scholarly journals Antibody binding epitope Mapping (AbMap) of two hundred antibodies in a single run

2019 ◽  
Author(s):  
Huan Qi ◽  
Mingliang Ma ◽  
Chuansheng Hu ◽  
Zhao-wei Xu ◽  
Fan-lin Wu ◽  
...  
Keyword(s):  
Crystal Structures ◽  
High Throughput ◽  
Epitope Mapping ◽  
List Type ◽  
Random Peptide Library ◽  
Random Peptide ◽  
Binding Interface ◽  
Conformational Epitopes ◽  
Binding Epitope ◽  
Multiple Species

AbstractEpitope mapping is essential for the understanding how an antibody works. Given millions of antibodies short of epitope information, there is an urgent need for high-throughput epitope mapping. Here we combined a commercial phage displayed random peptide library of 109 diversity with next generation sequencing to develop Antibody binding epitope Mapping (AbMap) technology. Over two hundred antibodies were analyzed in a single test and epitopes were determined for >50% of them. Strikingly, the antibodies were able to recognize different proteins from multiple species with similar epitopes. We successfully identified the epitopes of 14 anti-PD-1 antibodies, including Sintilimab (i.e., L128, A129 and P130), and confirmed that the binding epitopes of Nivolumab and Sintilimab are very close to the binding interface of PD-1 and PD-L1. The predicted conformational epitopes of Pembrolizumab and Nivolumab are consistent with their antibody-antigen co-crystal structures. AbMap is the first technology enables high-throughput epitope mapping.HighlightsThe first technology enables epitope mapping of two hundred antibodies in a single runLinear epitope was determined for >50% of the antibodiesDistinct epitopes of 14 anti-PD-1 antibodies, including Sintilimab, were determinedThe predicted conformational epitopes of Pembrolizumab and Nivolumab are consistent with the known antibody-antigen co-crystal structures

The binding epitope of sintilimab on PD-1 revealed by AbMap

2021 ◽  
Author(s):  
Mingliang Ma ◽  
Huan Qi ◽  
Chuansheng Hu ◽  
Zhaowei Xu ◽  
Fanlin Wu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Structural Analysis ◽  
Western Blot ◽  
Epitope Mapping ◽  
High Efficiency ◽  
Point Mutations ◽  
Therapeutic Antibodies ◽  
Binding Interface ◽  
Binding Epitope ◽  
Key Residues

Abstract PD-1 plays an important role as an immune checkpoint. Sintilimab is a newly approved PD-1 antibody for cancer immunotherapy with an unknown binding epitope on PD-1. In this study, to elucidate the molecular mechanism by which sintilimab blocks PD-1 activation, we applied Antibody binding epitope Mapping (AbMap) to identify the binding epitope of sintilimab. An epitope was successfully identified, i.e. SLAPKA, aa 127–132. By constructing a series of point mutations, the dominant residues S127, L128, A129, P130, and A132 of PD-1 were further validated by western blot analysis, biolayer interferometry, and flow cytometry. Structural analysis showed that the epitope is partially within the binding interface of PD-1 and PD-L1, and this epitope also partially overlaps with that of nivolumab and pembrolizumab. These results demonstrate that sintilimab can attenuate PD-1 activation by directly competing with the interaction between PD-1 and PD-L1 through binding with the key residues of the FG loop on PD-1. This study also demonstrates the high efficiency and accuracy of AbMap for determining the binding epitope of therapeutic antibodies.

scholarly journals Antibody binding epitope Mapping (AbMap) of hundred antibodies in a single run

2020 ◽  
pp. mcp.RA120.002314 ◽  
Author(s):  
Huan Qi ◽  
Mingliang Ma ◽  
Chuansheng Hu ◽  
Zhao-wei Xu ◽  
Fan-lin Wu ◽  
...  
Keyword(s):  
High Throughput ◽  
Epitope Mapping ◽  
Binding Capacity ◽  
Antibody Binding ◽  
Spike Protein ◽  
Binding Epitope ◽  
Order Of Magnitude ◽  
Linear Epitopes ◽  
Phage Displayed Peptide Library ◽  
Generation Sequencing

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the Binding Capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.

