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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

Dillon, Paul P. and Manning, Bernadette M. and Daly, Stephen J. and Killard, Anthony J. and O'Kennedy, Richard (2003) 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. Journal of Immunological Methods, 276 (1-2). pp. 151-161. ISSN 0022-1759

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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.

Item Type:Article (Published)
Refereed:Yes
Uncontrolled Keywords:recombinant antibody; morphine-3-glucuronide; ELISA; surface plasmon resonance; biosensor; inhibition assay;
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Biochemistry
Biological Sciences > Immunology
Physical Sciences > Chemistry
Biological Sciences > Biosensors
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Chemical Sciences
DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > National Centre for Sensor Research (NCSR)
Publisher:Elsevier
Official URL:http://dx.doi.org/10.1016/S0022-1759(03)00099-1
ID Code:51
Deposited On:08 Nov 2006 by DORAS Administrator. Last Modified 30 Jan 2009 11:53

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