scholarly journals Efficient Construction and Effective Screening of Synthetic Domain Antibody Libraries

2019 ◽  
Vol 2 (1) ◽  
pp. 17 ◽  
Author(s):  
Arghavan Solemani Zadeh ◽  
Alissa Grässer ◽  
Heiko Dinter ◽  
Maximilian Hermes ◽  
Katharina Schindowski
Keyword(s):  
Phage Display ◽  
Selection Process ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Effective Screening ◽  
Naturally Occurring ◽  
Domain Antibody ◽  
Efficient Construction ◽  
Antibody Libraries ◽  
Complementarity Determining Region

Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.

In vitro affinity maturation of HuCAL antibodies: complementarity determining region exchange and RapMAT technology

Immunotherapy ◽  
2009 ◽  
Vol 1 (4) ◽  
pp. 571-583
Author(s):  
Josef Prassler ◽  
Stefan Steidl ◽  
Stefanie Urlinger
Keyword(s):  
Monoclonal Antibodies ◽  
Affinity Maturation ◽  
Optimization Techniques ◽  
Human Diseases ◽  
Therapeutic Antibodies ◽  
Naturally Occurring ◽  
Human Immunoglobulins ◽  
Antibody Libraries ◽  
Complementarity Determining Region

Monoclonal antibodies gain ever-increasing importance in the treatment of human diseases across a broad range of indications. Diverse technologies currently exist, which are used to generate recombinant therapeutic antibodies that are basically indistinguishable from naturally occurring human immunoglobulins. We describe how human combinatorial antibody libraries are used together with unique optimization techniques to produce such therapeutically relevant proteins, for instance in the areas of oncology and inflammation.

scholarly journals Phage-display reveals interaction of lipocalin allergen Can f 1 with a peptide resembling the antigen binding region of a human γδT-cell receptor

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Matthias Habeler ◽  
Bernhard Redl
Keyword(s):  
Phage Display ◽  
Cell Receptor ◽  
Antigen Binding ◽  
Binding Region ◽  
Antigen Binding Site ◽  
Random Peptide Library ◽  
Random Peptide ◽  
Γδt Cell ◽  
High Throughput Technology ◽  
Can F 1

AbstractAlthough some progress has been achieved in understanding certain aspects of the allergenic mechanism of animal lipocalins, they still remain largely enigmatic. One possibility to unravel this property is to investigate their interaction with components of the immune system. Since these components are highly complex we intended to use a high-throughput technology for this purpose. Therefore, we used phage-display of a random peptide library for panning against the dog allergen Can f 1. By this method we identified a Can f 1 binding peptide corresponding to the antigen-binding site of a putative γδT-cell receptor. Additional biochemical investigations confirmed this interaction.

Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 259-270 ◽  
Author(s):  
Kostas Stamatopoulos ◽  
Chrysoula Belessi ◽  
Carol Moreno ◽  
Myriam Boudjograh ◽  
Giuseppe Guida ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Clinical Features ◽  
Lymphocytic Leukemia ◽  
Antigen Binding ◽  
Public Database ◽  
Antigen Binding Site ◽  
Clinical Correlations ◽  
Immunoglobulin Repertoire ◽  
Indolent Disease ◽  
Complementarity Determining Region

Abstract The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is biased and characterized by the existence of subsets of cases with closely homologous (“stereotyped”) complementarity-determining region 3 (CDR3) sequences. In the present series, 201 (21.9%) of 916 patients with CLL expressed IGHV genes that belonged to 1 of 48 different subsets of sequences with stereotyped heavy chain (H) CDR3. Twenty-six subsets comprised 3 or more sequences and were considered “confirmed.” The remaining subsets comprised pairs of sequences and were considered “potential”; public database CLL sequences were found to be members of 9 of 22 “potential” subsets, thereby allowing us to consider them also “confirmed.” The chance of belonging to a subset exceeded 35% for unmutated or selected IGHV genes (eg, IGHV1-69/3-21/4-39). Comparison to non-CLL public database sequences showed that HCDR3 restriction is “CLL-related.” CLL cases with selected stereotyped immunoglobulins (IGs) were also found to share unique biologic and clinical features. In particular, cases expressing stereotyped IGHV4-39/IGKV1-39-1D-39 and IGHV4-34/IGKV2-30 were always IgG-switched. In addition, IGHV4-34/IGKV2-30 patients were younger and followed a strikingly indolent disease, contrasting other patients (eg, those expressing IGHV3-21/IGLV3-21) who experienced an aggressive disease, regardless of IGHV mutations. These findings suggest that a particular antigen-binding site can be critical in determining the clinical features and outcome for at least some CLL patients.

scholarly journals Surprisingly Fast Interface and Elbow Angle Dynamics of Antigen-Binding Fragments

2020 ◽  
Vol 7 ◽  
Author(s):  
Monica L. Fernández-Quintero ◽  
Katharina B. Kroell ◽  
Martin C. Heiss ◽  
Johannes R. Loeffler ◽  
Patrick K. Quoika ◽  
...  
Keyword(s):  
Light Chain ◽  
Variable Region ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Antigen Specificity ◽  
Antigen Binding Site ◽  
Elbow Angle ◽  
Distinct Distribution ◽  
Complementarity Determining Region

