Genetic engineering of antibody fragments for the detection of illicit drugs

The monitoring of illicit drugs is now of major importance. Key areas of analysis include the detection of illicit use by drivers, workplace testing, forensic investigations and general monitoring to detect or reduce abuse. Thus, the development of a highly sensitive and reliable array-based assay for saliva, an assay-friendly matrix for detection of illicit drugs, would be of major benefit. This thesis describes the production and affinity maturation of antibodies for the detection of illicit drugs and their application in ELISA, Biacore surface plasmon resonance -based biosensor assays and, finally, in a multidrug-based assay. The first phase of this work focused on the development of a repertoire of antibody fragments against various illicit drugs including morphine-3-glucuronide (M3G), amphetamine and tetrahydrocannabinol (THC). Antibody libraries were generated from murine, avian and leporine immune systems. A number of antibody fragments were produced, purified and characterised by SDS-PAGE, Western blotting and ELISA. A pre-characterised, murine anti-M3G, whole IgG structure was used for conversion to scFv and Fab antibody fragments. This permitted SPR-based comparative studies regarding the validity of using either scFv or Fab fragments in current high throughput screening strategies. The Fab fragment revealed an apparent off rate 40-fold higher than that of the scFv, that did not translate to higher affinity for the target in question. A murine anti-amphetamine Fab fragment demonstrated a 3-fold higher affinity than that of the parent IgG clone. Diverse leporine immune libraries were utilised in the development of novel recombinant Fab and scFv fragments against amphetamine and THC. Subsequently the antibodies were solubly expressed in Escherichia coli, and fully characterised with respect to their binding capabilities on ELISA and Biacore-based analytical platforms. The next phase of the work involved the in-vitro affinity maturation of the anti-M3G Fab and anti-THC scFv fragments. The wild-type antibodies were used as templates for 22 construction of mutant recombinant antibody libraries. Initial mutagenesis strategies exploited error prone-PCR, DNA shuffling, light-chain shuffling and various methods of site-directed mutagenesis to generate improved affinity antibody fragments. Kunklestyle, site-directed mutagenesis, proved to be the most successful method for the generation of an anti-M3G Fab mutated library. The highest affinity anti-THC clones, characterised by ELISA, were carried forward for light-chain shuffling and subjected to specifically-tailored affinity selection conditions. Comparative gene and amino-acid sequence analyses were carried out on the wild-type and selected mutants and confirmed the presence of mutations in the selected CDR’s. The final phase of this research involved the incorporation of the antibodies onto multianalyte assay formats. Optimisations were carried out with respect to immobilisation strategies, surface chemistries, recombinant antibody fragment selection, use of drugprotein conjugates and detection strategies.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

Dublin City University

Year dc:date.issued

2009

Author dc:creator

• Townsend, Sue

• Biotechnology

Language dc:language

en