https://orcid.org/0000-0002-2110-4445
Figures
Abstract
Giardia lamblia (syn. intestinalis, duodenalis) is the causative agent of Giardiasis, a diarrheal disease of global medical importance, especially problematic in young children living in unhygienic, resource-constrained settings. Diagnostics of potential Giardia infections are generally done through classic light-microscopy stool examination. This is often insufficient, and ELISA-based fluorescence detection using costly proprietary reagents is employed. These reagents are often not affordable in contexts where they are needed the most, and this limits their use to resource-rich settings where Giardiasis is rarely problematic. To address these issues in medical equity while designing novel strategies to investigate the Giardia cyst wall, we report on the development and characterisation of alpaca derived single-domain antibodies, known as nanoantibodies, elicited against G. lamblia enriched cyst-wall preparations. We evaluated the effectiveness and binding capacity of twelve unique E. coli-produced recombinant nanoantibody sequences for Giardia cyst wall detection and provide proof of concept for the effectiveness and versatility of these protein domains.
Author summary
As a globally distributed intestinal parasite, Giardia lamblia causes 300 million cases of water-borne diarrheal disease per year, a scourge of underserved and neglected populations, and especially problematic in young children. Giardia detection relies on laborious microscopy-based analysis of faecal samples, often coupled to antibody-based detection of coproantigen using costly proprietary reagents which are not routinely affordable in the areas hardest hit by this parasitic infection. The availability of cost-effective and locally produced diagnostics tools for giardiasis would be of great advantage, especially in resource-limited settings. To begin to address these issues in global diagnostics equity, this work reports on the development and characterization of a set of twelve unique Nanoantibody (Nab) sequences, with specific and strong binding affinity to Giardia cysts both in vivo and in vitro. Nabs can be cheaply produced and scaled up without the need for expensive equipment, animal housing facilities and animal sacrifice. To our knowledge, the only other Nabs developed for parasite diagnostics target Trypanosoma cruzi, making this work a first in terms of novel diagnostic tools for intestinal parasites of global medical relevance.
Citation: Wirdnam CD, Schaerer T, Rubin M, Oberli A, Štefanić S, Hehl AB, et al. (2026) Functionally-derivatized Nanoantibodies as novel tools for Giardia lamblia cyst detection. PLoS Negl Trop Dis 20(3): e0013844. https://doi.org/10.1371/journal.pntd.0013844
Editor: Guilherme L. Werneck, Universidade do Estado do Rio de Janeiro, BRAZIL
Received: May 15, 2025; Accepted: December 6, 2025; Published: March 9, 2026
Copyright: © 2026 Wirdnam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The datasets supporting the conclusions of this article are included within the article (and its supplementary files).
Funding: Funding for this project was provided by the Stiftung für wissenschaftliche Forschung an der Universität Zürich (https://www.research.uzh.ch/de/funding/researchers/stwf.html) awarded to ABH, and by grant number #21A012 of the Novartis Foundation for Medical-Biological Research (https://www.stiftungmedbiol.novartis.com/) awarded to CF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The protozoan intestinal parasite Giardia lamblia (syn: intestinalis, duodenalis) is a protist intestinal parasite with clear zoonotic potential [1–3]. G. lamblia is the causative agent of Giardiasis, a diarrheal disease of global medical relevance, presenting with 300 million cases of diagnosed cases p.a. (a likely underestimate of the true extent of Giardia infection) [4] and one of the most common waterborne non-bacterial and non-viral diarrheal disease [5,6]. The species is present in at least eight so-called assemblages (A-H), or variants, with assemblages A and B commonly found in humans [7].
The transmission agent for Giardiasis is the parasite’s environmentally resistant form called the cyst. Giardia cysts remain viable and infectious even after prolonged periods (months) of environmental exposure. In contrast to the cyst, the flagellated trophozoite form is responsible for symptoms of Giardiasis (watery stool, cramping, bloating, nausea, malabsorption) [5] and, as a microaerophile, cannot withstand long-term exposure to air. Hence, only the cyst can survive outside a suitable host long enough to be detected, as both the transmission agent and diagnostic particle for Giardia detection.
Diagnosis of Giardiasis is still primarily light-microscopy based, with the operator scoring for cysts in fixed stool preparations on wet mounts. This method is generally sufficient but is often complemented by ELISA-based detection of coproantigen, specifically cyst wall protein 1 (CWP-1). CWP-1 is one of three abundant proteins that are expressed during parasite differentiation of the flagellated trophozoite form to the cyst form [8–10]. CWPs 1–3 are deposited in the cyst wall complexed to a unique polysaccharide, β1,3-linked-N-acetylgalactosamine [11] which renders the wall a formidable structure, resistant to mechanical and chemical insults, and essentially impermeable to most solvents. CWP-1 is detected using a monoclonal antibody (Mab) developed and licensed only by Waterborne Inc. and is included in diagnostic kits produced by different companies. This Mab is a costly proprietary product which has been extensively used off-label for basic research applications involving detection and visualization of Giardia cysts [10–27].
To develop cost-effective alternatives to the single proprietary Mab on the market for Giardia cyst/coproantigen detection, we embarked upon the generation of anti-Giardia cyst wall Nanoantibodies (Nabs) from alpacas. Nabs, also known as single-domain antibodies (sdAb) [28,29] correspond to the specific hypervariable domain (VHH; ca. 14kDa) derived from a special type of camelid single chain antibody.
Nab structure is composed of two β-sheets comprising four and five β-strands connected by loops and a disulphide bond (Cys23-Cys94). Surrounded by a conserved framework (FR1–4), three hypervariable regions (HV) located on loops H1-3 form a cluster at the NB N-terminus with a continuous surface. The complementarity-determining regions (CDR1–3) are responsible for antigen recognition and binding. Unlike paratopes for conventional antibody which are generally flat or concave, Nab paratopes are convex; this promotes binding and entry/interference with catalytic sites, making Nabs not only valuable detection but potential interference tools, thus presenting additional possibilities for therapeutic/pharmaceutical intervention [30–38].
Nabs are derived from targeted immunization of camelids with a specific antigen/epitope [32] or they can be generated synthetically as massive libraries of binders (known as Sybodies) and then screened for binding to targets of interest [28]. Here, we report on the generation, profiling, heterologous expression and testing/validation of 12 unique Nab sequences, derived from alpacas immunized with enriched in vitro-produced and fragmented Giardia cyst walls (Fig 1).
Twelve Nab sequences were cloned in vectors suitable for expression in E. coli periplasm as HA-tagged variants. Ten sequences were successfully expressed and characterized in terms of binding affinity, using immunoprecipitation, immunoblotting and immunofluorescence assays to antigens derived from extracts prepared in vitro from both encysting and non-encysting Giardia cells. Four Nabs were selected for a more in-depth analysis, and one was selected for derivatization by fluorophore coupling and tested for use as a detection tool for cysts in both in vitro generated suspensions and complex clinical reference faecal samples. Created in BioRender. Faso, C. (2025) https://BioRender.com/b4kdddf.
