Skip to content
naulon
Get started

No retraction, correction or concern found when last checked on Oct 25, 2026.

Biotechnological applications of recombinant single-domain antibody fragments

Ario de Marco

Microbial Cell Factories · 2011 · Review

doi:10.1186/1475-2859-10-44

Abstract

Background. Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications.

Results. The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic.

Conclusions. Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments.

Full text

From PubMed Central, U.S. National Library of Medicine, as current on Oct 25, 2026. Paragraph numbers are ours, so an answer can point at one.

Introduction

Conventional poly-and monoclonal antibodies are still indispensable reagents in basic research and diagnostics. Nevertheless, both of them have some shortcomings-most of all the batch-to-batch variability of the polyclonal antibodies and the elevated costs and long time necessary for the production of the monoclonal ones. Furthermore, their dimension is detrimental for some diagnostic and therapeutic applications since it limits the efficient penetration into solid tumors and the passage through the blood-brain barrier. All these reasons urged the development of strategies aimed at the production of alternative scaffolds and recombinant antibodies of smaller dimensions that could be easily selected, produced, and manipulated using standard molecular biology techniques.

Although a vast number of recombinant antibody structures has been proposed, the single chain (scFv) and the single domain (VHH, VH, and NAR V) formats are the most widespread for both research and industrial applications. Recombinant antibodies seem particularly promising as immunoconjugates and for activating the biosensor chip surfaces for detecting specific antigens. Specifically, VHHs succeed in targeting brain epitopes by transmigrating through the blood-brain barrier, can be used for tuning and detecting the activity of cell proteins in vivo, provide better diffusion in fixed cells in comparison to conventional antibodies, and can simplify the generation of anti-idiotypic antibodies suitable for vaccination.

The success of the technology makes it impossible to review the entire literature concerning the different classes of antibody fragments and their applications in biology. The modes of application of VHHs, even though recently reviewed, constantly increase in numbers with the publication of new reports describing innovative uses. Therefore, this paper will on the one side integrate information concerning the established biotechnological utility of single-domain antibodies with the most recent data. On the other side, it will overview the potentials of the different classes of single-domain antibodies that developed massively in the last two years, such as immunoaffinity, assisted crystallography, protein aggregation and activity tuning, and toxin inactivation, for which a systematic update is now necessary.

Unconventional single-domain antibodies

Camelidae single-chain antibodies and their recombinant VHH domains have ceased to be an exotic group with a merely theoretical interest and have become an established tool in biology and biotechnology. The number of publications that increases year in and year out testifies that an ever larger number of research groups work successfully with these molecules in different fields. Although not equally widespread, also the single chain antibodies of shark origin (IgNAR) have seen an increasing interest as a source of single-domain antibodies. In contrast, the scientific community is probably less aware of the qualities of other single-domain antibodies, the existence of which was for long time considered a simple anomaly (Figure 1).

VHH stability has been attributed to the mutation of the hydrophobic residues responsible for the interaction between VL and VH domains. However, there are single-domains, such as the human VH domain HEL4, that conserve conventional VH hallmarks but are stable even in the absence of the pairing VL partner. HEL4 crystal structure suggests that the reason of its stability is due to an interaction by which the CDR1 partially masks the hydrophobic residues otherwise exposed on the framework region. This structural arrangement resembles the stabilizing role of CDR3 residues observed in some VHHs, resulting in a loop partially bending over the framework and indicates that single-domain antibodies can display different conformational patterns to prevent the exposure of potentially destabilizing amino acids. Although useful, the necessity to renounce the loop flexibility that contributes to the framework stability constrains the theoretical variability of the paratope in terms of shape and residues. In this sense, a VH domain stabilized by the sole replacement of the hydrophobic residues involved in the VL interaction has been proposed since it would preserve the maximal structural adaptability of the CDR3 and offer an ideal scaffold for generating libraries with loops hypermutated with respect to both length and residue combination. At the same time, these libraries derived from human VHs would represent a source for selecting binders with no immunogenic response and, therefore, of great therapeutic interest.

For this purpose, it is relevant that individual VHs not only have structural features similar to those of the VHHs in terms of thermal stability and refolding efficiency, but also that they are perfectly functional in terms of antigen-binding characteristics, as initially reported by Ward et al.. The mouse VH domains isolated by panning against lysozyme had affinity in the 20 nM range for their antigen and the same binding affinity was calculated for other VH single-domain antibodies isolated successively from a semi-synthetic library. Another single-domain antibody (clone V86) was successfully used in several immunotechniques and its nature as a structurally independent domain was confirmed by the observation that its specificity and affinity for the antigen were almost completely lost when it was coupled with a VL domain for reconstituting a scFv.

Single-domains with VH signature were isolated also after panning of Camelidae libraries designed for sampling only VHHs and their presence was considered as the consequence of material contaminations during the amplification steps of the single-domain DNA. However, these VHs were functional and stable, indicating that they possessed structural features of independent single-domain antibodies.

Finally, specific classes of VH domains have been identified recently both in llama and mouse for which the presence of the light chains is entirely optional since they can either form conventional or equally functional but single-domain antibodies. The stability and functionality of independent VHs is maintained also when they are expressed as intrabodies.

The identification of stable VH domains has been recognized as a potential opportunity to widen the overall pool of functional single-domain antibodies. From this perspective, the protocol used for single-domain library preparation has been modified for obtaining mixed libraries containing the whole repertoire of both VH and VHH domains. Stable and antigen-specific VH domains were isolated by panning from these libraries.

In contrast to VHs, only few independent VL domains have been reported as maintaining the binding capacity and specificity of the complete antibody molecule. These result from the in vitro cleavage of functional IgG fragments, as in the case of the VL binder specific for the streptococcal protein L and of the VL-barnase immunotoxin that accumulates soluble and functional in E. coli cytoplasm.

Targeting bacteria and phages

Antibodies directed against pathogenic bacterial-specific surface antigens enable to identify them in, and remove them from, biological samples. The advantage of using fragment antibodies lies in the fact that they lack the Fc domain, an IgG conserved region recognized by related proteins expressed by bacteria belonging to different species. Consequently, conventional antibodies are not suitable for specifically identifying a bacterial sub-group inside a complex population, whereas single-domain antibodies constitute a reliable tool for these diagnostic applications. For instance, single-domain isolated from a VH library and directed towards the staphylococcal Protein A has been used in diagnostic applications based on both nanoparticles and nanoaggregate-embedded beads.

Antibodies can be used for preventing in vivo the attachment of pathogenic bacteria to the target cells by competing with them for the host cell receptors. The main limitation of such a strategy is that it is difficult to provide constantly the necessary amount of antibody because of the titer dilution and molecule clearance. The environment colonization with microbial cell factories that ensure the production and secretion of the competitor antibodies in situ represents a possibility to overcome this drawback, but IgG are not suitable for this application, in the way that antibody fragments are. Recombinant antibodies in scFv format raised against the streptococcal cell surface antigenI/II and transformed into lactobacilli were therefore used to infect the oral cavity of model rats to inhibit Streptococcus mutans, the agent responsible for dental caries. The method succeeded in decreasing the development of dental caries and resulted more effective when antibody production was induced by a constitutive promoter. Nevertheless, VHHs were successively preferred to scFvs because of their superior structural robustness and demonstrated their effectiveness in controlling the damage. Orally administrated, transformed lactobacilli were also successfully used for delivering anti-TNF nanobodies to the colon at a concentration sufficient to significantly reduce chronic colitis.

The small dimensions of VHHs allow also their fusion to relatively large tags without negative effects in terms of recombinant yields. Therefore, their fusion to glucose oxidase for inducing the production of active forms of oxygen with microbicidal effect in the oral cavity has been proposed. This approach may offer a synergistic effect due to the contribution of the direct competition plus the antimicrobial enzymatic activity, but its effectiveness remains uncertain in vivo.

Mutagenesis is another tool that can be exploited to improve the in vivo reliability of antibacterial VHHs by selecting variants with superior features for a critical condition. A set of anti-E. coli F4 fimbriae antibodies was isolated and the most promising clone was further characterized. Even though very effective in vitro, it protected poorly the piglets from intestinal infection since it was rapidly degraded. Therefore, its proteolytical stability was increased by mutagenesis for obtaining a significantly more effective binder suitable for oral treatment.

The control of the pathogenic bacterial infection can be also addressed by reducing the bacterial resistance to antibiotics. Microorganisms can survive in the presence of inhibitors since they produce enzymes that metabolize the antibiotics. This is the case of the beta-lactamase, a secreted enzyme that hydrolyzes ampicillin-like antibiotics. VHHs with beta-lactamase inhibitory effect have been isolated demonstrating the feasibility of selecting single-domain antibodies that could silence this resistance strategy, although no biotechnological application has followed so far.

Unique features of the single-domain antibodies can be illustrated by a further example. The addition of recombinant VHH raised against a phage tail protein involved in the host recognition prevented the infection of Lactococcus lactis cultures by the p2 bacteriophage. Subsequently, the lactobacilli have been engineered to produce directly phage-neutralizing VHHs at a rate sufficient for protecting themselves from infections. This method represents an interesting approach for vaccinating microorganisms of biotechnological interest with the scope of protecting them from harmful aggressions of biotic and abiotic origin.

Single-domain antibodies against fungi and protozoans

The potentials of single-domain antibodies have been only scarcely applied to fungi and protozoans. Nevertheless, those few projects have significantly contributed to the understanding of some general antibody structural features. It is the case of the antibodies raised against a cell wall protein of Malassezia furfur, a fungus implicated in dandruff. Since the selected VHH antibodies should be potentially included in a shampoo formulation, they had to resist to the harsh chemical conditions brought about by elevated concentrations of anionic and nonionic surfactants. Therefore, the panning washing conditions were adapted to represent the high-detergent content of shampoos. This approach enabled the recovery of VHHs with specifically increased stability under denaturing conditions and to identify the key role of arginine at position 44 for improving this characteristic.

IgNARs, the single-chain antibodies of shark origin, have been used for identifying variable domains of the malarial Apical Membrane Antigen 1 of Plasmodium falciparum. In sharks, IgNARs represent the antibody isotope responsible for antigen-driven immune response and the generation of high affinity antibodies as the consequence of rapid somatic hyper-mutation. Therefore, the authors simulated the natural mutational process by means of error prone PCR using conditions that introduced a single mutation in the whole sequence of the binder previously recovered by conventional panning. This approach allowed for the identification of more affine variants that differed from the original NAR V single-domain by having a less rigid structure, a feature that determined a better induced fit for the antibody-antigen complex and explained a slower dissociation rate of the mutants. Co-crystallization results indicated that the antibody's CDR3 loops penetrated deeply into hydrophobic clefts on the antigen surface and allowed for the identification of conserved residues that could be targeted preferentially in order to develop polymorphism-independent binders.

