Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries

Lou, Jianlong; Marzari, Roberto; Verzillo, Vittorio; Ferrero, Federica; Pak, Daniel T.S.; Sheng, Morgan; Yang, Chunrong; Sblattero, Daniele; Bradbury, Andrew

doi:10.1016/s0022-1759(01)00385-4

Cited by 63 publications

(36 citation statements)

References 40 publications

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“…As different selection strategies of the same antigen typically results in different antibody fragments with distinct applicative potential, 28 we modified the selection strategy for the second dysferlin fragment, DYSF2, by introducing one round of selection on PVDF-blotted antigen. Although all selected clones worked in immunofluorescent microscopy with H7 giving the strongest signal, only the entire F4 phage particle selected against DYSF1 could recognize dysferlin on Western blots of muscle.…”

Section: Discussionmentioning

confidence: 99%

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

et al. 2005

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Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limbgirdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.

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Section: Discussionmentioning

confidence: 99%

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

et al. 2005

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“…6, which is published as supporting information on the PNAS web site) was based upon methods described (15). Phage supernatants were screened by bacteriophage ELISA as described (16,17), where the biotinylated forms of 5H and IZN36 were immobilized onto 96-well ABGene, Surrey, U.K., streptavidin plates. For viral neutralization assays, immobilized metal ion affinity chromatography-purified soluble scFv fragments were prepared by using standard methods (18).…”

Section: In Vitro Isolation Of 5h͞i1-bmv-d5 (D5) Single-chain Variablmentioning

confidence: 99%

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

Miller¹,

Geleziunas

Bianchi³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

146

181

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HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H͞I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusioninhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a ''hot spot'' for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.envelope ͉ fusion ͉ prehairpin ͉ vaccine

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“…The amplified phages were used for a second cycle of selection as reported. 32,56 Deep Sequencing of cDNA Inserts After the second round of selection, colonies growing on agar plates were harvested, and the phagemid DNAs were isolated using a standard miniprep procedure. The cDNA inserts were processed for 454 sequencing according to the Titanium Rapid Library preparation method manual (Roche, Milano, Italy).…”

Section: Biopanning Proceduresmentioning

confidence: 99%

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

et al. 2015

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We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of a-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.

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Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries

Cited by 63 publications

References 40 publications

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

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