Following expression in E. coli, enrichment and extraction, we performed an in-depth investigation of each Nab’s binding preference using immunoprecipitation approaches coupled to tandem mass-spectrometry. Next, we selected the four most promising Nab candidate molecules for a more detailed identification of their preferred antigen, followed by fluorescence microscopy-based investigations on in vitro-cultured encysting Giardia cells and cysts to test their binding properties. Finally, we assessed the potential of a functionally-derivatized Nab for clinical application on reference clinical samples using microscopy-based detection workflows.
Results
Generation and cloning of twelve unique Nab sequences following alpaca immunization
Alpacas were immunized with suspensions of fragmented Giardia cyst walls, extensively washed to eliminate non-wall contaminants. Monitoring of the antibody response profile showed enrichment for IgG2 and IgG3 antibodies. cDNA from peripheral blood lymphocytes was used as a template to amplify the VHH repertoire and clone these sequences into a phagemid vector for display on filamentous phages. Positive selection of binders to the immobilized antigen fraction produced a total of 13 Nab sequence families with high affinity for fragmented cyst walls. These were cloned in a vector suitable for arabinose-induced expression in E. coli cells as periplasm-targeted cleavable HA-tagged fusions to 10x-His tagged Maltose-Binding Protein (MBP; Fig 2A). Each cloned Nab sequence was assigned a plasmid number and a no Nab negative control plasmid was additionally produced (Table 1).
A. Schematic of the expected protein product, including (from the N’ terminus) a periplasm targeting signal (pelB), a His-tagged MBP fusion protein, the cleavage site for protease HRV-3C, and the C-terminally HA-tagged Nab moiety. Created in BioRender. Faso, C. (2025) https://BioRender.com/ec3mcn9 B. Immunoblot analysis of crude lysates derived from induced (I) and uninduced (U) E. coli cells expressing each Nab fusion (plasmids 126-138), including Nab 139 as a no-Nab control. Predicted sizes for either the full-length fusion product (MBP-Nab-HA), His-tagged MBP alone (MBP) and HA-tagged Nab alone (Nab-HA) are indicated. All samples were separately probed using both anti-His and anti-HA antibodies.
Induction of expression for all Nab fusion constructs was tested (Fig 2B). Nabs 128 and 138 were found to be deficient and were not further pursued. Nab 128 had a deletion in the HA tag-encoding segment, while a closer analysis of the Nab 12-P-138 sequence revealed mutations at positions 52 (Phe→Gly) and 54 (Ala→Ser) previously reported as deleterious to overall stability [39]. Rescue of the Nab 138 sequence by site-directed mutagenesis was attempted but expression improved insufficiently.
Periplasmic extraction of Nab fusions, immobilisation on anti-HA agarose beads and proteolytic cleavage
Following periplasmic extraction and immobilisation of HA-tagged Nab fusion proteins on anti-HA agarose beads, a solution of Human Rhinovirus 3C (HRV-3C) protease at ~0.01 mg/ml was directly applied to release the Nab N-terminus, a requirement for efficient Nab binding. Immunoblot analysis of HA-tagged Nab fusions immobilized on anti-HA agarose beads shows that Nab trapping occurs as expected (Fig 3). Furthermore, both HA and His tag detection in samples pre and post HRV-3C protease treatment demonstrate efficient cleavage and release of the 10xHis-tagged MBP fusion partner in solution, while HA-tagged Nab remains trapped on beads. Incidentally, HRV-3C protease is also His-tagged and is likewise detected in the flowthrough.
Left panel: Immunoblot analysis using anti-HA tag detection of samples pre (n) and post (p) HRV-3C protease treatment for all indicated Nabs immobilised on anti-HA agarose beads through the C terminal HA tagged. Right panel: Immunoblot analysis using anti-His tag detection on supernatants/flowthroughs (Sup) from each Nab-agarose bead incubation reaction, post HRV-3C treatment. Bands for either the full-length fusion product (MBP-Nab), His-tagged MBP and HRV-3C alone (MBP and HRV-3C) and HA-tagged Nab alone (Nab-HA) are indicated by arrows.
Nab-mediated immunoprecipitation from G. lamblia extracts and target identification
Having established that all robustly expressed Nab sequences in E. coli can be immobilized and directly processed with HRV-3C on anti-HA agarose beads, N-terminally available Nab-decorated beads were used to immune-precipitate (IP) target antigen(s) from both non-encysting and encysting Giardia cell extracts from two independent biological replicates (S1–S3 Tables). Results from LC/MS analysis and calculation of relative iBAQ values (S4 Table) show that all selected Nabs, except for Nabs 127 and 137, identify almost exclusively encystation-related antigens such as CWPs and High Cystein non-variant cyst protein (Tables 2 and S4), generally with several fold enrichment when IPed from encysting as opposed to non-encysting extracts. Fold riBAQ enrichment was variable across biological replicates. However, and in contrast, IP using the no Nab control 139 captures several low-affinity binders in both non-encysting and encysting extracts, with no obvious preference for any antigen.
Immunoblotting of G. lamblia extracts using HA-tagged Nabs
Nabs generally perform best when used to detect native epitopes and often fail to detect linear ones [40,41]. Having established that several Nab sequences are capable of immunoprecipitating CWPs from encysting Giardia extracts, we tested whether these molecules could also function for detection in immunoblotting analyses in denaturing conditions. To do this, identical aliquots of encysting Giardia cell extract were subjected to denaturing gels, were blotted, and the resulting nitrocellulose membranes were cut in strips. These were incubated in HRC-3V treated periplasmic extracts derived from each Nab-producing E. coli line. Detection of the HA-tag showed that only Nabs 126, 127, 130, 133 and, to a lesser extent, 134, can detect denatured antigen, comparably to the anti-CWP1 Mab, currently the gold standard in Giardia cyst detection (Fig 4A).
A. Immunoblotting anti-HA analysis of Giardia extracts from 18h encysting cells using either the indicated Nab molecule or anti-CWP1 Mab-anti-Mouse-HRP shows that four Nab sequences can bind denatured encystation-related antigens. A Coomassie stained gel is included for equal loading quality control. B. Time course immunoblotting analysis of encysting Giardia extracts. N: non-encysting; 0-24: hours post induction of encystation. Each membrane was incubated with either with a Nab-enriched periplasmic extract (126, 127, 130, 133) and probed with αHA-HRP, or with α-CWP1 Mab/α-Mouse-HRP.