Single-domain antibodies can apparently also recognize effectively epitopes of parasite glycoproteins both in diagnostic settings and in vivo. It is the case of a VHH that binds the Ts14 protein of Taenia solium with sub-nanomolar affinity and of other VHHs specific for oligomannose residues of the variant surface glycoprotein from Tripanosoma b. rhodiense. Pan-reactive VHHs against Tripanosoma spp. were successively isolated for developing a rapid flow-cytometric diagnostic system for quantitative assess of the pathogen concentration in blood samples.

Strategies for virus detection and neutralization

Also in the case of viruses, recombinant antibodies have been conceived as an inexpensive alternative to IgGs for masking the virion proteins involved in the host receptor binding and thus impairing the subsequent infection in vivo. Single-domain antibodies possess a large protruding loop corresponding to the CDR3 and this structural feature represents an advantage over conventional antibodies in reaching the typically cryptic viral epitopes responsible for host recognition, similarly to what has been observed in the case of VHH competitive inhibition of the enzymatic catalytic site. On the other hand, each virion usually contains tens of such epitope copies. Although it is true that conventional IgGs have no direct access to such hidden epitopes, their large dimension can increase virus neutralization by steric hindrance. To overcome the dimension limitation of VHHs and improve their neutralizing capacity in vitro and in vivo, their mass was increased by conjugation with immunoglobulins or inducing their N-glycosylation by providing suitable residues and successive expression in yeast. The approach of defining the residues for N-glycosylation by mutagenesis should avoid specificity and affinity loss for the antigen due to paratope modification and reduced epitope accessibility. Nevertheless, the effectiveness of N-glycosylation for in vivo applications is still to be demonstrated because the large mannosylated carbohydrates synthetized by yeast are actively bound by liver receptors leading to fast VHH clearance.

The options for rendering single-domain fragments effective in human therapy described above are crucial in view of the fact that an increasing number of binders specific for viruses have been isolated, characterized, and successfully used in vitro and in animal models. For example, an effective diagnostic assay based on llama single-domains for identifying independently four different variants of the Marburg virus was delivered in three working weeks. In this case, the availability of a semi-synthetic library enabled the reduction of the time necessary for the selection procedure, providing the evidence that reliable diagnostic immunoassays based on recombinant antibodies can be optimized during the initial phase of an infective outbreak.

VHHs selected using the group A rotavirus-conserved VP6 inner capsid protein are yet another example of efficacy of antibody fragments in neutralizing the virus and in protecting mice from diarrhea, where conventional antibodies had a modest effect. Using a strategy similar to that used for bacterial pathogens, VHHs raised against rotavirus were expressed on the surface of lactobacilli and orally administrated in a mouse model. Severity and duration of the disease were significantly shortened as a consequence of a decrease in the viral load. An increase in the variety and availability of expression cassettes for the secretion or surface display of VHH antibodies should boost this approach for the protection from virus and bacteria infections.

Furthermore, VHHs and NAR Vs resulted effective in detecting poliovirus and inhibiting its replication in vitro, in binding vaccinia virus, and in preventing the assembly and secretion of hepatitis B virions in cellular models by the expression of engineered NAR V and VHH fragments directed against the cellular endoplasmic reticulum.

The inhibition of viral secretion using conventional antibodies has not been attempted in mammals because of the technical complexity, but it is becoming feasible using VHHs expressed as intrabodies. They can inhibit the multimerization of the HIV-1 Rev protein by binding to the target protein, sequestering it in the cytoplasm, and impairing the HIV-1 replication. The cell uptake of the same virus is impaired by the small drug AMD3100 that acts as an antagonist for the CD4-activated co-entry receptor CXCR4. Conventional antibodies with the same function had previously been selected with the aim of isolating receptor antagonists with longer half-life than AMD3100, but they did not block sufficiently the virus-receptor recognition. As an alternative, anti-CXCR4 VHHs have been selected by competitive elution with the natural binder CXCL12 and successively proved to compete with AMD3100. Such VHHs behave as competitive antagonists and potently inhibit the virus uptake. The strategy aimed at inhibiting the HIV-1 infection using VHHs that recognize envelope proteins and neutralize the virions by competing with the main cellular receptor CD4 is conceptually similar. Finally, nanobodies have been also used to bind intracellularly the multifunctional Mf1 domain of the IRF-1 tumor suppressor with the idea of regulating this viral infection-stimulated regulative protein.

Single-domain antibodies were also successfully used for preparing piezoimmunosensors for the detection of HIV-1 virions. The possibility of displaying at high density and controlling the orientation of these binders on the sensor surfaces strongly increases the method sensitivity.

Finally, the engineering of single-domain antibodies into multivalent anti-viral molecules have been proved to increase the neutralizing action against and to reduce the infectivity of Synctyal, Rabies and influenza viruses. The simultaneous targeting of different epitopes of the same virus seems a promising alternative to increased avidity provided by multivalent display of the same antibody.

Toxin identification and detoxification

The use of single-domain antibodies for toxin identification and neutralization has increased exponentially in the last two years. Two are the main reasons for the success, namely the possibility to use pre-immune libraries and the opportunity to have a scaffold structurally very stable, but extremely suitable for engineering application-specific modification.

VHH stability is crucial when harsh processing conditions, such as elevated temperatures, high detergent and reducing agent concentrations, or denaturing conditions, are used. Specifically, toxic prolamins can be effectively extracted for quantification only in the presence of a solvent containing ethanol, 2-mercaptoethanol and guanidinium chloride. Conventional antibodies do not withstand these denaturing conditions and, therefore, are not available for immunological test. On the contrary, it was possible to select solvent-compatible VHHs with increased stability due to an introduction of extra disulfide bonds that constrained the CDR3 flexibility. N-glycosylation is another parameter that allows the antibody stability tuning and can improve the antibody effectiveness in vivo. Similarly to what has been observed in the case of anti-virus single-domain antibodies, N-glycosylation increased the neutralizing capacity of VHHs against the E. coli heat-labile toxin in a cell model. Such an effect was attributed to the larger mass of the antibody that blocks simultaneously more than one of the five receptor-binding sites present on each toxin molecule.

Parallel panning of one-pot libraries yielded single domain antibodies in both VHH and NAR V format specific for ricin, cholera toxin, and staphylococcal enterotoxin B. These studies demonstrated that pre-immune libraries can be used for rapid generation of antibodies against a large number of harmful antigens and that the single-domain antibody stability is beneficial for increasing their shelf life in diagnostic applications. Furthermore, the troublesome low sensitivity of single domain antibodies in immunological tests due to their monovalency could be overcome by using phage-displayed and not purified antibodies. This method allows the signal amplification by detecting several copies of phage-coating proteins in every single antibody-toxin binding event. Although conventional IgGs are used preferentially for capturing toxins in microflow cytometry for multiplexed detection and in the preparation of metal nanoparticle resistors for cholera toxin detection, single-domain antibodies seem suitable for similar applications.

A very interesting application in which single-domain cannot be substituted by conventional antibodies has been recently reported. Salmonella SpvB toxin is secreted directly from the bacteria into the host cell cytoplasm and, consequently, is not accessible for extracellular antibodies. However, VHHs raised against the toxin can be expressed and maintain their functionality and specificity as intrabodies. They block the toxin at a molar ratio of 1:1 and prevent its pathological consequences at cellular level.

VHHs outperformed conventional antibodies also when used for the preparation of bivalent and bispecific constructs used for the in vivo neutralization of the AahI'/AahII scorpion toxins. Multivalency induced by VHH polymerization was exploited also to increase the neutralizing capacity of antibodies against the E. coli verotoxin 1, alpha-cobrotoxin, and C. difficile toxin A, whereas the reduced production costs and the high density at which VHH can be coupled to resin substrates made feasible the preparation of hemofiltration columns for removing toxin shock syndrome toxin 1 from plasma to alleviate staphylococcal-induced sepsis. Also an unconventional mouse VH resulting from an incomplete scFv antibody has been isolated and characterized because of its anti-toxin potentialities. In particular, it appeared to be a functional anti-idiotypic antibody for the HM-1 killer toxin with a higher affinity than scFv antibodies recovered from the same library for the neutralizing monoclonal antibody.

Toxins such as ricin and botulinum neurotoxin are classified as bioweapons and have been the object of a particular attention. In a project aimed at isolating anti-ricin antibodies, the panning protocol was first optimized using the toxin immobilized on the surface of microspheres. The isolated VHHs were as effective as conventional monoclonal antibodies in blocking the ricin biological activity, but were superior in terms of specificity.

The majority of the publications on the toxin topic concerns the Clostridium botulinum neurotoxin (BoNT) because it causes frequently fatal disease and there is no drug able to reverse the symptoms once the toxin has entered the neuron. The interest in developing single-domain antibodies against BoNT is primarily motivated by the stability of these molecules even when they are expressed in the cytoplasm as intrabodies, facilitating not only diagnostic, but also therapeutic applications. Flow cytometry sorting of pre-immune, yeast displayed antibodies in VHH format allowed the isolation of anti-BoNT single-domains against the toxin light chain that inhibited the toxin protease activity in vitro and had structural features compatible for their production in the host cell cytoplasm. Single-domain antibodies that inhibit the toxin protease activity by occupying the enzymatic cleft were also recovered from a mixed VH/VHH library, whereas VHHs isolated from an immune library retained their toxin inhibitory properties also when expressed in mammalian neuronal cell cytosol.

Hapten targeting

Small molecules are not expected to be targeted efficiently by single-domain antibodies since they posses a limited number of conformational epitopes suitable for recognition by protruding single-domain paratopes. Nevertheless, there are several examples showing that VHHs can be exploited for detecting haptens as different as herbicides, caffeine, mycotoxins, trinitrotoluene, steroids and therapeutic drugs for this antibody class can interact with its targets adopting different binding patterns. For instance, the CDR1 loop provides a strong interaction for the azo-dye Reactive Red 6, CDR2 and a framework residue contribute to the binding of Reactive Red 1, whereas a non-conventional, substrate-dependent dimerization mechanism is involved in the binding of caffeine and its metabolites. The stability of single-domain antibodies expressed in the cytoplasm has been used to induce their cellular accumulation as an effective tool for neutralizing the mycotoxin 15-acetyldeoxynivaleol in vivo.