Fab consist of a heavy and light chain and can be subdivided into a variable (VH and VL) and a constant region (CH1 and CL). The variable region contains the complementarity-determining region (CDR), which is formed by six hypervariable loops, shaping the antigen binding site, the paratope. Apart from the CDR loops, both the elbow angle and the relative interdomain orientations of the VH–VL and the CH1–CL domains influence the shape of the paratope. Thus, characterization of the interface and elbow angle dynamics is essential to antigen specificity. We studied nine antigen-binding fragments (Fab) to investigate the influence of affinity maturation, antibody humanization, and different light-chain types on the interface and elbow angle dynamics. While the CDR loops reveal conformational transitions in the micro-to-millisecond timescale, both the interface and elbow angle dynamics occur on the low nanosecond timescale. Upon affinity maturation, we observe a substantial rigidification of the VH and VL interdomain and elbow-angle flexibility, reflected in a narrower and more distinct distribution. Antibody humanization describes the process of grafting non-human CDR loops onto a representative human framework. As the antibody framework changes upon humanization, we investigated if both the interface and the elbow angle distributions are changed or shifted. The results clearly showed a substantial shift in the relative VH–VL distributions upon antibody humanization, indicating that different frameworks favor distinct interface orientations. Additionally, the interface and elbow angle dynamics of five antibody fragments with different light-chain types are included, because of their strong differences in elbow angles. For these five examples, we clearly see a high variability and flexibility in both interface and elbow angle dynamics, highlighting the fact that Fab interface orientations and elbow angles interconvert between each other in the low nanosecond timescale. Understanding how the relative interdomain orientations and the elbow angle influence antigen specificity, affinity, and stability has broad implications in the field of antibody modeling and engineering.

Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

2015 ◽  
Vol 21 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Nur Hidayah Hairul Bahara ◽  
Siang Tean Chin ◽  
Yee Siew Choong ◽  
Theam Soon Lim
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Selection Process ◽  
Antibody Library ◽  
Affinity Selection ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Variable Heavy ◽  
Vh Domain ◽  
Complementarity Determining Region ◽  
Selection Of

The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

Aggregation prone regions in antibody sequences raised against Vibrio cholerae: A bioinformatic approach

2020 ◽  
Vol 15 ◽  
Author(s):  
Zakia Akter ◽  
Anamul Haque ◽  
Md. Sabir Hossain ◽  
Firoz Ahmed ◽  
Md Asiful Islam
Keyword(s):  
Vibrio Cholerae ◽  
Binding Sites ◽  
Bioinformatic Analysis ◽  
Antigen Binding ◽  
Short Term ◽  
Protein Database ◽  
The Stability ◽  
Bioinformatic Approach ◽  
Self Aggregation ◽  
Complementarity Determining Region

Background: Cholera, a diarrheal illness causes millions of deaths worldwide due to large outbreaks. Monoclonal antibody used as therapeutic purposes of cholera are prone to be unstable due to various factors including self-aggregation. Objectives: In this bioinformatic analysis, we identified the aggregation prone regions (APRs) of different immunogens of antibody sequences (i.e., CTB, ZnM-CTB, ZnP-CTB, TcpA-CT-CTB, ZnM-TcpA-CT-CTB, ZnP-TcpA-CT-CTB, ZnM-TcpA, ZnP-TcpA, TcpA-CT-TcpA, ZnM-TcpA-CT-TcpA, ZnP-TcpA-CT-TcpA, Ogawa, Inaba and ZnM-Inaba) raised against Vibrio cholerae. Methods: To determine APRs in antibody sequences that were generated after immunizing Vibrio cholerae immunogens on Mus musculus, a total of 94 sequences were downloaded as FASTA format from a protein database and the algorithms such as Tango, Waltz, PASTA 2.0, and AGGRESCAN were followed to analyze probable APRs in all of the sequences. Results: A remarkably high number of regions in the monoclonal antibodies were identified to be APRs which could explain a cause of instability/short term protection of anticholera vaccine. Conclusion: To increase the stability, it would be interesting to eliminate the APR residues from the therapeutic antibodies in a such way that the antigen binding sites or the complementarity determining region loops involved in antigen recognition are not disrupted.

scholarly journals The mycoplasma surface proteins MIB and MIP promote the dissociation of the antibody-antigen interaction

2021 ◽  
Vol 7 (10) ◽  
pp. eabf2403
Author(s):  
Pierre Nottelet ◽  
Laure Bataille ◽  
Geraldine Gourgues ◽  
Robin Anger ◽  
Carole Lartigue ◽  
...  
Keyword(s):  
Surface Proteins ◽  
Mycoplasma Species ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Fab Fragment ◽  
Cryo Electron Microscopy ◽  
Antigen Complex ◽  
Antigen Interaction ◽  
Immunoglobulin Binding

Mycoplasma immunoglobulin binding (MIB) and mycoplasma immunoglobulin protease (MIP) are surface proteins found in the majority of mycoplasma species, acting sequentially to capture antibodies and cleave off their VH domains. Cryo–electron microscopy structures show how MIB and MIP bind to a Fab fragment in a “hug of death” mechanism. As a result, the orientation of the VL and VH domains is twisted out of alignment, disrupting the antigen binding site. We also show that MIB-MIP has the ability to promote the dissociation of the antibody-antigen complex. This system is functional in cells and protects mycoplasmas from antibody-mediated agglutination. These results highlight the key role of the MIB-MIP system in immunity evasion by mycoplasmas through an unprecedented mechanism, and open exciting perspectives to use these proteins as potential tools in the antibody field.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library

2021 ◽  
Vol 492 ◽  
pp. 112990
Author(s):  
Jothivel Kumarasamy ◽  
Samar Kumar Ghorui ◽  
Chandrakala Gholve ◽  
Bharti Jain ◽  
Yogesh Dhekale ◽  
...  
Keyword(s):  
Phage Display ◽  
Phage Display Library ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
T7 Phage Display ◽  
T7 Phage
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