Development of an immunoassay for ciprofloxacin based on phage-displayed antibody fragments.
The widespread use of ciprofloxacin in human, animal and plant health has raised an environmental problem, paralleled by several other antibiotics. The aim of this work is the development of a rapid and sensitive ELISA assay for ciprofloxacin, which can constitute an alternative to time-consuming HPLC methods. For this purpose, we worked with antibody fragments, instead of whole antibodies, and used magnetic beads as solid support. Ciprofloxacin was successfully immobilized onto this support with a carbodiimide-mediated reaction. A library of phage particles that express human single-chain antibodies at their surface was then screened with an optimized protocol. Several positive fragments were isolated and identified as being V(L) fragments. These were then fully characterized. A reproducible competitive ELISA was developed using the magnetic beads - ciprofloxacin as support and the phages displaying the V(L) fragment as recognition entity. This assay showed limits of detection and quantification of 9.3 nM and 33 nM, respectively. Also, competitive ELISAs with ciprofloxacin homologues and other molecules showed cross-reactivities lower than 12%.
Gomes
Francisco Bruno Moreira Brás
FB
CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal.
Riedstra
Symon
S
Ferreira
João Paulo Medeiros
JP
D000595:Amino Acid Sequence; D001667:Binding, Competitive / Q000276:immunology; D002234:Carbodiimides / Q000737:chemistry; D002939:Ciprofloxacin / Q000031:analogs & derivatives / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D000077422:Enrofloxacin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D024841:Fluoroquinolones / Q000276:immunology; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008863:Microspheres; D015242:Ofloxacin / Q000276:immunology; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
D002234:Carbodiimides; D024841:Fluoroquinolones; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies; D000077422:Enrofloxacin; D002939:Ciprofloxacin; D015242:Ofloxacin