Reagents for immunodetection, purification and bioseparation

As an affinity-based technique, immunopurification presents some theoretical advantages over chromatographic methods based on chemical and physical properties. It can simplify complex multi-step procedures to a single step protocol, reducing production costs and time. Consequently, it can improve yields and limit potential product degradation. Nevertheless, immunopurification performed with conventional antibodies often requests extreme elution conditions that can damage the purified product. The advantages offered by single-domain antibody fragments in immunochromatography have been demonstrated for the first time by Verheesen et al.. Their monomeric nature facilitated the elution of the target protein, whereas their physical stability and effective refolding allowed the regeneration of the column under harsh cleaning conditions for more than 2000 times. Mild elution conditions are necessary to preserve product structure and activity. From this perspective, panning VHH libraries has resulted as being an optimal solution since binders can be selected not only based on their specificity, but also on their elution features. In this way, unstable proteins such as serum factor VIII and alpha-1 antitrypsin have been immunopurified successfully using VHH antibodies for their capture and elution at neutral pH. In a comparative assay with longer antibody constructs and complete IgGs, affinity columns prepared with VHHs enabled higher yields, probably because of the higher density at which they are bound to the matrix. Such VHH-based affinity columns have found their applications for both purifying specific components from heterogeneous material and for depleting very abundant proteins from samples in which it is necessary to detect the variation of scarcely represented polypeptides. It is the case of the platforms developed for the removal of IgG and human serum albumin from plasma before processing the samples in proteomic assays and of the nanotrap for the purification of GFP-fused proteins from cell homogenates. VHH-based affinity chromatography resulted also as very effective in removing contaminants such as DNA and virus particles. At the same time, VHHs specific for adeno-associated viruses allowed for the simplification of the purification protocol of these molecules from five to one or two steps and for doubling of the final yields. Beside the published reports and filed patent applications, a further indication of the single-domain antibody reliability as reagents for immunopurification can be inferred by the list of VHH-based affinity resins developed by biotech companies.

The small dimension (15 KDa, 4 × 2.5 × 3 nm) of single-domain antibodies and the possibility to produce them as recombinant proteins fused to suitable tags make them inexpensive binders that can be easily oriented at high density on capture surfaces such as chips and biosensors or adapted to a large variety of applications such as purification, imaging, immunomicelle and liposome preparation, or immobilization on nanoparticle resistors for the detection of cancer markers and cholera toxin. A promising utilization strategy considers the VHH labeling via the 6xHis tag with metastable (99m) technetium for application in single-photon emission computed tomography (SPECT), a noninvasive monitoring technique for observing the tumor response to therapy, but nanobodies have been effectively labeled also with (68)Ga for immuno-positron emission tomography (PET). In both cases, their elevated tissue penetration and rapid clearance allow for optimal high-contrast, specific localization in vivo in a time significantly shorter than that enabled by using conventional IgGs. Finally, the use of single-domain antibodies double-tagged with gadolinium and near-infrared dye Cy5.5 allowed the simultaneous optical and magnetic resonance imaging of brain tumor vessels.

Single-domain antibody fragments as crystallography chaperones and tools for studying protein aggregation and activity regulation

The possibility of using antibodies to stabilize the conformation of proteins that undergo crystallization trials has been recognized since long time. Nevertheless, conventional antibodies and their fragments have limitations due to their bulky mass (IgG and Fab fragment) or low stability (scFv) and, therefore, other molecules have been proposed, such as affibodies, fibronectin, and DARPins. Single-domain antibodies seem a logical alternative since they can be produced inexpensively in short time, have a strong binding capacity, reduced mass, demonstrated capacity of providing "induced fit" to the antigen-antibody complex, and they improve crystal packing and X-ray phasing. Although VHHs are amenable for simple site-specific incorporation of SeMet into their scaffolds, the fact that co-crystallizing with a single-domain antibody improves the complementary anomalous dispersion data acquisition and gives an ideal template for molecular replacement represents a substantial advantage for structure resolution. Furthermore, VHHs are very flexible in terms of interface shapes and, therefore, can adapt to a large variety of epitopes and block intrinsically flexible regions of their antigens. VHHs have also been added directly to the solubilized membrane fraction for binding to and stabilizing the nitric oxide reductase membrane target protein during the purification that resulted in the recovery of the antigen-antibody complex. No condition giving rise to diffracting crystals were found with the enzyme alone, but crystals were obtained in the presence of the VHHs. It has also been demonstrated that VHHs and their corresponding antigens can be co-expressed recombinantly in the same host and co-purified using tags fused to the antibody sequence, simplifying the protocol for producing antigen-antibody complexes.

The VHH-dependent approaches led to the resolution of several structures in the last few years. VHHs seem to favor the formation of crystals of several different forms and to accelerate in a dramatic way the crystallization of recalcitrant protein complexes. In the case of the KREPA6 editosome protein in combination with the Nb5 and Nb15 VHHs, all crystal contacts are obtained by the single-domain co-chaperones, while the role of VHHs is mainly to keep the flexible loop connecting the N1 and N2 domains of GspD in a fixed conformation and to form stabilizing layers between layers of EpsI:EpsJ heterodimers. Furthermore, although CDR3 is usually the major component involved in the antigen binding and crystal chaperoning, a feature deriving probably from its structural flexibility, in some cases CDR2 and even framework 3 can become pivotal chaperoning elements.

In the case of a study of the G protein coupled β2 adrenoreceptor, the VHH characteristics were useful at different levels. Whereas the crystallization of the inactive state of different members belonging to such receptor class has been achieved, the efforts to obtain a crystal of the receptor active state were frustrated due to receptor instability in the absence of G proteins. On the other hand, the receptor-G protein complex is unstable in the presence of the detergent necessary for the receptor stabilization. Therefore, suitable agonists were necessary for this aim. A high-affinity VHH agonist of the G protein was successfully identified by panning, its binding to the receptor was stable, maintained it in its active form during the crystallization trial functioning as a substrate agonist, and allowed the structure resolution operating as a crystallization chaperone.

VHHs also proved as being particularly useful for studying the intermediates of aggregation-prone polypeptides such as in the case of prion protein and of the amyloidogenic fragments. Single domain antibodies of different origin enabled the elucidation of the variety of morphologically distinct Aβ aggregates, blocking their polymerization development into non-toxic intermediates, and the clarification of the plausible mechanism of amyloidogenic protein self-association. Specifically, domain swapping appears as a plausible self-association mechanism for amyloidogenic β2-microglobulin. Protein dimers undergoing swapping unmask amyloidogenic sequences that fold into two-stranded antiparallel β-sheet. Self-association of the sheet β-strands can provide the elongation mechanism by which large intermolecular β-sheets are built. The first X-ray crystallographic structure of the complete Aβ18-41 fragment has been obtained by an innovative approach that may be of general interest for the study of tricky short peptides. The sequence corresponding to the fragment has been cloned inside the CDR3 of an IgNAR V and the chimeric protein expressed. In such a way, amyloidogenic oligomerization was allowed through the direct contact of the exposed Aβ fragments, but its uncontrolled development prevented by the formation of an IgNAR cage. Crystallographic data confirmed the formation of an Aβ fragment-mediated tight tetramer in which the fine molecular mechanisms of the interaction could be observed and described.

The structures inferred from crystallographic datasets were also useful for the understanding of the mechanism by which a nanobody constrained the dehydrofolate reductase enzyme in its occluded form by binding it in a region adjacent to the active site. Such allosteric control of the enzyme activity raised hyperbolic inhibition kinetics and induced a conformational rearrangement of the whole enzyme structure. Structural data were also necessary to explain the allosteric inhibition mechanism of the botulinum neurotoxin induced by another VHH. In this case, it was possible to recognize that the antibody epitope corresponded to the α-exosite of the neurotoxin. This information has practical potentials since it identifies a target for the development of inhibitory small-molecule drugs.

Sometimes, the VHH-dependent allosteric enzyme modulation becomes extremely complex as illustrated by the case of the interaction between single-domains and the nucleoside hydrolase from Trypanosoma vivax. The same antibody inhibited the catalytic pathway, but increased the product release rate and showed inhibitory or simulating behavior according to the substrate affinity for the enzyme. Another example of a complex modulation has been provided by the anti-human protein kinase C for which two sets of VHHs were isolated by panning immune libraries. All the five isolated VHHs were specific exclusively for a single isozyme (PKCε), and surprisingly, whereas three of them increased the kinase activity in a concentration-dependent manner, the remaining two inhibited the isozyme. Taken together, these data indicate that VHHs possess not solely of a large array of paratope surfaces but that, beside their known capacity to block the enzyme active sites, they have alternative binding mechanisms involving different structural regions that are suitable for tuning their antigen-binding activity.

Technical considerations-Conclusions

The number of reports dealing with single-domain antibody applications in different fields clearly indicates that this class of immunoreagents represents an important tool for research as well as biotechnological uses (Table 1). Furthermore, the described examples show that single-domain antibodies are complementary rather than alternative with respect to conventional antibodies in the sense that perform functions not possible to accomplish with IgGs.

However, there are several technical issues that still need to be defined in a systematic way, at the recombinant antibody panning level as well as at the level of binder production strategies. For instance, the praxis indicates that one-pot libraries can deliver highly affine and specific antibodies, although somatic maturation was considered essential to obtain reliable binders. Moreover, it has been emphasized that non-immune library dimensions and display system were crucial for obtaining valid recombinant antibodies, but E. coli, yeast and ribosome display, even though successful at simplifying the panning and screening steps by means of flow-cytometric clone separation, did not perform better than small phage display libraries and recently even minimal libraries have proved to be potent means of selecting lead antibodies. For a long time, an elevated affinity has been considered an absolute necessity, but now we know that it is important for in vitro application, whereas it can correlate inversely with appropriate tumor penetration in vivo.

Finally, several applications in chromatography, bioseparation, crystallography, or therapy need large amounts of recombinant antibodies and their fusion derivates, but their production technology has not improved significantly, although innovations concerning their expression in both bacterial periplasm and in eukaryotic systems have been introduced. However, a breakthrough seems to have arrived now with the advent of a technology enabling the cytolasmic accumulation of disulfide bond-dependent proteins. This approach aims at inducing the disulfide bond formation by co-expressing a sulfhydryl oxidase instead of disrupting the reducing pathways in the cytosol and resulted in yields of VHH-fusion constructs in the range of several tens of mg/L culture.

Given the excellent qualitative performance of single-domain antibodies and their advantages in terms of structural features, stability, and production costs, a question remains to be answered as to why it has taken almost twenty years for them to become largely popular in the scientific community. Probably two reasons are the main responsible for this slow technology acknowledgment. The pretty restrictive intellectual property policy introduced by the inventor institutions with the "Hamers patents" that first defined the field has been extremely successful in boosting the spin-off companies funded to exploit the discovery, but it has probably prevented other actors from participating in the development. The second reason concerns the fact that for a long time there was only a minimal availability of those application-friendly tools for research and diagnostics that are necessary for making new technologies appealing to non-specialist researchers. With the original patents expiring in the next few years and the recent accessibility of new libraries, protocols, and vectors suitable for different immunoapplications, we expect an increase of the interest of both research and industry for this class of recombinant antibodies in the near future.

