Selection and identification of single domain antibody fragments from camel heavy‐chain antibodies

Ghahroudi, Mehdi Arbabi; Desmyter, Aline; Wyns, Lode; Hamers, R.; Muyldermans, Serge

doi:10.1016/s0014-5793(97)01062-4

Cited by 688 publications

(555 citation statements)

References 32 publications

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“…The sdAb library was constructed from RNA extracted from the peripheral blood lymphocytes using a two stage PCR as outlined by Gharhroudi et al [20]. Selection of target binding sdAbs was conducted using a standard biopanning protocol; two rounds of selection proved sufficient to enrich for target-binding sdAbs in all cases save BxpB, as judged by polyclonal phage ELISA, Figure 2.…”

Section: Selection Of Sdabs Targeting Spore Proteinsmentioning

confidence: 99%

“…Single domain antibodies (sdAbs) are recombinant antibodies derived from a novel class of immunoglobulins found in camelids referred to as heavy-chain only antibodies [7,8]. As the name implies, the parental antibody and its antigen binding domain is formed from only the heavy chain; a product of several mutations that eliminate interaction with the light chain subunit [9].…”

Section: Introductionmentioning

confidence: 99%

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Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

et al. 2013

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Abstract:To obtain thermostable immunoreagents specific for the spore form of Bacillus anthracis two llamas were immunized with a combination of six different recombinant proteins. These proteins BclA, gerQ, SODA1, SOD15, BxpB and the protein p5303 have all been shown as components of the B. anthracis spore and could potentially serve as targets for the detection of spores in multiplexed biosensors. Peripheral blood lymphocytes were used to construct a phage display library from which single domain antibodies (sdAbs) targeting each of the proteins were isolated. Unique sdAbs exhibiting nanomolar or better affinities for the recombinant proteins were obtained and most of the isolated sdAbs retained their ability to bind antigen after cycles of heating as determined by enzyme linked immunosorbent assay (ELISA). SdAbs targeting the BclA and gerQ proteins were able to successfully detect bacterial spores, whether broken or intact, using a direct ELISA; the sdAbs were specific, showing binding only to B. anthracis spores and not to other Bacillus species. Additionally, SODA1 and p5303 binding sdAbs detected spores in sandwich assays serving as both captures and tracers. Used in combination, sdAbs targeting B. anthracis proteins could be integrated into emerging biosensors to improve specificity in multiplex assays.

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Section: Selection Of Sdabs Targeting Spore Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

et al. 2013

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“…In recent years, antibody sources have been explored that give rise to soluble recombinant antibodies with a higher frequency. Among those are heavy-chain IgG antibodies from camelids, such as camels, dromedaries and llamas (Ghahroudi et al 1997). Remarkably, camelid heavy-chain IgGs do not involve a light-chain, and lack the first constant domain of the heavy chain, which is replaced by an exceptionally long hinge region (Hamers-Casterman et al 1993).…”

Section: Introductionmentioning

confidence: 99%

“…The variable part of these antibodies (referred to as the VHH domain) is a single immunoglobulin-folded protein domain of about 140 amino acids, which does not involve artificial linkers. Generally, VHH domains can be produced in large quantities in microbial recombinant systems as stable, water-soluble molecules (Ghahroudi et al 1997;Muyldermans 2001).…”

Section: Introductionmentioning

confidence: 99%

Stable recombinant alpaca antibodies for detection of Tulip virus X

Beekwilder¹,

Houwelingen²,

Beckhoven

et al. 2008

Eur J Plant Pathol

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For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at −20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°C.

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“…They can also tolerate incubation at 37 C for a long period (i.e. more than one week) in PBS [67] or even in plasma (i.e. no decrease in activity is observed after an incubation for 44 h in mouse plasma, [68]).…”

Section: Nanobodies Are Highly Stable Entitiesmentioning

confidence: 99%

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

2015

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Keywords:Variable domain of heavy-chain antibody Inhibition of protein misfolding and aggregation Protein misfolding diseases Amyloidoses V H H Nanobody a b s t r a c tThe deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer's disease, Parkinson's disease, prion diseases and type II diabetes. These disorders, referred to as amyloidoses, generally become apparent late in life. Their psycho-sociological and economic incidence in western societies will be therefore considerable in the coming decades due to the ageing of the population. Neither preventing nor curative treatments are available yet. These disorders constitute therefore a medical challenge of great importance. Thus, an extensive research is being carried out to understand, at the molecular level, (i) how amyloidogenic proteins misfold and convert from their soluble form into amyloid fibrils, and (ii) how these aggregates or some of their oligomeric precursor species are toxic. The formation of amyloid fibrils proceeds through a complex nucleation/polymerisation mechanism with the formation of various species, including small oligomers. In this review, we focus on how V H Hs or nanobodies, the antigen-binding domains of camelid heavy-chain antibodies, are being increasingly used to characterise each of the species formed on the pathway of fibril formation in terms of structure, stability, kinetics of formation and toxicity. We first introduce the characteristic features of nanobodies compared to those of conventional antibody fragments. Thereafter, we discuss how nanobodies, due to their unique properties, are used as probes to dissect the molecular mechanisms of misfolding and aggregation of six proteins associated with diseases, i.e. human lysozyme, b2-microglobulin, a-synuclein, prion, polyadenylate binding protein nuclear 1 and amyloid b-peptide. A brief general presentation of each disease and the associated peptide/protein is also provided. In addition, we discuss how nanobodies could be used as early diagnostic tools and as novel strategies to treat diseases associated with protein misfolding and aggregation.© 2015 Elsevier B.V. and Societe Française de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.Abbreviations: Ab, antibody; Ab, amyloid b-peptide; AD, Alzheimer's disease; ANS, anilino naphthalene-sulfonic acid; AP, alkaline phosphatase; APP, amyloid precursor protein; aSyn, a-synuclein; b2m, b2-microglobulin; BBB, bloodebrain barrier; CDR, complementarity determining region; C m , concentration of mid-denaturation; CR, Congo red; CSF, cerebrospinal fluid; DN6b2m, a truncated form of b2m lacking the six N-terminal amino acids; DLS, dynamic light scattering; DRA, dialysis-related amyloidosis; FR, framework; FTIR, Fourier transform infrared spectroscopy; Fv, variable fragment made of the VH and VL domains of conventional antibodies; GFP, green fluorescent protein; HCAb, heavy-chain antibody; H/D, hydrogen/deuterium; HSQC, heteronuclear s...

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Selection and identification of single domain antibody fragments from camel heavy‐chain antibodies

Cited by 688 publications

References 32 publications

Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

Stable recombinant alpaca antibodies for detection of Tulip virus X

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

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