For a more detailed analysis of the binding profile for Nabs 126, 127, 130 and 133, we prepared extracts from encysting Giardia cells over 24 hours post induction of encystation (hpie) and non-encysting control cells. Each extract was loaded on five identical denaturing gels. Each gel was blotted, and membranes were incubated with either HRC-3V treated periplasmic extracts containing Nabs 126, 127, 130 and 133, or anti-CWP-1 Mab (Fig 4B). All Nabs consistently detect only encystation-related antigens, with Nabs 126, 130 and 133 showing a similar binding profile to the anti-CWP-1 Mab. However, the binding profile for Nab 127 clearly differs, and is consistent with detection of an antigen subject to processing as encystation progresses.
Defining the exact CWP binding targets for Nabs 126, 127, 130 and 133
Nabs 126, 127, 130 and 133 were shown to bind epitopes present only in encysting Giardia cells, in both native and denatured conditions. LC/MS data also points to CWPs and High Cystein non-variant cyst protein as the most likely binding targets for the aforementioned Nabs. To address the specificity of Nab binding in a more rigorous and conclusive manner and to determine exact binding targets for Nabs 126, 127, 130 and 133, we engineered E. coli lines for recombinant CWP expression and extraction. ORFs coding for CWP-1 (GL50803_005638), CWP-2 (GL50803_005435), CWP-3 (GL50803_002421) were cloned in expression plasmid pBXNPHM3, in frame with a pelB sequence at the 5’ end and a His tag coding sequence at the 3’ end (Table 3 and S1–S5 Text). Additionally, His-tagged truncated versions of CWP-2 (CWP2-C and CWP2-N) were also generated and similarly cloned in plasmid pBXNPHM3 (S3 and S4 Text).
Following transformation and arabinose induction, selected bacterial clones were analysed for recombinant CWP production. Induction of periplasm-targeted CWP-3-His expression resulted in fragile bacteria which yielded an extremely viscous extract. Analysis by immunoblotting of periplasmic extracts shows that CWP-1-His is expressed and correctly targeted for secretion, as are products CWP-2-N-His and CWP3-His (Fig 5A).
A. Immunoblot analysis of His-tagged recombinantly produced CWPs detected with αHis-HRP Mab in the soluble periplasmic (left upper panel) and the insoluble (left lower panel) fraction. Right panel: Immobilization and detection of His-tagged CWPs on Ni-NTA columns. Extracts from E. coli transformed with plasmid 126 (Table 1; His-MBP-Nab 126) were included as a positive control for expression, extraction and chromatography. B. Immunoblot analysis of CWP pull-down using HA-tagged Nab-decorated agarose beads from periplasmic extracts containing His-tagged CWP1, CWP2-and CWP3. All bands detected using αHis-HRP Mab.
However, CWP-2-fl-His and CWP-2-C-His are not detected in periplasm and immunoblotting of the insoluble fraction demonstrates expression of CWP-2-fl-His, while CWP-2-C-His remained undetected (Fig 5A). Consistent with these data, soluble CWP capture in periplasmic extract using Ni-NTA beads was successful only for CWP1-His and CWP-2-N-His (Fig 5A). Surprisingly, although we had previously detected CWP-3-His, we were unable to capture it.
We then tested whether agarose-bead immobilised Nabs 126, 127, 130 and 133 would pull down their target antigen from CWP-containing periplasmic extracts. As a control for Nab binding, we included agarose beads incubated with periplasmic extract prepared from E. coli transformed with control plasmid 139 (no Nab control). Immunoblotting of the IP reaction followed by detection of the His tag reveals that all selected Nabs bind CWP-1-His specifically, with Nab 127 additionally binding to CWP-2-N-His, with no detectable binding in the corresponding control reactions (Fig 5B). The viscosity of the CWP-3-His preparation was such that it was pulled down by all Nabs, including the no Nab control.
Nab-enriched periplasmic extracts as tools for IFAs
To further validate our Nabs as tools for detection of Giardia cyst wall material, we tested periplasm extract derived from E. coli cells expressing Nabs 126, 127, 130 and 133 in immunofluorescence assays (IFA) on both non-encysting and 18 hpie fixed and permeabilized Giardia cells and in vitro-generated cysts (Fig 6). As a negative control, we included periplasm extract from E. coli line 139 (Fig 6, no Nab control). Confocal fluorescent microscopy shows that the selected Nabs, like anti-CWP-1 Mab, label both encysting cells and cysts. Consistently, in these conditions, none of the tested NBs label non-encysting cell samples, confirming the selectivity of Nabs 126, 127, 130 and 133 for cyst wall antigens.
Confocal microscopy imaging fixed cells from non-encysting, 18h-encysting and Giardia cyst in vitro cultures. Samples were simultaneously labelled with the indicated HA-tagged Nabs, followed by anti-Rat-FITC, anti-CWP1-TxRed and DAPI.
Derivatization of selected Nab molecules and application to clinical reference samples
The selected Nab sequences have been shown to bind Giardia cyst walls with high affinity. However, Nab detection as shown previously, relies on binding of the HA tag and therefore requires a secondary anti-HA fluorophore-conjugated antibody.
To simplify the workflow for implementation of the selected Nabs, thus making them more amenable to detection of Giardia cysts in a diagnostic setting, we labelled Nab 133 with HiLyte Fluor 488 (Nab 133-HF488). As proof of concept, the directly conjugated molecule Nab 133-HF488 was first tested for detection of in vitro-generated Giardia cysts. Nab 133-HF488 was able to bind a suspension of in vitro generated cysts at ca. 2ug/ml final concentration (Fig 7A).
Nab 133-HF488 detects both A. in vitro-derived Giardia cysts and B. cysts in human patient-derived complex clinical reference samples. DIC: differential interference contrast. Ref 1-10: human clinical reference stool samples, randomly-picked from a larger pool, and blind.
To determine whether Nab 133-HF488 was able to detect Giardia cysts against a complex and heavily contaminated background, we next tested performance of Nab 133-HF488 on a set of ten randomly-picked human clinical reference stool samples, from a larger pool of stored samples. These formalin-preserved specimens have previously been examined by wet mount microscopy under daily routine diagnostic conditions (accredited according to ISO 1025 norms) and five of these had been previously called out as positive for Giardia cysts.
Visual inspection of samples at a fluorescence microscope (Fig 7B) for the presence or absence of labelled particles resembling Giardia cysts, was recorded (Table 4). These data were then compared to diagnostics outcome for each unblinded clinical reference sample. Implementation of Nab 133-HF488 at ca. 2ug/ml final concentration correctly identified all positive samples, demonstrating how Nab 133-HF488 can detect Giardia cysts even in complex clinical samples containing debris and other contaminants, with overall minimal background signal.
Discussion
Diarrheal diseases caused by infectious agents are still amongst the leading causes of mortality in children under five years of age [42]. Given how many different types of pathogens (viruses, bacteria, parasites) can cause diarrheal disease, with few distinguishing hallmark symptoms, accurate diagnostic tools are especially needed to determine the nature of the infectious agent at play, thus defining the appropriate therapeutic course of action.