MONOCLONAL ANTIBODIES OF THE IgM AND IgG CLASS SPECIFIC FOR CROSSLINKED FIBRIN DEGRADATION PRODUCTS

1987 ◽  
Author(s):  
P J Gaffney ◽  
L J Creighton ◽  
A Curry ◽  
B MacMahon ◽  
R Thorpe
Keyword(s):  
Monoclonal Antibodies ◽  
Thrombolytic Therapy ◽  
Epitope Mapping ◽  
Degradation Products ◽  
Subcutaneous Route ◽  
Fibrin Degradation Products ◽  
Conformational Epitopes ◽  
Immunoglobulin Class Switching ◽  
Unique Specificity ◽  
D Dimer

Monoclonal antibodies (mabs) to crosslinked fibrin degradation products (XL-FDP) having the general formula D/Y[X]nY/D (known as X-oligomer) and D-D (known as D dimer) have been raised in balb/C mice by both a novel mtrasplenic and a conventional subcutaneous route of immunisation and by combinations of both these procedures. Mabs to X-oligomers (NIBn 52 and NIBn 123) obtained by an intrasplenic procedure have been demonstrated to crossreact only with X-oligomer in a 2-site ELISA procedure and not with D dimer or whole fibrinogen and have been shown to be of value m the examination of clinical material obtained from patients with various types of thrombosis and have also been useful in monitoring the efficacy of thrombolytic therapy. The X-oligomer mabs are immunoglobulins of the M class. It was demonstrated that their unique specificity for conformational epitopes on the large X-oligomer fragments does not reside in the IgM structure since alterative immunisation procedures have been used to generate mabs of the IgG class which have the same specificity. Using immunoglobulin class switching in culture rather than during immunisation was suggested by certain cell lines which produced both IgM and IgG specific for X-oligomer. This latter point needs rigorous validation.Immunoglobulin G type mabs to highly purified D dimer were raised by conventional subcutaneous immunisation of balb/C mice. One of these, NIBn-11, was found to crossreact with PVC-immobilised X-oligomer and D dimer but not with fibrinogen. However NIBn-11 did not bind to D dimer in a 2-site ELISA procedure while crossreactmg quite avidly with X-oligomer. This suggests that the D dimer epitope to which NIBn-11 is directed is expressed in some conformations and not m others and that these conformations are always expressed in the complex X-oligomer group of fragments. These mabs, whilst of value in measuring certain unique fibrin fragments m plasma, are useful in the epitope mapping of fibrinogen/fibrin and their plasmm-mediated

scholarly journals EM by EM: High-Efficiency Epitope Mapping using High-Throughput Electron Microscopy

2016 ◽  
Vol 22 (S3) ◽  
pp. 1080-1081
Author(s):  
Alberto Estevez ◽  
Colin Garvey ◽  
Claudio Ciferri
Keyword(s):  
Electron Microscopy ◽  
High Throughput ◽  
Epitope Mapping ◽  
High Efficiency

36. Random Peptide Library Based on a Spider Neurotoxin, and Utilization of the Library in in vitro Evolution Directed to GPCR Ligands

Toxicon ◽  
2012 ◽  
Vol 60 (2) ◽  
pp. 113 ◽  
Author(s):  
Tai Kubo ◽  
Seigo Ono ◽  
Tadashi Kimura ◽  
Suzuko Kobayashi ◽  
Tetsuro Kondo ◽  
...  
Keyword(s):  
Peptide Library ◽  
In Vitro Evolution ◽  
Random Peptide Library ◽  
Random Peptide

Application of a high-throughput laboratory method to assess litter decomposition rates in multiple-species experiments

2013 ◽  
Vol 57 ◽  
pp. 929-932 ◽  
Author(s):  
Pablo García-Palacios ◽  
Rubén Milla ◽  
Mónica Álvaro-Sánchez ◽  
Nieves Martín-Robles ◽  
Melchor Maestro
Keyword(s):  
Litter Decomposition ◽  
High Throughput ◽  
Laboratory Method ◽  
Decomposition Rates ◽  
Multiple Species

scholarly journals Structure-based drug design against Trypanosoma brucei methionyl-tRNA synthetase