Competing interests

The author declares that they have no competing interests.

References 201

  1. 1.

    Gebauer M Skerra A Engineered protein scaffolds as next-generation antibody therapeuticsCurr Opin Chem Biol2009 13245255

    Not in our library10.1016/j.cbpa.2009.04.627

  2. 2.

    Kontermann RE Alternative antibody formatsCurr Opin Mol Ther2010 12176183

    Not in our library

  3. 3.

    EditorialRecent patent applications in antibody fragmentsNat Biotechnol2009 2741

    Not in our library

  4. 4.

    Wesolowski J Alzogaray V Reyelt J Unger M Juarez K Urrutia M Cauerhff A Danquah W Rissiek B Scheuplein F Schwarz N Adriouch S Boyer O Seman M Licea A Serreze DV Goldbaum FA Haag F Koch-Nolte F Single domain antibodies: promising experimental and therapeutic tools in infection and immunityMol Microbiol Immunol2009 198157174

    Not in our library10.1007/s00430-009-0116-7

  5. 5.

    Schrama D Reisfeld RA Becker JC Antibody targeted drugs a cancer therapeuticsNat Rev2006 5147159

    Not in our library10.1038/nrd1957

  6. 6.

    Chames P Van Regenmortel M Weiss E Baty D Therapeutic antibodies: successes, limitations and hopes for the futureBrit J Pharmacol2009 157220233

    Not in our library10.1111/j.1476-5381.2009.00190.x

  7. 7.

    Nanduri V Bhunia AK Tu S-I Paoli GC Brewster JD SPR biosensor for the detection of L. monocytogenes using phage-displayed antibodyBiosens Bioelectron2007 23248252

    Not in our library10.1016/j.bios.2007.04.007

  8. 8.

    Abulrob A Sprong H Van Bergen en Henegouwen P Stanimirovic D The blood-brain barrier transmigrating single domain antibody: mechanism of transport and antigenic epitopes in human brain endothelial cellsJ Neurochem2005 9512011214

    Not in our library10.1111/j.1471-4159.2005.03463.x

  9. 9.

    Iqbal U Trojahn U Albaghdadi H Zhang J O'Connor-McCourt M Stanimirovic D Tomanek B Sutherland G Abulrob A Kinetic analysis of novel mono-and multivalent VHH-fragments and their application for molecular imaging of brain tumoursBr J Pharmacol2010 16010161028

    Not in our library10.1111/j.1476-5381.2010.00742.x

  10. 10.

    Jobling SA Jarman C The M-M Holmberg N Blake C Verhoeyen E Immunomodulation of enzyme function in plants by single-domain antibody fragmentsNat Biotechnol2003 217780

    Not in our library10.1038/nbt772

  11. 11.

    Klooster R Eman R le Duc Q Verheesen P Verrips T Roovers RC Post JA Selection and characterization of KDEL-specific VHH antibody fragments and their application in the study of ER resident protein expressionJ Immunol Meth2009 342112

    Not in our library10.1016/j.jim.2008.10.009

  12. 12.

    Schornack S Fuchs R Huitema E Rothbauer U Lipka V Kamoun S Protein mislocalization in plant cells using a GFP-binding chromobodyPlant J2009 60744754

    Not in our library10.1111/j.1365-313x.2009.03982.x

  13. 13.

    Kirchhofer A Helma J Schmidthals K Frauer C Cui S Karcher A Pellis M Muyldermans S Casas-Delucchi CS Cardoso MC Leonhardt H Hopfner KP Rothbauer U Modulation of protein properties in living cells using nanobodiesNat Struct Mol Biol2010 17133138

    Not in our library10.1038/nsmb.1727

  14. 14.

    Perrucchini C Pecorari F Bourgeois J-P Duyckaerts C Rougeon F Lafaye P Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodiesActa Neuropathol2009 118685695

    Not in our library10.1007/s00401-009-0572-6

  15. 15.

    Alvarez-Rueda N Ladjemi MZ Behar G Corgnac S Pugniere M Roquet F Bascoul-Mollevi C Baty D Pèlegrin A Navarro-Teulon I A llama single domain anti-idiotypic antibody mimicking HER2 as a vaccine: Immunogenicity and efficacyVaccine2009 2748264833

    Not in our library10.1016/j.vaccine.2009.05.067

  16. 16.

    Huang L Muyldermans S Saerens D Nanobodies®: proficient tools in diagnosticExpert Rev Mol Diagn2010 10777785

    Not in our library10.1586/erm.10.62

  17. 17.

    Barthelemy PA Raab H Appleton BA Bond CJ Wu P Wiesmann C Sidhu SS Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domainsJ Biol Chem2008 28336393554

    Not in our library10.1074/jbc.m708536200

  18. 18.

    Jespers L Schon O James LC Veprintsev D Winter G Crystal structure of HEL4, a soluble, refoldable human VH single domain with germ-line scaffoldJ Mol Biol2004 337893903

    Not in our library10.1016/j.jmb.2004.02.013

  19. 19.

    Bond CJ Marsters JC Sidhu SS A structure-based database of antibody variable domain diversityJ Mol Biol2003 332643655

    Not in our library10.1016/s0022-2836(03)00967-7

  20. 20.

    To R Hirama T Arbabi-Ghahroudi M MacKenzie R Wang P Xu P Ni F Tanha J Isolation of monomeric human VHs by a phage selectionJ Biol Chem2005 2804139541403

    Not in our library10.1074/jbc.m509900200

  21. 21.

    Chen W Zhu Z Feng Y Xiao X Dimitrov DS Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domainJ Mol Biol2008 382779789

    Not in our library10.1016/j.jmb.2008.07.054

  22. 22.

    Jespers L Schon O Famm K Winter G Aggregation-resistant domain antibodies selected on phage by heat denaturationNat Biotechnol2004 2211611165

    Not in our library10.1038/nbt1000

  23. 23.

    de Marco A A step ahead: combining protein purification and correct folding selectionMicrob Cell Fact2004 312

    Not in our library10.1186/1475-2859-3-12

  24. 24.

    Kortt AA Guthrie RE Hinds MG Power BE Ivancic N Caldwell JB Gruen LC Norton RS Hudson PJ Solution properties of Escherichia coli-expressed VH domain of anti-neuraminidase antibody NC41J Prot Chem1995 14167178

    Not in our library10.1007/bf01980329

  25. 25.

    Ward ES Güssow D Griffiths AD Jones PT Winter G Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coliNature1989 341544546

    Not in our library10.1038/341544a0

  26. 26.

    Reiter Y Schuck P Boyd LF Plaskin D An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interfaceJ Mol Biol1999 290685698

    Not in our library10.1006/jmbi.1999.2923

  27. 27.

    Cai X Garen A A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patientProc Natl Acad Sci USA1996 9362806285

    Not in our library10.1073/pnas.93.13.6280

  28. 28.

    Tanha J Dubue G Hirama T Narang SA MacKenzie CR Selection by phage display of llama conventional VH fragments with heavy chain antibody VHH propertiesJ Immunol Methods2002 26397109

    Not in our library10.1016/s0022-1759(02)00027-3

  29. 29.

    Monegal A Ami D Martinelli C Huang H Aliprandi M Francavilla C Ossolengo G de Marco A Immunological applications of single domain llama recombinant antibodies isolated from a naïve libraryProt Engineer Des Sel2009 22273280

    Not in our library10.1093/protein/gzp002

  30. 30.

    Zou X Osborn MJ Bolland DJ Smith JA Corcos D Hamon M Oxley D Hutchings A Morgan G Santos F Kilshaw PJ Taussig MJ Corcoran AE Brüggemann M Heavy chain-only antibodies are spontaneously produced in light chain-deficient miceJ Exp Med2007 20432713283

    Not in our library10.1084/jem.20071155

  31. 31.

    Deschacht N De Groeve K Vincke C Raes G De Baetselier P MuyldermansA novel promiscuous class of camelid single-domain antibody contributes to the antigen-binding repertoireJ Immunol2010 18456965704

    Not in our library10.4049/jimmunol.0903722

  32. 32.

    Tanaka T Lobato MN Rabbitts TH Single domain intracellular antibodies: A minimal fragment for direct in vivo selection of antigen-specific intrabodiesJ Mol Biol2003 33111091120

    Not in our library10.1016/s0022-2836(03)00836-2

  33. 33.

    Tanaka T Sewell H Waters S Phillips SE Rabbits TH Single domain intracellular antibodies from diverse libraries: emphasizing dual functions of LMO2 protein interactions using a single VH domainJ Biol Chem2011 28637073716

    Not in our library10.1074/jbc.m110.188193

  34. 34.

    Kastelic D Frkovič-Grazio S Baty D Truan G Komel R Pompon D A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markersJ Immunol Meth2009 3505462

    Not in our library10.1016/j.jim.2009.08.016

  35. 35.

    Thanongsaksrikul J Srimanote P Maneewatch S Choowongkomon K Tapchaisri P Makino S Kurazono H Chaicumpa W A VHH that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type AJ Biol Chem2010 28596579666

    Not in our library10.1074/jbc.m109.073163

  36. 36.

    Cossins AJ Harrison S Popplewell AG Gore MG Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein LProt Expr Purif2007 51253259

    Not in our library10.1016/j.pep.2006.07.013

  37. 37.

    Martsev SP Tsybovsky YI Stremovskiy OA Odintsov SG Balandin TG Arosio P Kravchuk ZI Deyev SM Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stabilityProtein Eng Des Sel2004 178593

    Not in our library10.1093/protein/gzh011

  38. 38.

    Ryan S Kell AJ van Faassen H Tay L-L Simard B MacKenzie R Gilbert M Tanha J Single-domain antibody-nanoparticles: Promising architectures for increased Staphylococcus aureus detection specificity and sensitivityBioconjugate Chem2009 2019661974

    Not in our library10.1021/bc900332r

  39. 39.

    Huang P-J Tay L-L Tanha J Ryan S Chau L-K Single-domain antibody-conjugated nanoaggregate-embedded beads for targeted detection of pathogenic bacteriaChem Eur J2009 1593309334

    Not in our library10.1002/chem.200901397

  40. 40.

    Harmsen MM van Solt CB Hoogendoom A van Zijederveld FG Niewold TA van der Meulen J Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoeaVet Microbiol2005 1118998

    Not in our library10.1016/j.vetmic.2005.09.005

  41. 41.