Giardiasis is a primarily water-borne diarrheal disease of global medical relevance. Given Giardia’s anthropo-zoonotic nature, wildlife, domesticated and companion animal reservoirs of proliferating and encysting Giardia cells add to human cyst discharge, thus ensuring continued cycles of infection in both humans and other animals. It is therefore of paramount importance that Giardiasis-related diagnostic tools be readily available and easily implementable, especially in communities where household water quality is at risk of contamination with both human and animal organic waste.
Although Giardia cyst identification is still widely done by microscopic investigation of formalin-preserved stool samples, final confirmation of the presence of intact Giardia cysts can only be achieved using a commercially available anti-CWP-1 Mab, licensed by the company Waterborne Inc. as part of commercial kits for detection of coproantigen. These products are not immediately affordable for most diagnostics labs in LMICs, the most likely to be faced with cases of both acute and chronic Giardiasis. Similarly, regular monitoring for the presence and concentration of Giardia cysts in water supplies could be more frequent and cost-effective when based on more affordable reagents.
Nabs can be produced at a fraction of the cost of Mabs and with little to no animal suffering; no animal sacrifice is needed, and camelids can be immunized several times and, with appropriate clearance periods, can be exposed to several types of antigen while being constantly monitored for adverse reactions. When successful, the outcome of the procedure is a set of DNA sequences that can be readily cloned in a plasmid of choice, expressed in E. coli and other systems, and banked for repeated and indefinite use. Nab production media is cheaply and easily made, and the required equipment for Nab extraction is limited to a thermically regulated shaking incubator and a centrifuge. This has huge potential for democratization of resources for diagnostics and research, with the only essential information being a simple email carrying sequence information.
For this reason, we sought to develop anti-cyst wall Nabs as a valid alternative to expensive Mabs, while appreciating that all affinity-based molecular detection tools, including antibodies and Nabs, might lead to both false negatives and false-positives and suffer from similar limitations. Twelve unique Nab sequences were derived from the immunization and phage display panning cycles of enrichment for high-affinity binding to cyst wall material. Of these sequences, ten were successfully produced as fusion proteins in E. coli and were shown to be effective tools for detection of encystation-specific antigens and main protein components of Giardia cyst walls CWP-1/3 and CWP-2. Exceptionally, four out of ten Nab sequences were able to bind both denatured protein epitopes in immunoblotting experiments and preserved epitopes in formaldehyde-fixed cells in IFAs. One of these Nab sequences was chosen to provide proof of concept for the maintenance of affinity to cysts when directly conjugated to a fluorophore, thus mimicking the proprietary anti-CWP-1 Mab fused to a red fluorophore. Importantly, the enrichment workflow involving agarose beads to trap epitope-tagged Nabs appears to not be absolutely required for Nab-based detection, since crude periplasmic extracts could be used to label cysts. However, derivatizing a complex periplasmic extract is challenging, given contaminants and non-target particles which would render the derivatization procedure highly inefficient. Finally, and perhaps most importantly, fluorescent Nab was able to bind both in vitro-generated cysts and Giardia cysts in complex clinical reference samples.
For our Nabs to be an even more attractive alternative to the anti-CWP-1-Mab, it will be important to determine whether they can be used to detect coproantigen in lateral flow immunoassay (LFIA) diagnostic tests [43] and whether they can perform robustly on mixed infection samples, both aspects currently under investigation. To our knowledge, the lateral flow test employing an anti-Trypanosoma Nab for plasma samples is, to date, the only reported approach employing a Nab for parasite detection and provides for an encouraging precedent [44,45].
Taken together, in this report we present a cost-effective and scalable production workflow to produce high affinity Nabs as alternatives to expensive proprietary Mabs for the detection, in both immunoblotting and IF assays, of Giardia cysts and their main components. In the context of 3R-inspired approaches to reduce animal sacrifice, coupled to the need to promote equitable practices in medical and diagnostics, Nabs provide an excellent alternative to conventional antibody-based approaches and promotes the implementation of 3R principles in the furthering of human and animal health [46–48].
One important limitation of this study is the small sample size of tested human clinical reference samples, and, in addition, the lack of inclusion of veterinary clinical reference samples. Both a larger and more diverse sample population are important parameters to consider in this kind of study. We acknowledge this limitation and further work with larger sample sizes should be performed to confirm the results of this study.
Materials and methods
Ethics statement
The immunizations of alpaca were conducted strictly according to the guidelines of the Swiss Animals Protection Law and were approved by the Cantonal Veterinary Office of Zurich, Switzerland (License No. ZH 172/2014). Anonymous reference human clinical stool samples used in this study are commercial quality control samples which do not require ethical approval for the purpose of this study.
Giardia cell culture for encystation and transfection
Giardia lamblia trophozoites of strain WB-A C6 (ATCC 50803) were axenically cultured according to previously established protocols [49–52]. Cells were grown in standard Giardia growth medium at 37°C and passaged every two to three days when cultures had reached confluency. The ‘high bile’ method for encystation was used to maximize cyst production [53]. Trophozoites were cultivated to confluency in a T-25 culture flask. The medium was poured off and replaced with high bile medium (media with 10mg/ml bovine bile, pH adjusted to 7.85) and cultivated for 48 hours at 37°C. The cysts were gently pelleted (5min at 300g, RT) and washed with 10mL PBS. The cysts were gently pelleted again (5min at 300g, RT), the supernatant was discarded, and the cysts were stored in water at 4°C. Alternatively, the 2-step method for inducing a gradual process of encystation was used to produce encysting trophozoites at different stages of encystation [20,54].
Generation of alpaca-derived Nab sequences
Generation of Nab candidate sequences against fragmented G. lamblia cyst walls was done by the Nanoantibody Service Facility (University of Zurich) using alpacas for immunization and subsequent selection of high-affinity Nab sequences. Briefly, in vitro-culture Giardia cysts were washed extensively with 1xPBS to remove medium contaminants, resuspended in tap water for 1 week and kept cold, pelletted at 600g for 5min and washed extensively in tap water. The cyst suspension was disrupted by sonication (Branson Sonic Power-Sonifier B-12) at power level 4 and 100W, twice 30sec (with 10–15sec break), incubated, and monitored for complete fragmentation. The ensuing solution was used to immunize alpacas four times, over two months. Their immune response was monitored for production of IgG2 and IgG3 antibodies, leading to isolation of peripheral blood lymphocytes, RNA extraction, reverse-transcription and selective amplification. Cloning of amplicons to generate a VHH immune collection in a phagemid-based library allowed for production of Nab-decorated phages after superinfection with a helper phage. Two rounds of panning were employed to identify affinity-matured Nab sequences, thus yielding 12 unique Nab sequences covered by patent application EP26160048.0.