2014 ◽  
Vol 70 (a1) ◽  
pp. C708-C708
Author(s):  
Cho Yeow Koh ◽  
Jasmine Nguyen ◽  
Sayaka Shibata ◽  
Zhongsheng Zhang ◽  
Ranae Ranade ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Trypanosoma Brucei ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protozoan Parasite ◽  
Trna Synthetase ◽  
Structure Based Drug Design ◽  
Chemical Structures ◽  
Ic50 Values ◽  
Methionyl Trna Synthetase

Infection by the protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, commonly known as sleeping sickness. The disease is fatal without treatment; yet, current therapeutic options for the disease are inadequate due to toxicity, difficulty in administration and emerging resistance. Therefore, methionyl-tRNA synthetase of T. brucei (TbMetRS) is targeted for the development of new antitrypanosomal drugs. We have recently completed a high-throughput screening campaign against TbMetRS using a 364,131 compounds library in The Scripps Research Institute Molecular Screening Center. Here we outline our strategy to integrate the power of crystal structures with high-throughput screening in a drug discovery project. We applied the rapid crystal soaking procedure to obtain structures of TbMetRS in complex with inhibitors reported earlier[1] to approximately 70 high-throughput screening hits. This resulted in more than 20 crystal structures of TbMetRS·hit complexes. These hits cover a large diversity of chemical structures with IC50 values between 200 nM and 10 µM. Based on the solved structures and existing knowledge drawn from other in-house inhibitors, the IC50 value of the most promising hit has been improved. Further development of the compounds into potent TbMetRS inhibitors with desirable pharmacokinetic properties is on-going and will continue to benefit from information derived from crystal structures.

scholarly journals Scoping review of the applications of peptide microarrays on the fight against human infections.

2021 ◽  
Author(s):  
Arthur Vengesai ◽  
Maritha Kasambala ◽  
Hamlet Mutandadzi ◽  
Tariro Mduluza-Jokonya ◽  
Takafira Mduluza ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Scoping Review ◽  
Epitope Mapping ◽  
Cell Epitope ◽  
Binding Assays ◽  
Peptide Microarray ◽  
Random Peptide ◽  
Peptide Microarrays ◽  
Scoping Reviews

Abstract Introduction This scoping review explores the use of peptide microarrays in the fight against infectious diseases. The research domains explored included the use of peptide microarrays in the mapping of linear B-cell and T cell epitopes, antimicrobial peptide discovery, immunosignature characterisation and disease immunodiagnostics. This review also provides a short overview of peptide microarray synthesis.   Methods Electronic databases were systematically searched to identify relevant studies. The review was conducted using the Joanna Briggs Institute methodology for scoping reviews and data charting was performed using a predefined form. The results were reported by narrative synthesis in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guidelines. Results Eighty-six articles from 100 studies were included in the final data charting process. The majority (93%) of the articles were published during 2010–2020 and were mostly from Europe (44%) and North America (34 %). The findings were from the investigation of viral (44%), bacterial (30%), parasitic (25%) and fungal (2%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (78%) were related to mapping B-cell linear epitopes, 10% were on diagnostics, 9% reported on immunosignature characterisation and 6% reported on viral and bacterial cell binding assays. Two studies reported on T-cell epitope profiling. Conclusion The most important application of peptide microarrays was found to be B-cell epitope mapping or antibody profiling to identify diagnostic and vaccine targets. Immunosignatures identified by random peptide microarrays were found to be applied in the diagnosis of infections and interrogation of vaccine responses. The analysis of the interactions of random peptide microarrays with bacterial and viral cells using binding assays enabled the identification of antimicrobial peptides. Peptide microarray arrays were also used for T-cell linear epitope mapping which may provide more information for the design of peptide-based vaccines and for the development of diagnostic reagents.

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