    Krüger C Hultberg A Marcotte H Hermans P Bezemer S Frenken LGJ Hammarström L Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental cariesAppl Microbiol Biotechnol2006 72732737

    Not in our library10.1007/s00253-006-0347-0

  42. 42.

    Krüger C Hu Y Pan Q Marcotte H Hultberg A Delwar D van Dalen PJ Pouwels PH Leer RJ Kelly CG van Dollenweerd C Ma JK Hammarström L In situ delivery of passive immunity by lactobacilli producing single-chain antibodiesNat Biotechnol2002 20702706

    Not in our library10.1038/nbt0702-702

  43. 43.

    Krüger C Hultberg A van Dollenweerd C Marcotte H Hammarström L Passive immunization by lactobacilli expressing single-chain antibodies against Streptococcus mutansMol Biotechnol2005 31221231

    Not in our library10.1385/mb:31:3:221

  44. 44.

    Vandenbroucke K de Haard H Beirnaert E Dreier T Lauwereys M Huyck L Van Huysse J Demetter P Steidler L Remaut E Cuvelier C Rottiers P Orally administrated L. lactis secreting an anti-TNF nanobody demonstrate efficacy in chronic colitisMucosal Immunol2010 34956

    Not in our library10.1038/mi.2009.116

  45. 45.

    Szynol A de Soet JJ Sieben-van Tuyl E Bos JW Frenken LG Bacterial effect of a fusion protein of llama heavy-chain antibodies coupled to glucose oxidase on oral bacteriaAntimicrob Agents Chemother2004 4833903395

    Not in our library10.1128/aac.48.9.3390-3395.2004

  46. 46.

    Harmsen MM van Solt CB van Zijederveld-van Bemmel AM Niewold TA van Zijederveld FG Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapyAppl Microbiol Biotechnol2006 72544551

    Not in our library10.1007/s00253-005-0300-7

  47. 47.

    Conrath KE Lauwereys M Galleni M Matagne A Frere JM Kinne J Wyns L Muyldermans S Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the CamelidaeAntimicrob Agents Chemoter2001 4528072812

    Not in our library10.1128/aac.45.10.2807-2812.2001

  48. 48.

    Ledeboer AM Bezemer S De Haard HJ Schaffers IM Verrips CT van Vliet Düsterhöft EM Zoon P Moineau S Frenken LG Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llamaJ Dairy Sci2002 8513761382

    Not in our library10.3168/jds.s0022-0302(02)74204-5

  49. 49.

    de Haard HJ Bezemer S Ledeboer AM Müller WH Boender PJ Moineau S Coppelmans MC Verkleij AJ Frenken LG Verrips CT Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infectionJ Bacteriol2005 18745314541

    Not in our library10.1128/jb.187.13.4531-4541.2005

  50. 50.

    Hultberg A Tremblay D de Haard HJ Verrips T Moineau S Hammarström L Marcotte H Lactobacilli expressing llama VHH fragments neutralize Lactococcus phagesBMC Biotechnol2007 758

    Not in our library10.1186/1472-6750-7-58

  51. 51.

    Dolk E van der Vaart M Hulsik DL Vriend G de Haard HJ Spinelli S Cambillau C Frenken L Verrips T Isolation of llama antibody fragments for prevention of dandruff by phage display in shampooAppl Environ Microbiol2005 71442450

    Not in our library10.1128/aem.71.1.442-450.2005

  52. 52.

    Nuttall SD Humbestone KS Krishnan UV Carmichael JA Doughty L Hattarki M Coley AM Casey JL Anders RF Foley M Irving RA Hudson PJ Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1Proteins2004 55187197

    Not in our library10.1002/prot.20005

  53. 53.

    Henderson KA Streltsov VA Coley AM Dolezal O Hudson PJ Batchelor AH Gupta A Bai T Murphy VJ Anders RF Foley M Nuttall SD Structure of an IgNAR-AMA1 complex: targetig a conserved hydrophobic cleft broadens malarial strain recognitionStructure2007 1514521466

    Not in our library10.1016/j.str.2007.09.011

  54. 54.

    Deckers N Saerens D Kanobana K Conrath K Victor B Wernery U Vercruysse J Muyldermans S Dorny P Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosisInt J Parasitol2009 39625633

    Not in our library10.1016/j.ijpara.2008.10.012

  55. 55.

    Stijlemans B Conrath K Cortez-Retamozo V Van Xong H Wyns L Senter P Revets H De Baetselier P Muyldermans S Magez S Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodiesJ Biol Chem2004 27912561261

    Not in our library10.1074/jbc.m307341200

  56. 56.

    Baral TN Magez S Stijlemans B Conrath K Vanhollebeke B Pays E Muyldermans S De Baetselier P Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factorNat Med2006 12580584

    Not in our library10.1038/nm1395

  57. 57.

    Saerens D Stijlemans B Baral TN Nguyen Thi GT Wernery U Magez S De Baetselier P Muyldermans S Conrath K Parallel selection of multiple anti-infectome nanobodies without access to purified antigensJ Immunol Meth2008 329138150

    Not in our library10.1016/j.jim.2007.10.005

  58. 58.

    Antoine-Moussiaux N Saerens D Desmecht D Flow cytometry enumeration of parasitemia and hematologic changes in trypanosome-infected miceActa Trop2008 107139144

    Not in our library10.1016/j.actatropica.2008.05.010

  59. 59.

    Parren PV Burton DR The antiviral activity of antibodies in vitro and in vivoAdv Immunol2001 77195262

    Not in our library10.1016/s0065-2776(01)77018-6

  60. 60.

    Lauwereys M Arbabi Ghahroudi M Desmyter A Kinne J Hölzer W De Genst E Wyns L Muyldermans S Potent enzyme inhibitors derived from dromedary heavy-chain antibodiesEMBO J1998 1735123520

    Not in our library10.1093/emboj/17.13.3512

  61. 61.

    Harmsen MM Van Solt CB Fijten HPD Van Setten MC Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulinsVaccine2005 2349264934

    Not in our library10.1016/j.vaccine.2005.05.017

  62. 62.

    Harmsen MM Van Solt CB Fijten HPD Van Keulen L Rosalia RA Weerdmeester K Cornelissen AH De Bruin MG Eblé PL Dekker A Passive immunization of guinea-pigs with llama single-domain antibody fragments against foot-and-mouth diseaseVet Microbiol2007 120193206

    Not in our library10.1016/j.vetmic.2006.10.029

  63. 63.

    Harmsen MM Van Solt CB Fijten HPD Enhancement of toxin-and virus-neutralizing capacity of single-domain antibody fragments by N-glcosylationAppl Microbiol Biotechnol2009 8410871094

    Not in our library10.1007/s00253-009-2029-1

  64. 64.

    Sherwood LJ Osborn LE Carrion R Patterson JL Hayhurst A Rapid assembly of sensitive antigen-capture assays for Marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level 4J Infect Dis2007 196S213S219

    Not in our library10.1086/520586

  65. 65.

    van der Vaart JM Pant N Wolvers D Bezemer S Hermans PW Bellamy K Sarker SA van der Logt CP Svensson L Verrips CT Hammarström L van Klinken BJ Reduction of morbidity of rotavirus induced diarrhoe in mice by yeast produced monovalent llama-derived antibody fragmentVaccine2006 2441304137

    Not in our library10.1016/j.vaccine.2006.02.045

  66. 66.

    Garaicoechea L Olichon A Marcoppido G Wigdorovitz A Mozgovoj M Saif L Surrey T Parreño V Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in miceJ Virol2008 8297539764

    Not in our library10.1128/jvi.00436-08

  67. 67.

    Pant N Hultberg A Zhao Y Svensson L Pan-Hammarström Q Johansen K Pouwels PH Ruggeri FM Hermans P Frenken L Boren T Marcotte H Hammarström L Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrheaJ Infect Dis2006 19415801588

    Not in our library10.1086/508747

  68. 68.

    Martin MC Pant N Ladero V Günaydin G Andersen KK Alvarez B Martínez N Alvarez MA Hammarström L Marcotte H Integrative expression system for delivery of antibody fragments by lactobacilliAppl Environ Microbiol2011 7721742179

    Not in our library10.1128/aem.02690-10

  69. 69.

    Thys B Schotte L Muyldermans S Wernery U Hassanzadeh-Ghassabeh G Rombaut B In vitro antiviral activity of single domain antibody fragments against poliovirusAntiviral Res2010 87257264

    Not in our library10.1016/j.antiviral.2010.05.012

  70. 70.

    Thys B Saerens D Schotte L De Bleeser G Muyldermans S Hassanzadeh-Ghassabeh G Rombaut B A simple quantitative affinity capturing assay of poliovirus antigens and subviral particles by single-domain antibodies using magnetic beadsJ Virol Meth2011 173300305

    Not in our library10.1016/j.jviromet.2011.02.023

  71. 71.

    Goldman ER Anderson GP Liu JL Delehanty JB Sherwood LJ Osborn LE Cummins LB Hayhurst A Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama libraryAnal Chem2006 7882458255

    Not in our library10.1021/ac0610053

  72. 72.

    Serruys B Van Houtte F Verbrugghe P Leroux-Roels G Vanlandschoot P Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in miceHepathol2009 493949

    Not in our library10.1002/hep.22609

  73. 73.

    Serruys B Van Houtte F Farhoudi-Moghadam A Leroux-Roels G Vanlandschoot P Production, characterization and in vitro testing of HBcAg-specific VHH intrabodiesJ Gen Virol2010 91643652

    Not in our library10.1099/vir.0.016063-0

  74. 74.

    Walsh R Nuttall S Revill P Colledge D Cabuang L Soppe S Dolezal O Griffiths K Bartholomeusz A Locarnini S Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabodyVirol2011 411132141

    Not in our library10.1016/j.virol.2010.12.034

  75. 75.

    Vercruysse T Pardon E Vanstreels E Steyaert J Daelemans D An intrabody based on a llama single-domain antibody targeting the N-terminal α-helical multimerization domain of HIV-1 Rev prevents viral productionJ Biol Chem2010 2852176821780

    Not in our library10.1074/jbc.m110.112490

  76. 76.

    Jähnichen S Blanchetot C Maussang D Gonzalez-Pajuelo M Chow KY Bosch L De Vrieze S Serruys B Ulrichts H Vandevelde W Saunders M De Haard HJ Schols D Leurs R Vanlandschoot P Verrips T Smit MJ CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cellsProc Natl Acad Sci USA2010 1072056520570

    Not in our library10.1073/pnas.1012865107

  77. 77.

