Health & Environmental Research Online (HERO)


Print Feedback Export to File
4898214 
Journal Article 
Enhancing recombinant antibody performance by optimally engineering its format 
Murphy, C; Stack, E; Krivelo, S; Breheny, M; Ma, H; O'Kennedy, R 
2018 
Yes 
Journal of Immunological Methods
ISSN: 0022-1759 
Elsevier B.V. 
463 
127-133 
English 
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. 
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 
Other
• Harmful Algal Blooms- Health Effects
     April 2021 Literature Search
          PubMed
          WOS
          Scopus
          Microcystins
               Date Limited
                    PubMed
                    WOS
               Not Date Limited
                    PubMed
                    WOS