Nab and cyst wall protein expression in E. coli, extraction, immobilization on beads, proteolytic cleavage and storage
Each Nab sequence was amplified, coupled to a 3’ HA-tag encoding sequence, and flanked with SapI (NEBiolabs) restriction sites for ligation in plasmid pBXNPHM3. This allowed for arabinose-induced protein expression in E. coli strain MC 1061 of C-terminally HA-tagged Nab (Nab-HA), preceded by the HRV-3C recognition site and a 10xHis-tagged MBP (Maltose Binding Protein), and targeted to the periplasm. Control plasmid 139 was generated by excision of the ccdB gene for negative selection in plasmid pBXNPHM3, followed by treatment with DNA Polymerase I Large (Klenow) Fragment (NEBiolabs) and blunt-end ligation. Cloning design was done using the free software Ape [55].
Selected positive clones were grown in 2ml ON culture in LB + Amp and 1ml was used to inoculate 100ml of 2xY medium + Amp. This culture was induced for expression with 2ml arabinose 1% at OD = 0.6 and incubated ON at 20°. The next day, cells were harvested at 4000g for 20min, resuspended in 20ml of DOC periplasm extraction buffer (100mM Tris pH 8, 0.15% Na deoxycholate) and incubated on a rotary shaker at 4° for at least 1h. Cells were pelleted as before, the supernatant was aliquoted in microtubes and spun at 16000g for 10 mins, cold. Supernatants were then pooled in a single 50ml tube and incubated ON with 80ul anti-HA agarose bead slurry (ThermoFisher) on a rotary shaker, cold. The next day, each 50ml tube was briefly spun (20sec) to pellet the agarose beads, the supernatant removed, and the beads washed once with 10ml of 1xPBS. Beads were transferred in 1ml of 1xPBS back to microtubes, washed once more with 1xPBS, pelleted and resuspended in a final volume of 0.5ml PBS. For immunoblotting analysis of pre-cleavage product, a 20ul aliquot was removed and stored at -20°C. The remaining solution was once more briefly spun to pellet the agarose beads, resuspended in 1xPBS-DTT 1mM to a final volume of 100ul, and treated with ~0.01 mg/ml HRV-3C protease (Sigma SAE0045) ON at 10°C. The next day, agarose beads were recovered with a brief spin, the supernatant was kept to test for MBP release, and beads were washed four times with 1xTBS-1% Triton. Beads were then resuspended in 0.5ml TBS, and a 20ul aliquot was removed and stored at -20°C for immunoblotting analysis of post-cleavage product. The rest of the bead suspension was stored at 4°C.
Native co-immunoprecipitation
Encysted and non-encysted Giardia trophozoites (ca. 4x107 cells) were grown to confluency, harvested by detachment on ice, pelleted at 900g for 10 minutes in the cold, washed once in 1xPBS, pelleted as previously done, and finally resuspended in 5ml RIPA-0.1% SDS buffer (+ 100µl PMSF 0.1M + 50µl protease inhibitor solution, all ThermoFisher). Cells in suspension were disrupted by sonication (Branson Sonic Power-Sonifier B-12) at power level 4 and 100W, twice 30sec (with 10–15sec break), incubated at 4° on a rotating shaker for ca. 2h, pelleted at max speed, 4°, 10min. The supernatant was transferred to a fresh vessel, filtered through Acrodisc MS filters (Pall MS-3301) and diluted 1:1 with fresh RIPA-1% Triton to yield a clarified and filtered soluble fraction derived from disruption of either non-encysting (N) or encysting (E) Giardia cells.
Freshly prepared Nab-decorated anti-HA agarose beads, prepared as described in this report (section “Nab expression in E. coli, extraction, immobilization on beads, proteolytic cleavage and storage”) were distributed equally in non-encysting and encysting pre-clarified and filtered soluble fractions, and incubated overnight on a rotary shaker at 4°. Beads were recovered by brief spinning (20sec), washed thrice with 10ml TBS + 0.1% Triton, resuspended in 1xPBS, transferred to a new vessel, and washed thrice more with 1ml 1xPBS. After a final brief spin, the supernatant was entirely removed and beads stored at -20°C until transfer to the MS core facility for LC/MS analysis.
Liquid Chromatography Mass Spectrometry (LC/MS) and co-IP data analysis
Mass spectrometry and protein identification was performed by the Core Facility for Proteomics & Mass Spectrometry of the University of Bern. In a first step the samples were resuspended in 8M Urea with 50mM Tris-HCl at pH 8 and then reduced at 37°C with DTT 0.1M with 100mM Tris-HCl at pH 8 and alkylated at 37°C in the dark with IAA 0.5M and 100mM Tris-HCl for 30min. The slurry was then diluted four times with 20mM Tris-HCl with 2mM CaCl2 before digestion overnight with 100ng sequencing grade trypsin (Promega). The samples were then centrifuged for peptide extraction from the supernatant which were then subject to liquid chromatography LC-MS (PROXEON coupled to a QExactive mass spectrometer, Thermo Fisher Scientific). µPrecolumn C18 PepMap100 (5μm, 100 Å, 300 μm × 5mm, Thermo Fisher Scientific, Reinach, Switzerland) was used to trap the peptides and then they were separated by backflush on a C18 column (5 μm, 100 Å, 75 μm × 15 cm, C18) by applying a 40min gradient of 5% acetonitrile to 40% in water, 0.1% formic acid, at a flow rate of 350 nl/min. Full Scan was set at a resolution of 70 000, an automatic gain control (AGC) target of 1E06, and a maximum ion injection time of 50ms. The following settings were applied with the data-dependent method for precursor ion fragmentation: resolution 17,500, AGC of 1E05, maximum ion time of 110ms, mass isolation window 2 m/z, collision energy 27, under fill ratio 1%, charge exclusion of unassigned and 1 + ions, and peptide match preferred, respectively. MaxQuant (v. 1.6.14.0) was used for MS data interpretation against a Giardia lamblia database (Giardiadb v. 47) using the default MaxQuant settings. MS hits were sorted by their abundance according to the intensity-based absolute quantification (iBAQ) values. Relative abundance (riBAQ) was then calculated from the total iBAQ for each protein hit (riBAQ = iBAQ/ΣiBAQ*100).
Immunoblotting analysis, immunofluorescence assays and microscopy
Immunoblotting analyses were performed in standard conditions. Briefly, cell pellets of both Giardia and E. coli were dissolved in SDS sample buffer and boiled for 3 minutes. Dithiothreitol (DTT) was added to a final concentration of 7.75 µg/ml before boiling. SDS-PAGE on 12% polyacrylamide gels and transfer to nitrocellulose membranes was done according to standard techniques. Nitrocellulose membranes were blocked in 5% dry milk/0.05% TWEEN-20/PBS and incubated with anti-tag antibodies coupled to HRP (anti-HA-HRP and anti-His-HRP; Thermofisher) at the appropriate dilution in blocking solution and membranes developed using Western Lightning Chemiluminescence Reagent (PerkinElmer Life Sciences, Boston, MA, USA). Data collection was done in a MultiImage Light Cabinet with AlphaEase FC software (Alpha Innotech, San Leonardo, CA, USA) using the appropriate settings.
