1 (1*10 9 cells). The pNVgfp02-10 library of variants utilizes constitutive promoters, and in this case, cell cultures were not induced with IPTG, but instead were grown for 5 hours at 37°C with shaking at 250 rpm. For anti-fibrinogen nanobody expression, cell cultures were induced with 1mM IPTG and incubated overnight at 37°C with shaking at 250 rpm before cells were harvested by centrifugation. Cells were washed twice and re-suspended in sterile phosphate buffered saline (PBS) pH 7.4 to a final OD600 of 1 (1*10 9 cells).

Nanobody quantification
The levels of cell-surface displayed nanobody were determined indirectly by measuring the were statically incubated for ~18 hours at room temperature. Assay results were documented using a photographic camera in one or more of the following photography setups: i) a microplate was placed on top of a dark surface, ii) a microplate was placed on top of a blue LED transilluminator without the use of blue light filter and iii) a microplate was placed on top of a blue LED transilluminator and the screen for blue light absorption was placed on top of the microplate. As for the results testing with hFib, a microplate was placed on top of a light box and photos were taken at three to six wells per frame using a mobile phone.

Fibrinogen mock samples
Human fibrinogen (hFib) (Plasminogen, vWF, and Fibronectin depleted; FIB3) was purchased from Enzyme Research Laboratories (USA) and diluted in PBS or in fibrinogen-deficient human plasma (Sekisui Diagnostics GmbH, Germany). For the plasma-based assays, fibrinogen stock solutions were made at ~ 5 mg/mL in plasma and then diluted to 1 mg/mL in PBS.

Mathematical model and computer simulations
The derivation of the mathematical model is described in Supplementary Note 3. The model was implemented in Matlab software using a third-party script (Garcia-Molla 2017a, b) for system simulations and sensitivity analysis.

Proof-of-concept for a new diagnostic assay system
For the detection of protein analytes, an assay format was adopted that resembles the format of the LAT. The assay design utilizes bacterial cells displaying a nanobody on their cell surface to facilitate analyte recognition in the extracellular environment ( Figure 1A)  nanobody on their cell surface have been incubated with monomeric GFP (negative control) or tdGFP (model biomarker) in microplates. The red box highlights wells with tdGFP concentrations that demonstrate high-levels of cell agglutination. In the main image, the microplate was back-illuminated with visible blue light for better contrast. In the secondary image, the same microplate was illuminated with the application of a GFP filter to demonstrate protein (GFP or tdGFP) concentrations.
For proof-of-concept experiments, E. coli cells were designed to express anti-GFP nanobody that is surface displayed through a -intimin anchor (Salema et al. 2013).

Modulating the dynamic range of the diagnostic assay
The use of bacterial agglutination assay as a new diagnostic technology would benefit from designs that allow versatility in the detection of disease biomarkers. While the design of the demonstrated prototype assay platform is inherently modular since the nanobody can be engineered to bind specifically to any target, different medical conditions may require detection of molecular targets at different physiological concentrations in biological samples (Assicot et al. 1993;Haverkate et al. 1997;NICE 2014;Ohman et al. 1996; Thompson et al. 2004).
To enable further understanding of our nanobody-induced cell agglutination system and allow for the development of tunable assays for specific medical applications, we adapted a previously described mathematical model of the agglutination process (Dolgosheina et al. 1992) to the specifics of our technology. Briefly, our mathematical model describes the  Table 1). The simulation predicted that the maximum value of normalized cell agglutination would occur at 7 nM tdGFP analyte ( Figure   2B). In practice, this means that these variables can be used to alter the concentration range of the zone of agglutination. Figure 2C shows the results of these simulations for both variables in their ten-fold perturbation scenarios. The prototype (v01) E. coli cells, which have been engineered to display an anti-GFP nanobody on their surface, were genetically modified to produce variants (v02-v10) that express different levels of anti-GFP nanobody. Genetic elements used to control the expression levels of the nanobody display vector are shown next to each variant. The bar chart shows the mean cell fluorescence of each cell variant population when labeled with saturating amounts of purified GFP. Cell fluorescence was recorded by flow cytometry, and error bars show standard deviation for three technical replicates. Images of cell agglutination assay in a microplate, with engineered E. coli cells displaying anti-GFP nanobody on their surface after incubation with various tdGFP concentrations. The microplate was back-illuminated with blue light. Control cells did not express any nanobody.

