Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments

Rafique, Abdur; Satake, Kiriko; Kishimoto, Seiji; Khan, Kamrul Hasan; Kato, Dai‐ichiro; Ito, Yuji

doi:10.1089/mab.2019.0027

Cited by 8 publications

(16 citation statements)

References 45 publications

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“…Previous studies demonstrated that NGS analysis combined with bio-panning is very powerful in the selection of speci c binders with desired properties [23] . In this study, 40,000 ~ 50,000 binders enriched after two rounds of panning were sequenced through NGS sequencing, and the top 200 most abundant sequences were selected and classi ed into four groups based on CDRH3 amino acid sequence (the most variable domain among VHHs) (Figure . 1).…”

Section: Discussionmentioning

confidence: 99%

Purification of SARS-CoV-2 RBD in Affinity Chromatography Using a Novel Nanobody Ligand

Zhu

Lin²,

Qiu³

et al. 2023

Preprint

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In the spike protein of SARS-CoV-2, the receptor-binding domain (RBD) contains multiple dominant neutralizing epitopes and can be used as an antigen for developing COVID-19 vaccines and neutral antibodies. Affinity chromatography is one of the most extensively used methods for rapid one-step protein purification. However, there is a lack of commercially available affinity ligands for RBD purification. Here, we report the rapid isolation of a nanobody suitable for purifying RBD as an affinity ligand from immune phage display libraries. After bio-panning, the enriched clones were sequenced on next-generation sequencing (NGS) platforms and classified into four groups based on the CDRH3 amino acid sequence. The representative sequences with high nanomolar affinities to RBD were further categorized into two groups via epitope binning analysis. Finally, from the two epitope bins, we found that SS3 showed easy elution under a mild eluting condition and could be used as a functional affinity ligand to purify RBD. These results also indicate that categorizing the bio-panned sequences via high-throughput sequencing (HTS) techniques followed by epitope binning represents a fast workflow to select specific binders with desired properties.

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Section: Discussionmentioning

confidence: 99%

Purification of SARS-CoV-2 RBD in Affinity Chromatography Using a Novel Nanobody Ligand

Zhu

Lin²,

Qiu³

et al. 2023

Preprint

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“…This conclusion is consistent with the basic concept of affinity maturation. Clone S1139 (L27626) in cluster 7 and clone L54 in cluster 33 were identified either by bio-panning or by a combination of bio-panning and NGS analysis (7,8). Both empirically identified clones had low antigen affinity and were localized to the root region of the phylogenetic tree.…”

Section: Discussionmentioning

confidence: 99%

“…Only the VHH clones in clusters 2, 5, 7, 10, 15, and 16 exhibited antigen binding ("hit clusters"). No antigen binding was detected for the clones in clusters 1, 3,6,8,9,11,12,13, or 14 ("miss clusters"). In SPR, clone S38 in cluster 4 bound aberrantly to the CL fragment.…”

Section: Propagation Of Vhh Sequences Through Immunizationmentioning

confidence: 99%

“…Seventy clusters and 1,678 sequences were identified in this experiment. Among them, we selected clusters EGFR-4, 8,9,11,14,19,20,23,24,25,34 and 46 (Figures 3C, 3D & S8) with wide range of maximum percentage appearance ranged from 0.3 to 2.7% (Figure S6B) by the same criteria as used for clusters prediction against IgG fragments. Of the 12 cluster predicted, 10 clusters contained antigen-binding clones, 5 of which showed antigen binding by both ELISA and SPR.…”

Section: Prediction Of Hit-clusters Using Sequence Data From Alpacas ...mentioning

confidence: 99%

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Prediction of antigen-responding VHH antibodies by tracking evolution of antibody along time course of immunization

Matsuda

Akazawa-Ogawa

Komaba

et al. 2021

Preprint

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It is difficult to link antibody repertoire expansion with changes in the physical characteristics of antigen-responding antibodies as correct light-chain and heavy-chain matching and conventional antibody production are laborious. Utilization of single-domain antibody solved these problems. A blood of immunized alpaca was collected weekly for three months for antibody repertoire analysis. The sequences processed to cluster and recombinant antibodies were generated to determine whether the clusters respond to antigens. The repertoire expansion in most of the antigen-responding clusters exhibited distinct patterns as compared to that of antigen irrelevant clusters. In addition, the sequences at the tips and root in the molecular phylogenetic cluster tree have strong and weak antigen affinity, respectively. These features may be utilized to predict clusters of antigen-binding antibodies and physical characteristics of antibodies.

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“…One bottleneck problem of the phage‐based detection of microbial toxins is the preparation of antibody analogs. In addition to the most commonly used immunoglobulin G (IgG), several types of antibody analogs have been designed based on PDTs, such as the single‐chain variable fragments of antibodies (Hanna et al., 2020; Muyldermans, 2021; Rafique et al., 2019; Ren et al., 2020). The production of VHHs often uses large animals and phages.…”

Section: Limitations Of Phage‐based Detection Technologiesmentioning

confidence: 99%

Phage‐based technologies for highly sensitive luminescent detection of foodborne pathogens and microbial toxins: A review

Guo

et al. 2022

Comp Rev Food Sci Food Safe

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Foodborne pathogens and microbial toxins are the main causes of foodborne illness. However, trace pathogens and toxins in foods are difficult to detect. Thus, techniques for their rapid and sensitive identification and quantification are urgently needed. Phages can specifically recognize and adhere to certain species of microbes or toxins due to molecular complementation between capsid proteins of phages and receptors on the host cell wall or toxins, and thus they have been successfully developed into a detection platform for pathogens and toxins. This review presents an update on phage‐based luminescent detection technologies as well as their working principles and characteristics. Based on phage display techniques of temperate phages, reporter gene detection assays have been designed to sensitively detect trace pathogens by luminous intensity. By the host‐specific lytic effects of virulent phages, enzyme‐catalyzed chemiluminescent detection technologies for pathogens have been exploited. Notably, these phage‐based luminescent detection technologies can discriminate viable versus dead microbes. Further, highly selective and sensitive immune‐based assays have been developed to detect trace toxins qualitatively and quantitatively via antibody analogs displayed by phages, such as phage‐ELISA (enzyme‐linked immunosorbent assay) and phage‐IPCR (immuno‐polymerase chain reaction). This literature research may lead to novel and innocuous phage‐based rapid detection technologies to ensure food safety.

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Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments

Cited by 8 publications

References 45 publications

Purification of SARS-CoV-2 RBD in Affinity Chromatography Using a Novel Nanobody Ligand

Purification of SARS-CoV-2 RBD in Affinity Chromatography Using a Novel Nanobody Ligand

Prediction of antigen-responding VHH antibodies by tracking evolution of antibody along time course of immunization

Phage‐based technologies for highly sensitive luminescent detection of foodborne pathogens and microbial toxins: A review

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