    Forsman A Beirnaert E Aasa-Chapman MMI Hoorelbeke B Hijazi K Koh W Tack V Szynol A Kelly C McKnight A Verrips T de Haard H Weiss RA Llama antibody fragments with cross-subtype human immuodeficency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120J Virol2008 821206912081

    Not in our library10.1128/jvi.01379-08

  78. 78.

    Koh WW Steffensen S Gonzalez-Pajuelo M Hoorelbeke B Gorlani A Szynol A Forsman A Aasa-Chapman MM de Haard H Verrips T Weiss RA Generation of a family-specific phage library of llama single chain antibody fragments that neutralize HIV-1J Biol Chem2010 2851911619124

    Not in our library10.1074/jbc.m110.116699

  79. 79.

    Hinz A Hulsik DL Forsman A Koh WW Belrhali H Gorlani A de Haard H Weiss RA Verrips T Weissenhorn W Crystal structure of the neutralizing llama VHH D7 and its mode of HIV-1 gp120 interactionPLoSOne2010 5e10482

    Not in our library10.1371/journal.pone.0010482

  80. 80.

    Möller A Pion E Narayan V Ball KL Intracellular activation of interferon regulatory factor-1 by nanobodies to the multifunctional (Mf1) domainJ Biol Chem2010 2853834838361

    Not in our library10.1074/jbc.m110.149476

  81. 81.

    Ferreira GN Encarnacao JM Rosa L Rodrigues R Beyner R Barrento S Pedro L Aires da Silva F Gonçalves J Recombinant single-chain variable fragment and single domain antibody piezoimmunosensors for detection of HIV-1 virion infectivity factorBiosens Bioelectron2007 23384392

    Not in our library10.1016/j.bios.2007.04.022

  82. 82.

    Ibañez LI De Filette M Hultberg A Verrips T Temperton N Weiss RA Vandevelde W Schepens B Vanlandschoot P Saelens X Nanobodies with in vitro neutralizing activity protect mice against H5N1 influenza virus infectionJ Infect Dis2011 20310631072

    Not in our library10.1093/infdis/jiq168

  83. 83.
    Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules · 2011
    No notice
  84. 84.

    Olichon A Schweizer D Muyldermans S de Marco A Heating represents a rapid purification method for recovering correctly folded thermo tolerant VH and VHH domainsBMC Biotechnol2007 77

    Not in our library10.1186/1472-6750-7-7

  85. 85.

    Dong J Thompson AA Fan Y Lou J Conrad F Ho M Pires-Alves M Wilson BA Stevens RC Marks JD A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic α-exosite binding regionJ Mol Biol2010 39711061118

    Not in our library10.1016/j.jmb.2010.01.070

  86. 86.

    Doňa V Urrutia M Bayardo M Alzogaray V Goldbaum FA Chirdo FG Single domain antibodies are specially suited for quantitative determination of gliadins under denaturing conditionsJ Agric Food Chem2010 58918926

    Not in our library10.1021/jf902973c

  87. 87.

    Liu JL Anderson GP Goldman ER Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display libraryBMC Biotechnol2007 778

    Not in our library10.1186/1472-6750-7-78

  88. 88.

    Liu JL Anderson GP Delehanty JB Baumann R Hayhurst A Goldman ER Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark libraryMol Immunol2007 441771783

    Not in our library10.1016/j.molimm.2006.07.299

  89. 89.

    Goldman ER Anderson GP Bernstein RD Swain MD Amplification of immunoassays using phage-displayed single domain antibodiesJ Immunol Meth2010 352182185

    Not in our library10.1016/j.jim.2009.10.014

  90. 90.

    Kim JS Anderson GP Erickson JS Golden JP Nasir M Ligler FS Multiplexed detection of bacteria and toxins using a microflow cytometerAnal Chem2009 8154265432

    Not in our library10.1021/ac9005827

  91. 91.

    Loyprasert S Hedström M Thavarungkul P Kanatharana P Mattiasson B Sub-attomolar detection of cholera toxin using a label-free capacitive immunosensorBiosens Bioelectron2010 2519771983

    Not in our library10.1016/j.bios.2010.01.020

  92. 92.

    Slocik JM Kim SN Auvil T Goldman ER Liu J Naik RR Single domain antibody templated nanoparticle resistors for sensingBiosens Bioelectron2010 2519081913

    Not in our library10.1016/j.bios.2010.01.004

  93. 93.

    Alzogaray V Danquah W Aguirre A Urrutia M Berguer P Garcia Vescovi E Haag F Koch-Nolte F Goldbaum FA Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimuriumFASEB J2011 25526234

    Not in our library10.1096/fj.10-162958

  94. 94.

    Hmila I Ben-Abderrazek Ben-Abdallah R Saerens D Benlasfar Z Conrath K Ayeb ME Muyldermans S Bouhaouala-Zahar B VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI'Mol Immunol2008 4538473856

    Not in our library10.1016/j.molimm.2008.04.011

  95. 95.

    Ben-Abderrazek Ben-Abdallah R Hmila I Vincke C Benlasfar Z Pellis M Dabbek H Saerens D El Ayeb M Muyldermans S Bouhaouala-Zahar B Identification of potent nanobodies to neutralize the most poisonous polypeptide from scorpion venomBiochem J2009 424263272

    Not in our library10.1042/bj20090697

  96. 96.

    Hmila I Saerens D Ben-Abderrazek Ben-Abdallah R Vincke C Abidi N Benlasfar Z Govaert J El Ayeb M Bouhaouala-Zahar B Muyldermans S A bispecific nanobody to provide full protection against lethal scorpion envenomingFASEB J2010 2434793489

    Not in our library10.1096/fj.09-148213

  97. 97.

    Conrath EK Lauwereys M Wyns L Muyldermans S Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructsJ Biol Chem2001 27673467350

    Not in our library10.1074/jbc.m007734200

  98. 98.

    Zhang J Tanha J Hirama T Khieu NH To R Tong-Sevinc H Stone E Brisson JR MacKenzie CR Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibodyJ Mol Biol2004 3354956

    Not in our library10.1016/j.jmb.2003.09.034

  99. 99.

    Stone E Hirama T Tanha J Tong-Sevinc H Li S MacKenzie CR Zhang J The assembly of single domain antibodies into bispecific decavalent moleculesJ Immunol Meth2007 3188894

    Not in our library10.1016/j.jim.2006.10.006

  100. 100.

    Stone E Hirama T Chen W Soltyk AL Brunton J MacKenzie CR Zhang J A novel pentamer versus pentamer approach to generating neutralizers of verotoxin 1Mol Immunol2007 4424872491

    Not in our library10.1016/j.molimm.2006.10.020

  101. 101.

    Stewart CS MacKenzie CR Hall JC Isolation, characterization and pentamerization of alpha-cobrotoxin specific single-domain antibodies from a naïve phage display library: preliminary findings for antivenom developmentToxicon49699709

    Not in our library10.1016/j.toxicon.2006.11.023

  102. 102.

    Hussack G Arbabi-Ghahroudi M van Faassen H Songer JG Ng KK Mackenzie R Tanha J Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell-receptor binding domainJ Biol Chem2011 28689618976

    Not in our library10.1074/jbc.m110.198754

  103. 103.

    Adams H Brummelhuis W Maassen B van Egmond N El Khattabi M Detmers F Specific immuno capturing of the staphylococcal superantigen toxic-shock syndrome toxin-1 in plasmaBiotechnol Bioeng2009 104143151

    Not in our library10.1002/bit.22365

  104. 104.

    Brummelhuis W Joles JA Stam JC Adams H Goldschmeding R Detmers FJ El Khattabi M Maassen BT Verrips CT Braam B Llama heavy-chain antibody fragments efficiently remove toxic shock syndrome toxin 1 from plasma in vitro but not in experimental porcine septic shockShock2010 34125132

    Not in our library10.1097/shk.0b013e3181cdc556

  105. 105.

    Kabir ME Krishnaswamy S Miyamoto M Furuichi Y Komiyama T An altered camelid-like single domain anti-idiotypc antibody fragment of HM-1 killer toxin: acts as an effective antifungal agentAppl Microbiol Biotechnol2011 90553564

    Not in our library10.1007/s00253-011-3123-8

  106. 106.

    Anderson GP Matney R Liu JL Hayhurst A Goldman ER Multiplexed fluid array screening of phage displayed anti-ricin single domain antibodies for rapid assessment of specificityBiotechniques2007 43806811

    Not in our library10.2144/000112600

  107. 107.

    Anderson GP Liu JL Hale ML Bernstein RD Moore M Swain MD Goldman ER Development of antiricin single domain antibodies toward detection and therapeutic reagentsAnal Chem2008 8096049611

    Not in our library10.1021/ac8019398

  108. 108.

    Anderson GP Bernstein RD Swain MD Zabetakis D Goldman ER Binding kinetics of antiricin single domain antibodies and improved detection using a B chain specific binderAnal Chem2010 8272027207

    Not in our library10.1021/ac100961x

  109. 109.

    Goldman ER Anderson GP Conway J Sherwood LJ Fech M Vo BH Liu JL Hayhurst A Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complexAnal Chem2008 8085838591

    Not in our library10.1021/ac8014774

  110. 110.

    Conway JO Sherwood LJ Collazo MT Garza JA Hayhurst A Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagentsPLoSOne2010 5e8818

    Not in our library10.1371/journal.pone.0008818

  111. 111.

    Swain MD Anderson GP Zabetakis D Bernstein RD Liu JL Sherwood LJ Hayhurst A Goldman ER Llama-derived single-domain antibodies for the detection of botulinum A neurotoxinAnal Bioanal Chem2010 398339348

    Not in our library10.1007/s00216-010-3905-3

  112. 112.

    Tremblay JM Kuo C-L Abeijon C Sepulveda J Oyler G Hu X Jin MM Shoemaker CB Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteasesToxicon2010 56990998

    Not in our library10.1016/j.toxicon.2010.07.003

  113. 113.

    Yau KYF Groves MAT Li S Sheedy C Lee H Tanha J MacKenzie CR Jermutus L Hall JC Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display libraryJ Immunol Meth2003 281161175

    Not in our library10.1016/j.jim.2003.07.011

  114. 114.

    Sheedy C Yau KY Hirama T MacKenzie CR Hall JC Selection, characterization, and CDR shuffling of naïve llama single-domain antibodies selected against auxin and their cross reactivity with auxinic herbicides from four chemical familiesJ Agric Food Chem2006 5436683678

    Not in our library10.1021/jf060219i

  115. 115.

    Ladenson RC Crimmins DL Landt Y Ladenson JH Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragmentAnal Chem2006 7845014508

    Not in our library10.1021/ac058044j

  116. 116.

