Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

Djender, Selma; Schneider, Aurélie; Beugnet, Anne; Crépin, Ronan; Desrumeaux, Klervi; Romani, Chiara; Moutel, Sandrine; Perez, Franck; Marco, Ario de

doi:10.1186/preaccept-1444588381355050

Cited by 17 publications

(30 citation statements)

References 37 publications

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“…Nanobodies directed against HER2 were selected from a naïve llama phage‐display library applying the protocol described previously for another pre‐immune library . The phages were directly incubated with HER2‐negative cells (MCF10A) to deplete the VHHs specific to common epitopes, and the unbound fraction was successively challenged with SKBR3 cells (HER2‐positive).…”

Section: Introductionmentioning

confidence: 64%

“…Nanobodies are antibody fragments which correspond to the variable region of the heavy‐chain‐only antibodies (VHH) of Camelidae origin. They know a constantly growing interest because of their small mass (14 kDa), high stability and simplicity in being mutated and functionalized . In this study, we show that an anti‐human epidermal growth factor receptor 2 (HER2) nanobody isolated from a pre‐immune library recognized also the canine protein homologue DER2.…”

Section: Introductionmentioning

confidence: 99%

“…) indicating a strong interaction between nanobody and antigen. EM1 was successively subcloned into vectors which enabled its expression as green fluorescent protein (GFP)‐EM1 and Fc‐EM1 fusion proteins with the idea of producing application‐friendly reagents to use in flow cytometry and immunofluorescence . The reconstitution of an IgG‐like antibody by the fusion of the single‐domain variable region (VHH) with an Fc domain has several advantages.…”

Section: Introductionmentioning

confidence: 99%

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Engineered cross‐reacting nanobodies simplify comparative oncology between humans and dogs

Mazzega

Marco

2017

Vet Comparative Oncology

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Antibodies cross-reacting with homologue antigens in different species would be essential reagents for the development of comparative oncology studies. In comparison with conventional immunoglobulin Gs, recombinant nanobodies (single-domain variable regions of heavy-chain only antibodies of Camelidae origin) can be easily isolated in vitro and engineered into a variety of reagents with optimized characteristics for different research and clinical applications. We recovered an anti-human epidermal growth factor receptor 2 (anti-HER2) nanobody from a naïve llama library by direct panning on whole cells and expressed it fused to Fc and green fluorescent protein. These immunoreagents were assessed by flow cytometry and immunofluorescence with both human and canine cells overexpressing HER2 and its canine homologue dog epidermal growth factor receptor 2. The reported data illustrate the potential of using this class of antibodies in comparative oncology and suggest some development perspectives enabled by in vitro panning of pre-immune nanobody libraries.

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Section: Introductionmentioning

confidence: 64%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Engineered cross‐reacting nanobodies simplify comparative oncology between humans and dogs

Mazzega

Marco

2017

Vet Comparative Oncology

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“…The PCR products were recloned into PET-28a vector using EcoRI/SalI restriction sites to contain a His-tag and transferred into BL21 (DE3) competent cells. Transformed cells were selected in the presence of kanamycin (50 mg/mL), made competent again, and further transformed with the constructs cloned in the pFH255 vector [26,27]. Bacteria were grown in LB/K/chloramphenicol up to an OD 600 of 0.6 and induced with a certain concentration of IPTG and arabinose for 16ß20 H at 20 • C. After centrifugation for 20 Min at 15,000g, the soluble VHHs were purified from the supernatant by His-tag protein purification column and identified by 10% SDS-PAGE.…”

Section: Selection Of Hif-1α-specific Vhhmentioning

confidence: 99%

Selection by phage display of nanobodies directed against hypoxia inducible factor‐1α (HIF‐1α)

Fan

Liu

et al. 2015

Biotech and App Biochem

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Hypoxia, which promotes tumor invasion and metastasis, is a common phenomenon in solid tumors. Hypoxia generally leads to a higher expression level of hypoxia inducible factor-1 (HIF-1) in tumors (cells) relative to normal tissues (cells). Given the unique expression of HIF-1α in human cancers and its vital importance in mediating hypoxic adaptation, we have identified 20 different HIF-1α-specific nanobodies by using a llama-derived nonimmune phage display library. PAS-B domain of HIF-1α (HIF-1α-PAS-B) has been used as an antigen. Nanobody (VHH16) was selected from these 20 nanobodies by phage enzyme-linked immunosorbent assay. The preliminary analysis of biological activity demonstrates that VHH16 can specifically bind to HIF-1α with high affinity. VHH16 is the first nanobody that specifically binds to HIF-1α-PAS-B as well. We suggest here that VHH16 is useful in disease diagnosis and also has potential in medical applications.

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“…Their modular structure allows increasing their avidity by multimerization 6 , they can be fused to tracer enzymes to produce recombinant conjugates 7,8 , be biotinylated in vivo 9,10 , and some approaches have been developed for their en masse screening 11 . The metabolic biotinylation of nanobodies is particularly attractive for immunoassay or biosensor development because of the oriented immobilization that can be attained on avidin/streptavidin coated surfaces 12 , and this method of immobilization has been adapted for the selection of immunoassay-ready nanobody pairs against biothreats 13 .…”

Section: Introductionmentioning

confidence: 99%

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Rossotti¹,

Pírez²,

González-Techera³

et al. 2015

Anal. Chem.

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Single domain heavy-chain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

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Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

Cited by 17 publications

References 37 publications

Engineered cross‐reacting nanobodies simplify comparative oncology between humans and dogs

Engineered cross‐reacting nanobodies simplify comparative oncology between humans and dogs

Selection by phage display of nanobodies directed against hypoxia inducible factor‐1α (HIF‐1α)

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

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