In-Cell Assembly of scFv from Human Thyroid- Infiltrating B Cells

Chapal, Nicolas; Bouanani, Majida; Embleton, M. J.; Navarro-Teulon, Isabelle; Biard-Piechaczyk, Martine; Pau, Bernard; Péraldi-Roux, Sylvie

doi:10.2144/97233rr03

Cited by 12 publications

(11 citation statements)

References 17 publications

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“…Although we previously verified that the C and V primers are able to amplify different genes (24), no -chain type was seen in any of our anti-TPO scFvs. Additional controls have since been done and demonstrate the low efficiency of the C Ext For primer used in the oligonucleotide pool for the RT-PCR and PCR1 (data not shown).…”

Section: Genes Encoding Tpo-specific In-cell Scfvsmentioning

confidence: 67%

“…The isolation and purification of the CD19 ϩ B cells as well as the in-cell PCR procedure have been described in detail (24). The three samples were amplified and cloned separately and then pooled for panning on TPO.…”

Section: Construction Of the In-cell Combinatorial Librarymentioning

confidence: 99%

“…One million human thyroid-infiltrating B CD19 ϩ lymphocytes were subjected to in-cell PCR in association with the Cre-loxP recombination system to obtain in vivo pairing of V H and V L genes (24). The scFv genes (V H -linker-V L ) were ligated into the pHEN1 vector and used to transform E. coli XL1b cells resulting in 10 5 clones.…”

Section: Selection Of Anti-tpo Scfv From In-cell Librarymentioning

confidence: 99%

“…To address this issue, we previously applied an in-cell PCR protocol, originally described by Embleton et al (23), to human thyroid-infiltrating CD19 ϩ B cells to obtain in vivo V H /V L gene pairing information (24). In the present study, we report the isolation and characterization of recombinant anti-TPO scFvs generated from this in-cell combinatorial library.…”

mentioning

confidence: 99%

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Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation

Chapal¹,

Péraldi-Roux²,

Bresson³

et al. 2000

The Journal of Immunology

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In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves’ disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vλ1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves’ disease patients tested were able to strongly inhibit (60–100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.

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Section: Genes Encoding Tpo-specific In-cell Scfvsmentioning

confidence: 67%

Section: Construction Of the In-cell Combinatorial Librarymentioning

confidence: 99%

Section: Selection Of Anti-tpo Scfv From In-cell Librarymentioning

confidence: 99%

mentioning

confidence: 99%

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Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation

Chapal¹,

Péraldi-Roux²,

Bresson³

et al. 2000

The Journal of Immunology

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“…Toutefois, les techniques classiques de banque combinatoire, où les associations des chaînes VH et VL des immunoglobulines se font de façon aléatoire, pouvaient ne pas refléter le répertoire pathologique du patient [57]. Ainsi, la technique de PCR intracellulaire, décrite par Embleton et al [17], a été appliquée aux lymphocytes B humains infiltrant la thyroïde et a conduit à l'obtention d'anticorps recombinants mimant les aAc pré-sents dans les sérums de patients [9]. Cette technique permet l'amplification et la recombinaison entre les segments des gènes VH et VL à l'intérieur même des lymphocytes B conduisant ainsi à des associations VH/VL représentatives de la situation in vivo.…”

Section: Sélection D'anticorps Recombinants Monoclonaux Humains Anti-unclassified

Les autoanticorps antithyroperoxydase : de la construction de banques combinatoires de fragments d'anticorps à la localisation de la région immunodominante de la thyroperoxydase

Rebuffat

Bresson

Péraldi-Roux

2005

Immuno-analyse & Biologie Spécialisée

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Pairing of T‐cell receptor chains via emulsion PCR

et al. 2013

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Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains -both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.Keywords: Antibodies r Drug design/discovery r Molecular biology r NGS r TCR Additional supporting information may be found in the online version of this article at the publisher's web-site

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In-Cell Assembly of scFv from Human Thyroid- Infiltrating B Cells

Abstract: The construction of a large library of single-chain Fv (scFv)

Cited by 12 publications

References 17 publications

Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation

Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation

Les autoanticorps antithyroperoxydase : de la construction de banques combinatoires de fragments d'anticorps à la localisation de la région immunodominante de la thyroperoxydase

Pairing of T‐cell receptor chains via emulsion PCR

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