    Alvarez-Rueda N Behar G Ferré V Pugniere M Roquet F Gastinel L Jacquot C Aubry J Baty D Barbet J Birklé S Generation of llama single-domain antibodies against methotrexate, a prototypcal haptenMol Immunol2007 4416801690

    Not in our library10.1016/j.molimm.2006.08.007

  117. 117.

    Doyle PJ Arbabi-Ghahroudi M Gaudette N Furzer G Savard ME Gleddie S Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenolMol Immunol2008 4537033713

    Not in our library10.1016/j.molimm.2008.06.005

  118. 118.

    Anderson GP Goldman ER TNT detection using llama antibodies and a two-step competitive fluid array immunoassayJ Immunol Meth2008 3394754

    Not in our library10.1016/j.jim.2008.08.001

  119. 119.

    Doyle PJ Saeed H Hermans A Gleddie SC Hussack G Arbabi-Ghahroudi M Seguin C Savard ME Mackenzie CR Hall JC Intracellular expression of a single domain antibody reduces cytotoxicity of 15-acetyldeoxynivalenol in yeastJ Biol Chem2009 2843502935039

    Not in our library10.1074/jbc.m109.045047

  120. 120.

    Kobayashi N Oyama H Nakao M Kanda T Banzono E Kato Y Karibe T Nishio T Goto J "Cleavable" hapten-biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragmentsAnal Biochem2009 387257266

    Not in our library10.1016/j.ab.2009.01.004

  121. 121.

    Koide A Tereshko V Uysal S Margalef K Kossiakoff AA Koide S Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratopeJ Mol Biol2007 373941953

    Not in our library10.1016/j.jmb.2007.08.027

  122. 122.

    Spinelli S Frenken LGJ Hermans P Verrips T Brown K Tegoni M Cambillau C Camelid heavy-chain variable domains provide efficient combining sites to haptensBiochemistry2000 3912171222

    Not in our library10.1021/bi991830w

  123. 123.

    Spinelli S Tegoni M Frenken L van Vliet C Cambillau C Lateral recognition of a dye hapten by a llama VHH domainJ Mol Biol2001 311123129

    Not in our library10.1006/jmbi.2001.4856

  124. 124.

    Sonneson GJ Horn JR Hapten-induced dimerization of a single domain VHH camelid antibodyBiochemistry2009 4866936695

    Not in our library10.1021/bi900862r

  125. 125.

    Verheesen P ten Haaft MR Lindner N Verrips CT de Haard JJW Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatographyBiochim Biophys Acta2003 16242128

    Not in our library10.1016/j.bbagen.2003.09.006

  126. 126.

    McCue JT Selvitelli K Walker J Application of a novel affinity adsorbent for the capture and purification of Factor VIII compoundJ Chromatog A2009 126178247830

    Not in our library10.1016/j.chroma.2009.09.045

  127. 127.

    Detmers F Hermans P. Jiao J-A McCue JT Novel affinity ligands provide for highly selective primary captureBioProcess Internatl2010 85054

    Not in our library

  128. 128.

    Aliprandi M Sparacio E Pivetta F Ossolengo G Maestro R de Marco A The availability of a recombinant anti-SNAP antibody in VHH format amplifies the application flexibility of SNAP-tagged proteinsJ Biomed Biotechnol2010 ID 658954

    Not in our library10.1155/2010/658954

  129. 129.

    Franco EJ Sonneson GJ DeLegge TJ Hofstetter H Horn JR Hofstetter O Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatographyJ Chromatogr B2010 878177186

    Not in our library10.1016/j.jchromb.2009.06.017

  130. 130.

    Beyer T Serum-free production and purification of chimeric IgA antibodiesJ Immunol Meth2009 3462627

    Not in our library10.1016/j.jim.2009.05.002

  131. 131.

    Kuroiwa Y Kasinathan P Sathiyaseelan T Jiao JA Matsushita H Sathiyaseelan J Wu H Mellquist J Hammitt M Koster J Kamoda S Tachibana K Ishida I Robl JM Antigen-specific human polyclonal antibodies from hyperimmunized cattleNature Biotechnol2009 27173181

    Not in our library10.1038/nbt.1521

  132. 132.

    Klooster R Maassen BTH Stam JC Hermans PW ten Haaft MR Detmers FJM de Haard HJ Post JA Verrips TC Improved anti-IgG and HAS affinity ligands: Clinical application of VHH antibodies technologyJ Immunol Meth2007 324112

    Not in our library10.1016/j.jim.2007.04.005

  133. 133.

    Rothbauer U Zolghadr K Muyldermans S Schepers A Cardoso MC Leonhardt H A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteinsMol Cel Proteomics2008 7282289

    Not in our library10.1074/mcp.m700342-mcp200

  134. 134.

    Kubala MH Kovtun O Alexandrov K Collins BM Structural and thermodynamic analysis of the GFP:GFP-nanobody complexProtein Sci2010 192382401

    Not in our library10.1002/pro.519

  135. 135.

    Low D O'Leary R Pujar NS Future of antibody purificationJ Chromatog B2007 8484863

    Not in our library10.1016/j.jchromb.2006.10.033

  136. 136.

    Hellström M Ruitenberg MJ Pollett MA Ehlert EM Twisk J Verhaagen J Harvey AR Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injectionGene Ther2008 16521532

    Not in our library10.1038/gt.2008.178

  137. 137.

    Smith RH Levy JR Kotin RM A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cellsMol Ther2009 1718881896

    Not in our library10.1038/mt.2009.128

  138. 138.

    Capture Selecthttp://www.captureselect.com

    Not in our library

  139. 139.

    Allele Biotechnologyhttp://www.allelebiotech.com

    Not in our library

  140. 140.

    Bossi S Ferranti B Martinelli C Capasso P de Marco A Antibody-mediated purification of co-expressed antigen-antibody complexesProt Expr Purif2010 725558

    Not in our library10.1016/j.pep.2010.01.003

  141. 141.

    Saerens D Huang L Bonroy K Muyldermans S Antibody fragments as probe in biosensor developmentSensors2008 846694686

    Not in our library10.3390/s8084669

  142. 142.

    Reulen SWA van Baal I Raats JMH Merckx M Efficient, chemioselective synthesis of immunomicelles using single-domain antibodies with C-terminal thioesterBMC Biotechnol2009 966

    Not in our library10.1186/1472-6750-9-66

  143. 143.

    Oliveira S Schiffelers RM van der Veeken J van der Meel R Vongpromek R van Bergen en Henegouwen PM Storm G Roovers RC Downregulation of EGFR by a novel multivalent nanobody-liposome platformJ Control Release2010 145165175

    Not in our library10.1016/j.jconrel.2010.03.020

  144. 144.

    Even-Desrumeaux K Baty D Chames P Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generationMol BioSyst2010 622412248

    Not in our library10.1039/c005279e

  145. 145.

    Huang L Reekmans G Saerens D Friedt JM Frederix F Francis L Muyldermans S Campitelli A Van Hoof C Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assaysBiosens Bioelectron2005 21483490

    Not in our library10.1016/j.bios.2004.11.016

  146. 146.

    Saerens D Frederix F Reekmans G Conrath K Jans K Brys L Huang L Bosmans E Maes G Borghs G Muyldermans S Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensingAnal Chem2005 7775477555

    Not in our library10.1021/ac051092j

  147. 147.

    Huang L Gainkam LO Caveliers V Vanhove C Keyaerts M De Baetseliers P Bossuyt A Revets H Lahoutte T SPECT imaging with 99mTc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expressionMol Imaging Biol2008 10165175

    Not in our library10.1007/s11307-008-0133-8

  148. 148.

    Gainkam LO Huang L Caveliers V Keyaerts M Hernot S Vaneycken I Vanhove C Revets H De Baetselier P Lahoutte T Comparison of the biodistribution and tumor targeting of two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole SPECT/micro-CTJ Nucl Med2008 49788795

    Not in our library10.2967/jnumed.107.048538

  149. 149.

    De Groeve K Deschacht N De Koninck C Caveliers V Lahoutte T Devoogdt N Muyldermans S De Baetselier P Raes G Nanobodies as tools for in vivo imaging of specific immune cell typesJ Nucl Med2010 51782789

    Not in our library10.2967/jnumed.109.070078

  150. 150.

    Tchouate Gainkam LO Keyaerts M Caveliers V Devoogdt N Vanhove C Van Grunsven L Muyldermans S Lahoutte T Correlation between epidermal growth factor receptor-specific nanobody uptake and tumor burden: A tool for noninvasive monitoring of tumor response to therapyMol Imaging Biol2010 PMID: 20865332

    Not in our library10.1007/s11307-010-0428-4

  151. 151.

    Tchouate Gainkam LO Caveliers V Devoogdt N Vanhove C Xavier C Boerman O Muyldermans S Bossuyt A Lahoutte T Localization, mechanism and reduction of renal retention of technetium-99m labeled epidermal growth factor receptor-specific nanobody in miceContrast Media Mol Imaging2010 68592

    Not in our library10.1002/cmmi.408

  152. 152.

    Vaneycken I Devoogdt N Van Gassen N Vincke C Xavier C Wernery U Muyldermans S Lahoutte T Caveliers V Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancerFASEB J2011 PMID: 21478264

    Not in our library10.1096/fj.10-180331

  153. 153.

    Vosjan MJ Perk LR Roovers RC Visser GW Stigter-van Walsum M van Bergen En Henegouwen PM van Dongen GA Facile labeling of an anti-epidermal growth factor receptor nanobody with (68)Ga via a novel bifunctional desferal chelate for immuno-PETEur J Nucl Med Mol Imaging2011 38753763

    Not in our library10.1007/s00259-010-1700-1

  154. 154.

    Yokota T Milenic DE Whitiow M Schlom J Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin formsCancer Res1992 5234023408

    Not in our library

  155. 155.

    Tijink BM Laeremans T Budde M Stigter-van Walsum M Dreier T de Haard HJ Leemans CR van Dongen GA Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular nanobody technologyMol Cancer Ther2008 722882297

    Not in our library10.1158/1535-7163.mct-07-2384

  156. 156.

    Iqbal U Albaghdadi H Nieh MP Tuor UI Mester Z Stanimirovic D Katsaras J Abulrob A Small unilamellar vesicles: a platform technology for molecular imaging of brain tumorsNanotechnology2011 22195102

    Not in our library10.1088/0957-4484/22/19/195102

  157. 157.

    Amit AG Mariuzza RA Phillips SE Poljak RJ Three-dimensional structure of an antigen-antibody complex at 2.8 Å resolutionScience1986 233747753

    Not in our library10.1126/science.2426778

  158. 158.

    Hunte C Michel H Crystallization of membrane proteins mediated by antibody fragmentsCurr Opin Struct Biol2002 12503508

    Not in our library10.1016/s0959-440x(02)00354-8

  159. 159.