Immunofluorescence assays were performed as described in previous studies [49–52]. Briefly, cells were grown to confluency in 12ml Nunc polystyrene culture tubes (Thermo Fisher Scientific) and then cooled on ice to detach for 30min. Tubes were hit on a soft surface to detach all cells and then centrifuged at 900g for 10 minutes. The cell pellet was washed in PBS (phosphate-buffered saline) and transferred to 1.5ml Eppendorf tubes where the cells were fixed for a minimum of one hour or overnight in 3% formaldehyde (Sigma) in PBS. After fixation, samples were quenched in 0.1M Glycine in PBS for 5 minutes before permeabilization in 1ml of 2% BSA (bovine serum albumin) +0.2% Triton-X-100 in PBS, for 20 minutes at room temperature and finally, blocking in 1 ml of 2% BSA/PBS and incubated at 4°C for >2 h. Cells/cysts were then incubated for 1.5hrs at RT with periplasmic extracts derived from selected HA-tagged Nab-expressing E. coli lines, including no-Nab control extract derived from E. coli transformed with and induced to express plasmid Nab 139. Samples were washed twice in 1% BSA + 0.05% TX-100 in PBS and then incubated in rat-derived monoclonal anti-HA antibody coupled to FITC (dilution 1:250; clone 3F10 Sigma/Roche) and anti-CWP1-TxRed Mab (1:50, A300TXR-20X, Waterborne.Inc) in the same conditions. Following two washes as previously done, cells/cysts were then carefully resuspended in ca. 30 µl Vectashield (Reactolab) containing 4′-6-diamidino-2-phenylindole (DAPI) as a nuclear DNA label. Cells were imaged at a Leica SP8 confocal microscope generally at full cell diameter.
Fluorophore labelling of nanobodies
MC 1061 E. coli transformed with plasmid 133 were grown in 25ml LB media + 100µg/ml Ampicillin ON at 37°C. The overnight culture was used to inoculate 4L of 2x YT media + 100µg/ml Ampicillin and then grown at 37°C until the culture reached an OD600 of 0.6. Nanoantibody expression was induced by adding 1% arabinose to the culture and then incubated ON at 20°C. Afterwards, the bacteria were pelleted, the supernatant discarded and then resuspended in DOC-buffer (100mM Tris-HCl pH 8.0, 0.15 Sodium deoxycholate) for Nab 133 extraction. The bacteria were incubated for 4 hours at 4°C on a rotary shaker and then pelleted for 30min at 4200g at 4°C. The supernatant was then incubated with 2ml of HisPur Ni-NTA Superflow Agarose beads (ThermoFischer) ON at 4°C on a rotary shaker. The beads were spun down and resuspended in 50ml Imidazole wash buffer (50mM Tris-HCl pH 8.0, 300mM NaCl, 20mM Imidazole). Two more washing steps with 50ml Imidazole Wash Buffer followed. The beads were then washed two times (with 25ml and 50ml) in wash buffer 0.1M NaHCO3 pH 8.3 and incubated with 100µl (1mg/100µl DMSO) HiLyte Fluor 488 succinimidyl ester (Anaspec) in 2ml of NaHCO3 wash buffer. The dye was incubated with the beads for 3.5h in the dark at RT on a rotary shaker. Afterwards, the labelled beads were pelleted, the supernatant was removed, and the labelled beads resuspended in 50ml Imidazole wash buffer to be stored at 4°C before the purification step.
Labelled beads were then washed four times with His-Wash buffer (50mM Tris-HCl pH 8.0, 300mM NaCl, 20mM Imidazole, 0.1mM EDTA, 1mM PMSF). After that last wash step the His-Wash buffer was removed and labelled Nab 133 molecules were eluted by adding 7ml His-elution buffer (50mM Tris-HCl pH 8.0, 50mM NaCl, 300mM Imidazole, 0.1mM EDTA, 1mM PMSF) and incubating for 1h at RT in the dark on a rotary shaker. The supernatant was saved and then elution process was repeated one more time and the supernatant after incubation saved again. The eluted Nabs were concentrated in a 50ml concentration tube (Pall) by centrifugation for 100min at max. speed, 4°C. The concentrate was washed by adding 10ml PBS and centrifugation was repeated for 20min at max. speed, 4°C. This washing step was repeated and centrifugation was again applied for ca. 90min at max. speed, 4°C to achieve a final volume of 1.5ml labelled Nab 133-HF488. Azide was added to the labelled nanobodies at a final concentration of 0.01% for storage at 4°C in the dark.
Fluorescence microscopy analysis of in vitro generated cysts and complex clinical reference samples
Unfixed cysts produced in vitro were pelleted for 5min at 600g and RT, the supernatant was removed and the pellet washed once with 500µl PBS. After the centrifugation, the supernatant was removed and then 200µl labelled Nab 133-HF488 (undiluted was added to half of the cysts and 200µl Nab 133-HF488 (4µL stock in 196 µl PBS, 1:50, ca. 2µg/ml) to the other half. The cysts were labelled for 45min in the dark at RT and then pelleted for 5min at 600g and RT, the supernatant was removed and the pellet washed once with 500µl PBS. After centrifugation most of the supernatant was removed and a few µl of both samples were put on slides for fluorescent microscopy (Zeiss AxioImager M1 fluorescent microscope).
Ten complex human clinical reference stool samples (IFIK-University of Bern) were incubated with Nab 133-HF488. 50µl of each reference sample were added to 450µl PBS for washing and then pelleted for 5min at 600g and RT, the supernatant was removed and the pellet washed once with 500µl PBS. After the centrifugation, the supernatant was removed and then 200µl Nab 133-HF488 (4µl stock in 196 µl PBS, 1:50) was added to the pellets of the reference samples. The reference samples were labelled for 45min in the dark at RT and further processed as described above for labelling of in vitro-generated cysts.
Supporting information
S1 Dataset. Nab_IP MS data.