Bioassay development towards use as a real-world diagnostic test
Human Fibrinogen (hFib) is a glycoprotein with normal blood plasma levels between 1.5 and 4.5 mg/mL. hFib plays a role in blood coagulation when it is converted into fibrin by thrombin protease (Lowe et al. 2004). Elevated levels of fibrinogen in plasma samples are commonly used as a biomarker for determining the risk of cardiovascular disorders in humans (Willis et al. 2004), whereas low levels of fibrinogen in plasma are associated with blood disorders such as disseminated clotting (Lowe et al. 2004). hFib is a heterohexamer, consisting of two dimers of three different polypeptide chains and therefore it is inherently a multivalent antigen.
Therefore, we decided to develop a diagnostic test for hFib as a first real-world use case of our new bacterial LAT platform.
We began by constructing two cell lines that surface-display two different anti-hFib nanobody constructs (NbFib1 and NbFib2) with different epitope recognition sites (Salema et al. 2016a).
Each nanobody was expressed on the cell surface using the same display vector, and anti-hFib nanobody display was confirmed by flow cytometry and fluorescent microscopy analyses (Supplementary Figure 5). Additionally, we used our mathematical model with updated parameter values specific to the hFib system to predict the range of analyte concentrations at which the agglutination zone would be observed ( Figure 4A).
We then verified the designed diagnostic test with purified samples of hFib protein in a microplate agglutination assay format. For each of the two cell lines with nanobodies specific against different hFib epitopes, the assay produced bacterial agglutination zones at the predicted analyte concentration ranges. The detection limit was the lowest for the cell line displaying the NbFib1 nanobody at ~50 pM hFib in the reaction; the detection limit for the assay using the NbFib2 nanobody was ~230 pM ( Figure 4B). We speculate that these shifts can be attributed to differences in nanobody expression (Supplementary Figure 5).
Importantly, a control reaction using cells that display anti-GFP nanobodies did not exhibit any agglutination, providing further evidence that specific interactions between analyte and nanobody elements drive the crosslinking effect.
The availability of nanobodies that bind to distinct epitopes in hFib provided the opportunity to test whether the limit of detection could be further decreased by increasing the propensity for crosslinking through the availability of multiple binding sites ( Figure 4B). Indeed, a mixture of the cells expressing NbFib1 and NbFib2, the two that individually had the lowest limit of detection, resulted in a further decrease in the limit of detection to 10 pM hFib.
Finally, to test the robustness of the assay to potential sample matrix effects, we compared the assay results using samples of hFib reconstituted in fibrinogen-deficient human plasma with those reconstituted in phosphate-buffered saline (PBS). As shown in Figure 4C, there was no observable difference between samples prepared in PBS or human plasma samples, suggesting that the assay is robust to the sample background and can be used with real patient samples without interference.

CONCLUSIONS
In this paper, we describe the development of a biological equivalent to the LAT, an assay widely used in medical diagnostics. Our bioassay includes cells that are genetically engineered to display nanobodies on their surface, analogous to the latex particles coated with antibodies in LATs. The presence of the analyte induces bacterial cross-linking, which is visible to the naked eye and enables facile detection of the target analyte in a manner similar to the readout of the LAT. This expands the repertoire of the types of analytes that can be

NbFib1
NbFib2 NBFib1&2 A sensed using whole-cell biosensors to include large molecules such as protein biomarkers or disease agents, which cannot easily be transported into the cell.
Whole-cell biosensors have become an alternative promising tool for the detection of analytes in diverse applications, as they are easy to manipulate, simple and low-cost. Our platform adds a new tool to this arsenal for the extracellular detection of protein analytes. Additionally, our platform is unique in enabling analyte detection without requiring an active cell metabolism.
This contrasts with most previous solutions where the output signal is dependent on active cellular metabolism and/or gene expression for reporter protein production (Courbet et al. 2015;Daringer et al. 2014;Ostrov et al. 2017). Lastly, the platform can potentially offer large cost savings to assay manufacturers as the cells autonomously express and surface display nanobodies which removes the need for the costly steps of antibody production, purification and attachment to latex particles surface.
The design of our biological LAT is inherently modular and is readily extensible to the detection of other analytes of interest by exchanging the nanobody that is surface displayed through simple molecular cloning techniques. There are numerous developed nanobodies described in the literature (Harmsen and De Haard 2007). Alternatively, phage-display libraries can be used to select new nanobodies for different targets or to enhance specificity (Tanha et al. 2002;Verheesen et al. 2006)or immunization, e.g. of camels, llamas, or sharks, can be used to isolate new nanobodies if desired (van der Linden et al. 2000). Thus, the technology described here could be used as a platform for the development of a range of assays for different target analytes. For inherently multivalent targets, a nanobody that binds to a single epitope is all that is necessary to develop a new assay. Monomeric targets can be detected by mixing two cell populations expressing nanobodies that bind to different epitopes.
Additionally, we have shown that the dynamic range of this diagnostic platform can be easily tuned for the requirement of the particular application, and very low limits of detection are easily achievable. In particular, the limit of detection of our biological LAT assay for hFib was over 29,000-fold lower than that of a piezoelectric agglutination sensor (Chen et al. 2010) and about 13-fold lower than that of an amperometric immunosensor (Campuzano et al. 2014). Using our mathematical model in combination with the developed library of variants of nanobody expression, new diagnostic tests can be conveniently designed for target analyte concentrations at will.
An important consideration for the adoption of new technologies is ease-of-use and seamless integration into existing professional workflows. Our biological LAT platform is underscored by the simplicity of its design and gives a visual readout that is easy to interpret. In addition, it fits within existing clinical diagnostic frameworks since the LAT is used in clinical laboratories to detect a number of pathogens including H. influenzae (Heikkilä et al. 1987), N. meningitidis (Leinonen and Herva 1977), P. and S. pneumoniae (Smith and Washington 1984) and therefore, it could readily be adopted by existing personnel with minimal training required to implement it.