Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama

Linden, Reinier van der; Geus, B. de; Stok, W.; Bos, Wil; Wassenaar, Dick van; Verrips, Theo; Frenken, Leon

doi:10.1016/s0022-1759(00)00188-5

Cited by 124 publications

(70 citation statements)

References 21 publications

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“…A reason for this may be that the staining reagents raised against ruminant Ig fail if there is broad epitope diversity (50). Despite this setback, camelids readily produce Ag-specific Abs in H2 and H2L2 configuration, and there is no indication that mixed molecules are expressed (16,51). Unfortunately, there is no information about pre-B cell development in camelids or whether an H chain without C H 1 can associate with a surrogate L chain to form the pre-BCR necessary to progress B cell development.…”

Section: Discussionmentioning

confidence: 99%

Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse

Zou

Smith

Nguyen

et al. 2005

The Journal of Immunology

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Section: Discussionmentioning

confidence: 99%

Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse

Zou

Smith

Nguyen

et al. 2005

The Journal of Immunology

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“…9,15 The VHHs were produced in Saccharomyces cerevisiae via batch fermentation. 12 VHHs were purified using a 5-mL ProteinA column (Hi-Trap, Pharmacia …”

Section: Fragments Used In This Studymentioning

confidence: 99%

“…12 This chemical antigen, the azo-dye RR6, has the advantage that it is not altered by the extreme conditions used in these experiments. CD spectroscopy was used to determine if the VHH-R2 fragment was completely unfolded at high temperatures and whether addition of the antigen at high temperatures could induce complex formation of the VHH-R2 fragment and its antigen.…”

Section: Introductionmentioning

confidence: 99%

Induced refolding of a temperature denatured llama heavy‐chain antibody fragment by its antigen

et al. 2005

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In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80°C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80°C. Interestingly, addition of the dye to the denatured VHH-R2 at 80°C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibodyantigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications. Proteins 2005;59:555-564.

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“…2, central part). They are synthetised by Camelidae (including camels, llamas and dromedaries) in addition to conventional antibodies and their proportion depends on the species: HCAbs represent 50% of the IgG in dromedaries and camels and 25e45% in llamas [32,34,35]. The antigenbinding domain of HCAbs is naturally reduced to a single domain of 130 amino acids (~14 kDa) and it therefore corresponds to the smallest available natural antigen-binding fragment; it is referred to as nanobody, V H H or single-domain antibody (sdAb).…”

Section: Unique Properties Of Nanobodiesmentioning

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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Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama

Cited by 124 publications

References 21 publications

Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse

Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse

Induced refolding of a temperature denatured llama heavy‐chain antibody fragment by its antigen

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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