MS-based analysis of replicate (n = 2) immunoprecipitation experiments using HA-tagged Nabs 127–139 as handles on either non-encysting (N samples) or encysting (E samples) Giardia cell extracts. Data is presented across three tabs (protein groups 1–3). iBAQ values for every identified protein are reported as replicate couples (N1 and E1, N2 and E2) and listed for each Nab in separate tabs. Total iBAQ values are included for each sample, included an overall fold enrichment value for total iBAQ in encysting vs. non-encysting samples.
https://doi.org/10.1371/journal.pntd.0013844.s001
(XLSX)
S2 Dataset. Comparative analysis for all Nab IP datasets.
riBAQ values for each Nab-mediated immunoprecipitation experiment using either non-encysting (N samples) or encysting (E samples) Giardia cell extracts, for each replicate. Fold enrichment in encysting vs. non-encysting extracts are calculated for each detected protein.
https://doi.org/10.1371/journal.pntd.0013844.s002
(XLSX)
S1 Text. Sequence information (as.txt file) for plasmid P178-pBX_CWP1 created for E. coli-based inducible expression of recombinant CWP1.
https://doi.org/10.1371/journal.pntd.0013844.s003
(TXT)
S2 Text. Sequence information (as.txt file) for plasmid P179-pBX_CWP2 created for E. coli-based inducible expression of recombinant CWP2.
https://doi.org/10.1371/journal.pntd.0013844.s004
(TXT)
S3 Text. Sequence information (as.txt file) for plasmid P180-pBX_CWP2-N created for E. coli-based inducible expression of recombinant CWP2 C-terminal truncation.
https://doi.org/10.1371/journal.pntd.0013844.s005
(TXT)
S4 Text. Sequence information (as.txt file) for plasmid P181-pBX_CWP2-C created for E. coli-based inducible expression of recombinant CWP2 N-terminal truncation.
https://doi.org/10.1371/journal.pntd.0013844.s006
(TXT)
S5 Text. Sequence information (as.txt file) for plasmid P182-pBX_CWP3 created for E. coli-based inducible expression of recombinant CWP3.
https://doi.org/10.1371/journal.pntd.0013844.s007
(TXT)
S4 Table. Comparative analysis for all Nab IP datasets.
https://doi.org/10.1371/journal.pntd.0013844.s011
(XLSX)
Acknowledgments
We thank the Microscopy Imaging Center (MIC) of the University of Bern, for training and access to confocal microscopy facilities. We thank the Proteomics and Mass Spectrometry Core Facility (PMSCF) of the Department for Biomedical Research at the University of Bern for generating all proteomics data.
References
- 1. Barbosa AD, Egan S, Feng Y, Xiao L, Ryan U. Cryptosporidium and Giardia in cats and dogs: what is the real zoonotic risk? Curr Res Parasitol Vector Borne Dis. 2023;4:100158. pmid:38089689
- 2. Li J, Qin H, Li X, Zhang L. Role of rodents in the zoonotic transmission of giardiasis. One Health. 2023;16:100500. pmid:36844973
- 3. Ryan U, Cacciò SM. Zoonotic potential of Giardia. Int J Parasitol [Internet]. 2013;43:943–56. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0020751913001720
- 4. Bourli P, Eslahi AV, Tzoraki O, Karanis P. Waterborne transmission of protozoan parasites: a review of worldwide outbreaks - an update 2017-2022. J Water Health [Internet]. 2023 [cited 2023 Oct 31];21. Available from: https://pubmed.ncbi.nlm.nih.gov/37902200/
- 5. Ali Q, Ahmed S, Aamer S, Iqbal N, Mamoon N. Giardiasis: an overlooked cause of recurrent abdominal pain. Cureus. 2021.
- 6. Einarsson E, Ma’ayeh S, Svärd SG. An up-date on Giardia and giardiasis. Curr Opin Microbiol. 2016;34:47–52. pmid:27501461
- 7. Zajaczkowski P, Lee R, Fletcher-Lartey SM, Alexander K, Mahimbo A, Stark D, et al. The controversies surrounding Giardia intestinalis assemblages A and B. Curr Res Parasitol Vector Borne Dis. 2021;1:100055. pmid:35284870
- 8. Faso C, Konrad C, Schraner EM, Hehl AB. Export of cyst wall material and Golgi organelle neogenesis in Giardia lamblia depend on endoplasmic reticulum exit sites. Cell Microbiol. 2013;15(4):537–53. pmid:23094658
- 9. Faso C, Bischof S, Hehl AB. The proteome landscape of Giardia lamblia encystation. PLoS One. 2013;8(12):e83207. pmid:24391747
- 10. Ebneter JA, Heusser SD, Schraner EM, Hehl AB, Faso C. Cyst-Wall-Protein-1 is fundamental for Golgi-like organelle neogenesis and cyst-wall biosynthesis in Giardia lamblia. Nat Commun. 2016;7:13859. pmid:27976675
- 11. Gerwig GJ, van Kuik JA, Leeflang BR, Kamerling JP, Vliegenthart JFG, Karr CD, et al. The Giardia intestinalis filamentous cyst wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology. 2002;12(8):499–505. pmid:12145190
- 12. Lujá N, Mowatt MR, Conrad JT, Bowers B, Nash TE. Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats implications for secretory granule formation and protein assembly into the cyst wall. J Biol Chem [Internet]. 2023. Available from: www.jbc.org
- 13. Samuelson J, Bushkin GG, Chatterjee A, Robbins PW. Strategies to discover the structural components of cyst and oocyst walls. Eukaryot Cell. 2013;12(12):1578–87. pmid:24096907
- 14. Touz MC, Nores MJ, Slavin I, Carmona C, Conrad JT, Mowatt MR, et al. The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia. J Biol Chem. 2002;277(10):8474–81. pmid:11773053
- 15. Samuelson J, Robbins P. A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia. Trends Parasitol. 2011;27(1):17–22. pmid:20934911
- 16. Sun C-H, McCaffery JM, Reiner DS, Gillin FD. Mining the Giardia lamblia genome for new cyst wall proteins. J Biol Chem. 2003;278(24):21701–8. pmid:12686559
- 17. Erlandsen SL, Macechko PT, van Keulen H, Jarroll EL. Formation of the Giardia cyst wall: studies on extracellular assembly using immunogold labeling and high resolution field emission SEM. J Eukaryot Microbiol. 1996;43(5):416–29. pmid:8822813
- 18. Huang Y-C, Su L-H, Lee GA, Chiu P-W, Cho C-C, Wu J-Y, et al. Regulation of cyst wall protein promoters by Myb2 in Giardia lamblia. J Biol Chem. 2008;283(45):31021–9. pmid:18768462
- 19. Krtková J, Thomas EB, Alas GCM, Schraner EM, Behjatnia HR, Hehl AB. Rac regulates Giardia lamblia encystation by coordinating cyst wall protein trafficking and secretion. mBio. 2016;7.
