Detection of the cyanobacterial toxin, microcystin-LR, using a novel recombinant antibody-based optical-planar waveguide platform

• Published in: Biosensors & bioelectronics Vol. 67; pp. 708 - 714
• Main Authors: Murphy, Caroline, Stack, Edwina, Krivelo, Svetlana, McPartlin, Daniel A, Byrne, Barry, Greef, Charles, Lochhead, Michael J, Husar, Greg, Devlin, Shauna, Elliott, Christopher T, O'Kennedy, Richard J
• Format: Journal Article
• Language: English
• Published: England 15.05.2015
• Subjects: Antibodies; Assaying; Biosensing Techniques; Biosensors; Cartridges; Cyanobacteria - chemistry; Format; Fragmentation; Fresh Water - analysis; Immunoassay; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - immunology; Microcystins - chemistry; Microcystins - isolation & purification; Recombinant; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Surface Plasmon Resonance; Toxins; Microcystin; Immunoassay; Recombinant Antibody; Cyanobacteria; Fluorescent planar wave-guide
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2014.10.039

Microcystins are a major group of cyanobacterial heptapeptide toxins found in freshwater and brackish environments. There is currently an urgent requirement for highly-sensitive, rapid and in-expensive detection methodologies for these toxins. A novel single chain fragment variable (scFv) fragment was generated and is the first known report of a recombinant anti-microcystin avian antibody. In a surface plasmon resonance-based immunoassay, the antibody fragment displayed cross-reactivity with seven microcystin congeners (microcystin-leucine-arginine (MC-LR) 100%, microcystin-tyrosine-arginine (MC-YR) 79.7%, microcystin-leucine-alanine (MC-LA) 74.8%, microcystin-leucine-phenylalanine (MC-LF) 67.5%, microcystin-leucine-tryptophan (MC-LW) 63.7%, microcystin-arginine-arginine (MC-RR) 60.1% and nodularin (Nod) 69.3%, % cross reactivity). Following directed molecular evolution of the parental clone the resultant affinity-enhanced antibody fragment was applied in an optimized fluorescence immunoassay on a planar waveguide detection system. This novel immuno-sensing format can detect free microcystin-LR with a functional limit of detection of 0.19 ng mL(-1)and a detection range of 0.21-5.9 ng mL(-1). The assay is highly reproducible (displaying percentage coefficients of variance below 8% for intra-day assays and below 11% for inter-day assays), utilizes an inexpensive cartridge system with low reagent volumes and can be completed in less than twenty minutes.