Type

Journal Article

Authors

Richard O'Kennedy
Anthony J. Killard
Stephen J. Daly
Bernadette M. Manning
Paul P. Dillon

Subjects

Biochemistry

Topics
biosensor biotechnology morphine 3 glucuronide biochemistry elisa inhibition assay chemistry immunology recombinant antibody biosensors surface plasmon resonance

Production of a recombinant anti-morphine-3-glucuronidenext term single-chain variable fragment (scFv) antibody for the development of a “real-time” biosensor-based immunoassay (2003)

Abstract A recombinant single-chain variable fragment (scFv) antibody to morphine-3-glucuronide (M3G) was produced using genetic material obtained from the spleen cells of mice immunised with a morphine-3-glucuronide-bovine serum albumin (M3G-BSA) conjugate. Immunoglobulin light (VL) and heavy (VH) chain genes were amplified and cloned into pAK vectors for generation of recombinant antibody fragments in Escherichia coli. A competition ELISA assay was developed in PBS to characterise the ability of the antibody fragments to recognise free drug and the detection limits were found to be as low as 3 ng ml−1. Surface plasmon resonance-based inhibition immunoassays were developed. The recombinant antibody was pre-incubated with various concentrations of free drug followed by injection over a morphine-3-glucuronide-thyroglobulin (M3G-THY) immobilised surface. The response of antibody binding to the surface of the chip was inversely proportional to the amount of free drug in solution. Regeneration conditions for antibody binding to the surface were optimised resulting in a binding-regeneration capacity of at least 30 cycles. The inhibition assay for M3G was tested with assay ranges between 3 and 195 ng ml−1 and 3 and 97 ng ml−1 in PBS and urine, respectively.
Collections Ireland -> Dublin City University -> Publication Type = Article
Ireland -> Dublin City University -> Subject = Physical Sciences: Chemistry
Ireland -> Dublin City University -> Status = Published
Ireland -> Dublin City University -> DCU Faculties and Centres = DCU Faculties and Schools: Faculty of Science and Health: School of Chemical Sciences
Ireland -> Dublin City University -> Subject = Biological Sciences: Biochemistry
Ireland -> Dublin City University -> Subject = Biological Sciences: Immunology
Ireland -> Dublin City University -> DCU Faculties and Centres = Research Initiatives and Centres: National Centre for Sensor Research (NCSR)
Ireland -> Dublin City University -> Subject = Biological Sciences: Biotechnology
Ireland -> Dublin City University -> DCU Faculties and Centres = DCU Faculties and Schools: Faculty of Science and Health: School of Biotechnology
Ireland -> Dublin City University -> Subject = Biological Sciences: Biosensors

Full list of authors on original publication

Richard O'Kennedy, Anthony J. Killard, Stephen J. Daly, Bernadette M. Manning, Paul P. Dillon

Experts in our system

1
R O'Kennedy
Dublin City University
Total Publications: 197
 
2
Anthony J. Killard
Dublin City University
Total Publications: 73