Enhancing recombinant antibody performance by optimally engineering its format.
| Title: | Enhancing recombinant antibody performance by optimally engineering its format. |
|---|---|
| Authors: | Murphy C; School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Electronic address: caroline.s.murphy@dcu.ie.; Stack E; School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.; Krivelo S; School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.; Breheny M; School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.; Ma H; School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.; O'Kennedy R; School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; Hamad Bin Khalifa University and Research, Development and Innovation, Qatar Foundation, Doha, Qatar. |
| Source: | Journal of immunological methods [J Immunol Methods] 2018 Dec; Vol. 463, pp. 127-133. Date of Electronic Publication: 2018 Oct 12. |
| Publication Type: | Journal Article; Research Support, Non-U.S. Gov't |
| Language: | English |
| Journal Info: | Publisher: Elsevier Country of Publication: Netherlands NLM ID: 1305440 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1872-7905 (Electronic) Linking ISSN: 00221759 NLM ISO Abbreviation: J Immunol Methods Subsets: MEDLINE |
| Imprint Name(s): | Publication: Amsterdam : Elsevier; Original Publication: Amsterdam, North-Holand |
| MeSH Terms: | Single-Chain Antibodies*/genetics ; Single-Chain Antibodies*/immunology ; Protein Engineering*; Antibody Specificity/*genetics ; Bacterial Toxins/*immunology ; Marine Toxins/*immunology ; Microcystins/*immunology; Recombinant Proteins/genetics ; Recombinant Proteins/immunology ; Amino Acid Substitution ; Cyanobacteria Toxins ; Humans ; Mutagenesis ; Mutation, Missense |
| 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.; (Copyright © 2018 Elsevier B.V. All rights reserved.) |
| Contributed Indexing: | Keywords: Antibody enhancement; Antibody-binding site; Computer-aided molecular design; Microcystin-LR; Recombinant antibody; Small-molecule |
| Substance Nomenclature: | 0 (Bacterial Toxins); 0 (Cyanobacteria Toxins); 0 (Marine Toxins); 0 (Microcystins); 0 (Recombinant Proteins); 0 (Single-Chain Antibodies); 77238-39-2 (microcystin) |
| Entry Date(s): | Date Created: 20181016 Date Completed: 20190924 Latest Revision: 20211204 |
| Update Code: | 20260130 |
| DOI: | 10.1016/j.jim.2018.10.005 |
| PMID: | 30321550 |
| Database: | MEDLINE |
Journal Article; Research Support, Non-U.S. Gov't