- 20. Konrad C, Spycher C, Hehl AB. Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia. PLoS Pathog. 2010;6(4):e1000835. pmid:20386711
- 21. Chen Y-H, Su L-H, Huang Y-C, Wang Y-T, Kao Y-Y, Sun C-H. UPF1, a conserved nonsense-mediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia. PLoS One. 2008;3(10):e3609. pmid:18974834
- 22. Chatterjee A, Carpentieri A, Ratner DM, Bullitt E, Costello CE, Robbins PW, et al. Giardia cyst wall protein 1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. PLoS Pathog. 2010;6(8):e1001059. pmid:20808847
- 23. Castillo-Romero A, Leon-Avila G, Wang CC, Perez Rangel A, Camacho Nuez M, Garcia Tovar C, et al. Rab11 and actin cytoskeleton participate in Giardia lamblia encystation, guiding the specific vesicles to the cyst wall. PLoS Negl Trop Dis. 2010;4(6):e697. pmid:20532229
- 24. Hehl AB, Marti M, Kö P. Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Mol Biol Cell. 2000.
- 25. Davis-Hayman SR, Hayman JR, Nash TE. Encystation-specific regulation of the cyst wall protein 2 gene in Giardia lamblia by multiple cis-acting elements. Int J Parasitol. 2003;33(10):1005–12. pmid:13129521
- 26. Erlandsen SL, Bemrick WJ, Schupp DE, Shields JM, Jarroll EL, Sauch JF, et al. High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging. J Histochem Cytochem. 1990;38(5):625–32. pmid:2332623
- 27. McCaffery JM, Faubert GM, Gillin FD. Giardia lamblia: traffic of a trophozoite variant surface protein and a major cyst wall epitope during growth, encystation, and antigenic switching. Exp Parasitol. 1994;79(3):236–49. pmid:7525336
- 28. Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021;288(7):2084–102. pmid:32780549
- 29. Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013;82:775–97. pmid:23495938
- 30. De Meyer T, Muyldermans S, Depicker A. Nanobody-based products as research and diagnostic tools. Trends Biotechnol. 2014;32(5):263–70. pmid:24698358
- 31. Jovčevska I, Muyldermans S. The therapeutic potential of nanobodies. BioDrugs. 2020;34(1):11–26. pmid:31686399
- 32. Salvador JP, Vilaplana L, Marco MP. Nanobody: outstanding features for diagnostic and therapeutic applications. Anal Bioanal Chem. 2019:1703–13.
- 33. Ahmad MI, Amorim CG, Abu Qatouseh LF, Montenegro MCBSM. Nanobody-based immunosensor for the detection of H. pylori in saliva. Biosens Bioelectron. 2024;260:116423. pmid:38810413
- 34. Gao S, Yang W, Zheng X, Wang T, Zhang D, Zou X. Advances of nanobody-based immunosensors for detecting food contaminants. Trends Food Sci Technol. 2025;156:104871.
- 35. Ahmad MIA, Amorim CG, Abu Qatouseh LF, Montenegro MCBSM. “Nanobody-based immunodiagnostics: a systematic review of nanobody integration in diagnostics and deep insight into electrochemical immunoassays”. Microchem J. 2024;196:109628.
- 36. Mustafa MI, Mohammed A. Nanobodies: a game-changer in cell-mediated immunotherapy for cancer. SLAS Discov. 2023;28(8):358–64. pmid:37634615
- 37. Kolkman JA, Law DA. Nanobodies - from llamas to therapeutic proteins. Drug Discov Today Technol. 2010;7(2):e95–146. pmid:24103724
- 38. Sroga P, Safronetz D, Stein DR. Nanobodies: a new approach for the diagnosis and treatment of viral infectious diseases. Future Virol. 2020;15(3):195–205.
- 39. Dingus JG, Tang JCY, Amamoto R, Wallick GK, Cepko CL. A general approach for stabilizing nanobodies for intracellular expression. Elife. 2022;11:e68253. pmid:36416528
- 40. Beghein E, Gettemans J. Nanobody technology: a versatile toolkit for microscopic imaging, protein-protein interaction analysis, and protein function exploration. Front Immunol. 2017 [cited 2025 Apr 3];8:276923. Available from: www.frontiersin.org
- 41. Pardon E, Laeremans T, Triest S, Rasmussen SGF, Wohlkönig A, Ruf A, et al. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014;9(3):674–93. pmid:24577359
- 42.
GBD_2021_Booklet_FINAL_2024.05.16.
- 43. Mabey D, Peeling RW, Ustianowski A, Perkins MD. Diagnostics for the developing world. Nat Rev Microbiol. 2004;2(3):231–40. pmid:15083158
- 44. Pinto Torres JE, Goossens J, Ding J, Li Z, Lu S, Vertommen D, et al. Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections. Sci Rep. 2018;8(1):9019. pmid:29899344
- 45. Stijlemans B, De Baetselier P, Caljon G, Van Den Abbeele J, Van Ginderachter JA, Magez S. Nanobodies as tools to understand, diagnose, and treat African trypanosomiasis. Front Immunol. 2017 [cited 2025 Mr 26];8. Available from: https://pubmed.ncbi.nlm.nih.gov/28713367/
- 46. Su Q, Shi W, Huang X, Yin S, Yang X, Lu X. Recent advances of nanobody applications in diagnosis and detection. MedComm – Biomater Appl. 2023;2(3):e54.
- 47. Alexander E, Leong KW. Discovery of nanobodies: a comprehensive review of their applications and potential over the past five years. J Nanobiotechnol. 2024;22(1):661. pmid:39455963
- 48. Jin BK, Odongo S, Radwanska M, Magez S. Nanobodies: a review of diagnostic and therapeutic applications. Int J Mol Sci [Internet]. 2023;24. Available from: https://pubmed.ncbi.nlm.nih.gov/36983063/
- 49. Morf L, Spycher C, Rehrauer H, Fournier CA, Morrison HG, Hehl AB. The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes. Eukaryot Cell. 2010;9(10):1566–76. pmid:20693303
- 50. Zumthor JP, Cernikova L, Rout S, Kaech A, Faso C, Hehl AB. Static clathrin assemblies at the peripheral vacuole-plasma membrane interface of the parasitic protozoan Giardia lamblia. PLoS Pathog. 2016;12(7):e1005756. pmid:27438602
- 51. Cernikova L, Faso C, Hehl AB. Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia. PLoS Pathog. 2020;16(2):e1008317. pmid:32092130
- 52. Pipaliya SV, Santos R, Salas-Leiva D, Balmer EA, Wirdnam CD, Roger AJ, et al. Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata. BMC Biol. 2021;19(1):167. pmid:34446013
- 53. Kane AV, Ward HD, Keusch GT, Pereira EA. In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency. J Parasitol. 1991.
- 54. Morf L, Spycher C, Rehrauer H, Fournier CA, Morrison HG, Hehl AB. The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes. Eukaryot Cell [Internet]. 2010 [cited 2024 Sep 28];9:1566–76. Available from: https://pubmed.ncbi.nlm.nih.gov/20693303/
- 55. Davis MW, Jorgensen EM. ApE, A Plasmid Editor: a freely available DNA manipulation and visualization program. Front Bioinform. 2022;2.