Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens

Kobayashi, N.; Karibe, Tsuyoshi; Goto, Junichi

doi:10.1016/j.ab.2005.09.016

Cited by 14 publications

(13 citation statements)

References 39 publications

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“…We have already undertaken modification of the present CAP into more efficient and feasible procedures. To surely recover scFv mutants bound on the antigen-immobilized solid phases with extremely high affinity, we might employ the dissociation-independent methods such that we previously reported 14 , 42 , 43 .…”

Section: Discussionmentioning

confidence: 99%

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

Kiguchi

Oyama

Morita

et al. 2020

Sci Rep

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"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 10 5 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32–63-fold improved K a values (> 10 10 M −1 ), compared with the wild-type scFv ( K a = 3.8 × 10 8 M −1 ), none of which could be recovered via conventional panning procedures operated for the entire library.

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Section: Discussionmentioning

confidence: 99%

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

Kiguchi

Oyama

Morita

et al. 2020

Sci Rep

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“…These methods sometimes are not so effective due to tight antigen-antibody binding and attachment to the solid support of the tubes or plates. To optimize the method of phage-display panning and also to recover high-affinity anti-idiotypic phage antibodies from immobilized antigens, different elution conditions have been used [16,17]. Each antigen-antibody interaction requires careful fine-tuning of elution conditions to produce antigen-specific scFv antibodies [17,18].…”

Section: Introductionmentioning

confidence: 99%

“…To optimize the method of phage-display panning and also to recover high-affinity anti-idiotypic phage antibodies from immobilized antigens, different elution conditions have been used [16,17]. Each antigen-antibody interaction requires careful fine-tuning of elution conditions to produce antigen-specific scFv antibodies [17,18]. We are trying to establish an improved, optimized subtractive panning method to select antigen-specific antibodies more efficiently.…”

Section: Introductionmentioning

confidence: 99%

An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody

et al. 2009

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BackgroundPhage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection ofrecombinant antibodies.ResultsTo produce more selective and specific anti-idiotypic scFv-antibodies from a cDNA library, constructed from HM-1 killer toxin (HM-1)-neutralizing monoclonal antibodies (nmAb-KT), the method was modified by using an elution buffer supplemented with HM-1 that shares structural and functional similarities with the active site of the scFv antibody. Competitive binding of HM-1 to nmAb-KT allowed easy and quick dissociation of scFv-displayed phages from immobilized nmAb-KT to select specific anti-idiotypic scFv antibodies of HM-1. After modified panning, 80% clones (40/50) showed several times higher binding affinity to nmAb-KT than regular panning. The major populations (48%) of these clones (scFv K1) were genotypically same and had strong cytocidal activity against Saccharomyces and Candida species. The scFv K1 (Kd value = 4.62 × 10-8 M) had strong reactivity toward nmAb-KT, like HM-1 (Kd value = 6.74 × 10-9 M) as judged by SPR analysis.ConclusionThe scFv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning. A simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. Our method offers an attractive platform to discover potential therapeutic candidates.

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“…Liquid-phase magnetic-bead screening has numerous advantages, including specificity, stability, accuracy, and complete exposure of the antigen. Less antigen is required for liquid-phase screening than solid-phase screening [ 22 , 23 ]. In this study, we screened the antibody library using liquid-phase screening.…”

Section: Discussionmentioning

confidence: 99%

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b

Qin

Duan

Pei

et al. 2022

Journal of Pharmaceutical Analysis

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Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens

Cited by 14 publications

References 39 publications

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b

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