Type

Journal Article

Authors

Richard O'Kennedy
Hui Ma
Mark Breheny
Svetlana Krivelo
Edwina Stack
Caroline Murphy

Subjects

Engineering

Topics
computational analysis performance characteristics recombinant antibodies nucleic acids environmental monitoring heavy chain illicit drugs performance engineering

Enhancing recombinant antibody performance by optimally engineering its format. (2018)

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 utilized 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 CDRL3 and FR3 were mutated in vitro and sensitivity and binding profiles were examined. It was identified 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 nvolved 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 antibody/small-molecule binding.
Collections Ireland -> Dublin City University -> PubMed

Full list of authors on original publication

Richard O'Kennedy, Hui Ma, Mark Breheny, Svetlana Krivelo, Edwina Stack, Caroline Murphy

Experts in our system

1
R O'Kennedy
Dublin City University
Total Publications: 211
 
2
Hui Ma
Dublin City University
Total Publications: 15
 
3
Edwina Stack
Dublin City University
Total Publications: 6
 
4
Caroline Murphy
Dublin City University
Total Publications: 15