    Ermolenko DN Zherdev AV Dzantiev BB Antibodies as specific chaperonesBiochemistry (Moscow)2004 6912331238

    Not in our library10.1007/s10541-005-0069-4

  160. 160.

    Hogbom M Eklund M Nygren PA Nordlund P Structural basis for recognition by an in vitro evolved affibodyProc Natl Acad Sci USA2003 10031913196

    Not in our library10.1073/pnas.0436100100

  161. 161.

    Lipovšek D Lippow SM Hackel BJ Gregson MW Cheng P Kapila A Wittrup KD Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III doman and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodiesJ Mol Biol2007 36810241041

    Not in our library10.1016/j.jmb.2007.02.029

  162. 162.

    Sennhauser G Grütter MG Chaperone-assisted crystallography with DARPinsStructure2008 1614431453

    Not in our library10.1016/j.str.2008.08.010

  163. 163.

    Tereshko V Uysal S Koide A Margalef K Koide S Kossiakoff AA Toward chaperone-assisted crystallography: Protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffoldProt Sci2008 1711751187

    Not in our library10.1110/ps.034892.108

  164. 164.

    Dolk E van Vliet C Perez JM Vriend G Darbon H Ferrat G Cambillau C Frenken LG Verrips T Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigenProteins2005 59555564

    Not in our library10.1002/prot.20378

  165. 165.

    Stanfield RL Dooley H Verdino P Flajnik MF Wilson IA Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit bindingJ Mol Biol2007 367358372

    Not in our library10.1016/j.jmb.2006.12.045

  166. 166.

    Loris R Marianovsky I Lah J Laeremans T Engelberg-Kulka H Glaser G Muyldermans S Wyns L Crystal structure of the intrinsically flexible addition antidote MazEJ Biol Chem2003 2782825228257

    Not in our library10.1074/jbc.m302336200

  167. 167.

    Conrath K Pereira AS Martins CE Timoteo CG Tavares P Spinelli S Kinne J Flaudrops C Cambillau C Muyldermans S Moura I Moura JJ Tegoni M Desmyter A Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductaseProt Sci2009 18619628

    Not in our library10.1002/pro.69

  168. 168.

    Korotkov KV Pardon E Steyaert J Hol WG Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobodyStructure2009 17255265

    Not in our library10.1016/j.str.2008.11.011

  169. 169.

    Lam AY Pardon E Korotkov KV Hol WG Steyaert J Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificusJ Struct Biol2009 166815

    Not in our library10.1016/j.jsb.2008.11.008

  170. 170.

    Wu M Park Y-J Pardon E Turley S Hayhurst A Deng J Steyaert J Hol WG Structures of a key interaction protein from the Trypanosoma brucei editosome in complex with single domain antibodiesJ Struct Biol2010 174124136

    Not in our library10.1016/j.jsb.2010.10.007

  171. 171.

    Rasmussen SGF Choi H-J Fung JJ Pardon E Casarosa P Chae PS Devree BT Rosenbaum DM Thian FS Kobilka TS Schnapp A Konetzki I Sunahara RK Gellman SH Pautsch A Steyaert J Weis WI Kobilka BK Structure of a nanobody-stabilized active state of the β2 adrenoceptorNature2011 469175180

    Not in our library10.1038/nature09648

  172. 172.

    Koide S Engineering of recombinant crystallization chaperonesCurr Opin Struct Biol2009 19449457

    Not in our library10.1016/j.sbi.2009.04.008

  173. 173.

    Abskharon RN Soror SH Pardon E El Hassan H Legname G Steyaert J Wohlkonig A Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion proteinActa Crystallogr Sect F Struct Biol Cryst Commun2010 6616441646

    Not in our library10.1107/s1744309110042168

  174. 174.

    Dumoulin M Last AM Desmyter A Decanniere K Canet D Larsson G Spencer A Archer DB Sasse J Muyldermans S Wyns L Redfield C Matagne A Robinson CV Dobson CM A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozymeNature2003 424783788

    Not in our library10.1038/nature01870

  175. 175.

    Habicht G Haupt C Friedrich RP Hortschansky P Sachse C Meinhardt J Wieligmann K Gellermann GP Brodhun M Götz J Halbhuber KJ Röcken C Horn U Fändrich M Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Abeta protofibrilsProc Natl Acad Sci USA2007 1041923219237

    Not in our library10.1073/pnas.0703793104

  176. 176.

    Lafaye P Achour I England P Duyckaerts C Rougeon F Single-domain antibodies recognize selectively small oligomeric forms of amyloid beta, prevent Abeta-induced neurotoxicity and inhibit fibril formationMol Immunol2009 46695704

    Not in our library10.1016/j.molimm.2008.09.008

  177. 177.

    Kasturirangan S Li L Emadi S Boddapati S Schulz P Sierks MR Nanobody specific for oligomeric beta-amyloyd stabilizes nontoxic formNeurobiol Aging2010 PMID: 21067847

    Not in our library10.1016/j.neurobiolaging.2010.09.020

  178. 178.

    Kasturirangan S Boddapati S Sierks MR Engineered proteolytic nanobodies reduce Abeta burden and ameliorate Abeta-induced cytotxicityBiochemistry2010 4945014508

    Not in our library10.1021/bi902030m

  179. 179.

    Domanska K Vanderhaegen S Srinivasan V Pardon E Dupeux F Marquez JA Giorgetti S Stoppini M Wyns L Bellotti V Steyaert J Atomic structure of a nanobody-trapped domain-swapped dimmer of an amyloidogenic {beta}2-microglobulin variantProc Natl Acad Sci USA2011 10813141319

    Not in our library10.1073/pnas.1008560108

  180. 180.

    Streltsov VA Varghese JN Masters CL Nuttal SD Crystal structure of the amylod-{beta} p3 fragment provides a model for oligomeric formation in Alzheimer's diseaseJ Neurosci2011 3114191426

    Not in our library10.1523/jneurosci.4259-10.2011

  181. 181.

    Oyen D Srinivasan V Steyaert J Barlow JN Constraining enzyme conformational change by an antibody leads to hyperbolic inhibitionJ Mol Biol2011 407138148

    Not in our library10.1016/j.jmb.2011.01.017

  182. 182.

    Barlow JN Conrath K Steyaert J Substrate-dependent modulation of enzyme activity by allosteric effector antibodiesBiochim Biophys Acta2009 179412591268

    Not in our library10.1016/j.bbapap.2009.03.019

  183. 183.

    Paalanen MM Ekokoski E El Khattabi M Tuominen RK Verrips CT Boonstra J Blanchetot C The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilonEur J Pharm Sci2011 42332339

    Not in our library10.1016/j.ejps.2010.12.012

  184. 184.

    De Genst E Silence K Decanniere K Conrath K Loris R Kinne J Muyldermans S Wyns L Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodiesProc Natl Acad Sci USA2006 10345864591

    Not in our library10.1073/pnas.0505379103

  185. 185.

    Verheesen P Roussis A de Haard HJ Groot EH Stam JC den Dunnen JT Frants RR Verkleij AJ Theo Verrips C van der Maarel SM Reliable and controllable antibody fragment selections from camelid non-immune libraries for target validationBiochim Biophys Acta2006 176413071319

    Not in our library10.1016/j.bbapap.2006.05.011

  186. 186.

    Simmons DP Streltsov VA Dolezal O Hudson PJ Coley AM Foley M Proll DF Nuttall SD Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structureProteins2008 71119130

    Not in our library10.1002/prot.21663

  187. 187.

    Veggiani G Ossolengo G Aliprandi M Cavallaro U de Marco A Single-domain antibodies that compete with the natural ligand Fibroblast Growth Factor block the internalization of the Fibroblast Growth Factor Receptor1Biochem Biophys Res Comm2011 408692696

    Not in our library10.1016/j.bbrc.2011.04.090

  188. 188.

    Daugherty PS Olsen MJ Iverson BL Georgiou G Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surfaceProt Engineer1999 12613621

    Not in our library10.1093/protein/12.7.613

  189. 189.

    Feldhaus MJ Siegel RW Opresko LK Coleman JR Weaver Feldhaus JM Yeung YA Flow-cytometry isolation of human antibodies from a nonimmune Saccharomices cerevisiae surface display libraryNat Biotechnol2003 21163170

    Not in our library10.1038/nbt785

  190. 190.
    Applications of cell sorting in biotechnology · 2006
    Not checked
  191. 191.

    Zahnd C Amstutz P Plückthun A Ribosome display: selecting and evolving proteins in vitro that specifically bind to a targetNat Methods2007 4269279

    Not in our library10.1038/nmeth1003

  192. 192.

    Mao H Graziano JJ Chase TMA Bentley CA Bazirgan OA Reddy NP Song BD Smider VV Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimizationNat Biotechnol2010 2811952202

    Not in our library10.1038/nbt.1694

  193. 193.

    Thurber GM Schmidt MM Wittrup KD Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearanceAdv Drug Deliv Rev2008 6014211434

    Not in our library10.1016/j.addr.2008.04.012

  194. 194.

    Rudnick SI Adams GP Affinity and avidity in antibody-based tumor targetingCancer Biother Radiopharm2009 24155160

    Not in our library10.1089/cbr.2009.0627

  195. 195.

    Paal M Heel T Schneider R Auer B A novel Ecotin-Ubiquitin-Tag (ECUT) for efficient, soluble peptide production in the periplasm of Escherichia coliMicrob Cell Fact2009 87

    Not in our library10.1186/1475-2859-8-7

  196. 196.
    Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli · 2009
    No notice
  197. 197.

    Joosten V Lokman C van den Hondel CAMJJ Punt PJ The production of antibody fragments and antibody fusion proteins by yeast and filamentous fungiMicrob Cell Fact2003 21

    Not in our library10.1186/1475-2859-2-1

  198. 198.

    Hatahet F Nguyen VD Salo KEH Ruddock LW Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coliMicrob Cell Fact2010 967

    Not in our library10.1186/1475-2859-9-67

  199. 199.
    Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli · 2011
    No notice
  200. 200.

    Veggiani G de Marco A Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidaseProt Expr Purif2011 PMID: 201. 21421053

    Not in our library10.1016/j.pep.2011.03.005

  201. 201.

    Bradbury ARM Sidhu S Dübel S McCafferty J Beyond natural antibodies: the power of in vitro display technologiesNat Biotechnol2011 29245254

    Not in our library10.1038/nbt.1791

Cited by papers we hold 2

Reads

Read by people on this page

None yet.

Checked or read by AI through naulon

None yet.

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.

Are you an author?

Is this your paper?

Claim it to add your note and be paid when agents cite it. Free.