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Citation
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HERO ID
4898214
Reference Type
Journal Article
Title
Enhancing recombinant antibody performance by optimally engineering its format
Author(s)
Murphy, C; Stack, E; Krivelo, S; Breheny, M; Ma, H; O'Kennedy, R
Year
2018
Is Peer Reviewed?
Yes
Journal
Journal of Immunological Methods
ISSN:
0022-1759
Publisher
Elsevier B.V.
Volume
463
Page Numbers
127-133
Language
English
PMID
30321550
DOI
10.1016/j.jim.2018.10.005
Web of Science Id
WOS:000453496300015
Abstract
Antibody-based sensors are now widely used in therapeutics, diagnostics, and in environmental monitoring. Recombinant antibodies are becoming integral parts of such devices due to their reported high affinities, their capacity for engineering to achieve highly defined performance characteristics and the fact that their production can be optimized to a significant degree. To aid as a model for the identification of important analyte binding residues within the antibody sub-structure and elucidate the docking characteristics of small molecules such as metabolites, illicit drugs, biotherapeutics (proteins, peptides and nucleic acids) or toxins towards the antibody, herein we report the binding of the harmful cyanobacterial-toxin, microcystin-leucine-arginine (MC-LR) to a single chain fragment variable (scFv) antibody fragment. Analysis of the binding of MC-LR to this scFv was used to identify key residues of interest and to show how 'freely-available' and 'easily-accessible' computer-based webservers can be utilised to initiate an investigation into the binding characteristics of interacting molecules. In this study, a detailed investigation of the sub-structure of the anti-MC-LR scFv was carried out and antibody/small-molecule binding interactions were analyzed. The profile elucidated using computational analysis revealed amino acids of importance in the complementarity determining region light chain region 3 (CDRL3) and framework region 3 (FR3) of the heavy chain. Important amino acid residues within in CDRL3 and FR3 were mutated in vitro and sensitivity and binding profiles were examined. It was found that phenylalanine (F) at position 91 and aspartate (D) at position 92 of the light chain region, and arginine (R) at position 66 in framework region 3 (FR3) of the heavy chain were identified to be involved in binding. The introduction of an auxiliary antibody domain to the variable heavy and variable light (scFv) to ascertain its influence on stability and binding was also examined. The strategy adopted provided a deeper knowledge of scFv sub-structure and identified the regions and amino acids essential to the antibody/small-molecule binding interactions.
Keywords
Antibody enhancement; Antibody-binding site; Computer-aided molecular design; Microcystin-LR; Recombinant antibody; Small-molecule; arginine; asparagine; aspartic acid; glutamic acid; immunoglobulin heavy chain; immunoglobulin light chain; lysine; microcystin LR; phenylalanine; recombinant antibody; serine; single chain fragment variable antibody; tyrosine; bacterial toxin; cyanobacterial toxin; marine toxin; microcystin; recombinant protein; single chain fragment variable antibody; amino acid sequence; antibody combining site; antibody engineering; antibody structure; antigen binding; Article; binding affinity; complementarity determining region; computer aided design; enzyme linked immunosorbent assay; immobilized metal affinity chromatography; in vitro study; molecular docking; nonhuman; point mutation; priority journal; sequence alignment; sequence homology; site directed mutagenesis; surface plasmon resonance; amino acid substitution; antibody specificity; genetics; human; immunology; missense mutation; mutagenesis; protein engineering; Amino Acid Substitution; Antibody Specificity; Bacterial Toxins; Humans; Marine Toxins; Microcystins; Mutagenesis; Mutation, Missense; Protein Engineering; Recombinant Proteins; Single-Chain Antibodies
Tags
Other
•
Harmful Algal Blooms- Health Effects
April 2021 Literature Search
PubMed
WOS
Scopus
Microcystins
Date Limited
PubMed
WOS
Not Date Limited
PubMed
WOS
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