Antibody fragments as probe in biosensor development

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed

Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains

BACKGROUND: Recombinant antibodies from Camelidae (VHHs) are potentially useful tools for both basic research and biotechnological applications because of their small size, robustness, easy handling and possibility to refold after chemio-physical denaturation. Their heat tolerance is a particularly interesting feature because it has been recently related to both high yields during recombinant expression and selective purification of folded protein. RESULTS: Purification of recombinant RE3 VHH by heat treatment yielded the same amount of antibody as purification by affinity chromatography and negligible differences were found in stability, secondary structure and functionality. Similar results were obtained using another class of thermotolerant proteins, the single domain VH scaffold, described by Jespers et al. [8]. However, thermosensitive VHs could not withstand the heat treatment and co-precipitated with the bacterial proteins. In both cases, the thermotolerant proteins unfolded during the treatment but promptly refolded when moved back to a compatible temperature. CONCLUSION: Heat treatment can simplify the purification protocol of thermotolerant proteins as well as remove any soluble aggregate. Since the re-folding capability after heat-induced denaturation was previously correlated to higher performance during recombinant expression, a unique heating step can be envisaged to screen constructs that can provide high yields of correctly-folded proteins

Structural basis for the high specificity of a Trypanosoma congolense immunoassay targeting glycosomal aldolase

Background : Animal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay's high specificity. Methodology/Principal findings : The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay. Conclusions/Significance : The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays

Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies : African trypanosomes as paradign

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immunotoxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody:: beta-lactamase fusion activating a prodrug

Development of a nanobody-based amperometric immunocapturing assay for sensitive and specific detection of Toxocara canis excretory-secretory antigen

Introduction Human Toxocariasis (HT) is a zoonosis that, despite of its wide distribution around the world, remains poorly diagnosed. The identification of specific IgG immunoglobulins against the Toxocara canis Excretory-Secretory antigen (TES), a mix of glycoproteins that the parasite releases during its migration to the target organs in infected patients, is currently the only laboratory tool to detect the disease. The main drawbacks of this test are the inability to distinguish past and active infections together with lack of specificity. These factors seriously hamper the diagnosis, follow-up and control of the disease. Aim To develop an amperometric immunocapturing diagnostic assay based on single domain immunoglobulins from camelids (nanobodies) for specific and sensitive detection of TES. Methods After immunization of an alpaca (Vicugna pacos) with TES, RNA from peripheral blood lymphocytes was used as template for cDNA amplification with oligo dT primers and library construction. Isolation and screening of TES-specific nanobodies were carried out by biopanning and the resulting nanobodies were expressed in Escherichia coli. Two-epitopes amperometric immunocapturing assay was designed using paramagnetic beads coated with streptavidin and bivalent nanobodies. Detection of the system was carried out with nanobodies chemically coupled to horseradish peroxidase. The reaction was measured by amperometry and the limit of detection (LOD) was compared to conventional sandwich ELISA. Results We obtained three nanobodies that specifically recognize TES with no-cross reactivity to antigens of Ascaris lumbricoides and A. suum. The LOD of the assay using PBST20 0.05% as diluent was 100 pg/ml, 10 times more sensitive than sandwich ELISA. Conclusion Sensitive and specific detection of TES for discrimination of active and past infections is one of the most difficult challenges of T. canis diagnosis. The main advantage of our system is the use of two different nanobodies that specifically recognize two different epitopes in TES with a highly sensitive and straightforward readout. Considering that the amounts of TES available for detection in clinical samples are in the range of picograms or a few nanograms maximum, the LOD found in our experiments suggests that the test is potentially useful for the detection of clinically relevant cases of HT

Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.Instituto de BiotecnologíaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; ChinaFil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; BélgicaFil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Single domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge

Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NAspecific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity.Fil: Cardoso, Francisco Miguel. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Ibañez, Lorena Itatí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Van Den Hoecke, Silvie. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: De Baets, Sarah. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Smet, Anouk. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Roose, Kenny. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Schepens, Bert. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Descamps, Francis J.. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Fiers, Walter. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Muyldermans, Serge. Structural Biology Research Center; Bélgica. Vrije Universiteit Brussel. Laboratory of Cellular and Molecular Immunology; BélgicaFil: Depicker, Ann. VIB. Department of Plant Systems Biology; Bélgica. Department of Biotechnology and Bioinformatics; BélgicaFil: Saelens, Xavier. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; Bélgic