Currently, many methods are available for the analysis of drugs of abuse in urine but they all have their drawbacks. Thus, the purpose of this research was to overcome some of these drawbacks by developing multi-analyte detection systems based on sequential or spatial techniques and immunoassays. The first system was based on a spatial technique and involved a simple indirect competitive ELISA format. This produced relatively rapid multi-analyte dip-strip ELISAs for benzoylecgonine (BE), methadone (MET) and morphine (MOR). Various enzyme-labelled antibodies, substrates and filters were investigated. A multi-analyte dip-strip assay was developed based on cellulose nitrate filters, alkaline phosphatase labelled anti-mouse second antibody and nitro blue tetrazolium / 5-bromo-4-chloro-3- indoyl phosphate (NBT /BCIP) substrate. The resulting assays gave a simple 'yes/no' result when drug was present or absent from a sample at concentrations of 1.45 f,lg ml-I , 1.55 f,lg ml-I and 1.43 f,lg ml-I for BE, MET and MOR respectively. Limitations however were encountered that caused the concentrations to be above the accepted cutoff levels for these three drugs of abuse. The second system was based on a sequential technique and involved a flow-injection nnmunoassay (FIlA). Various monoclonal antibodies, fluorotracers and immobilisation methods were investigated. For morphine, a novel simple FIlA was developed which is based on competition between a mixture of a fluorescein derivative of the drug and morphine in flow over low affinity monoclonal morphine antibodies immobilised on a N-hydroxysuccinimidyl chloroformate activated agarose immunoreactor. With this system, a split peak profile (unbound and retarded fractions) was observed under isocratic conditions with the retarded peak disappearing and the unbound peak increasing in peak height/area as the concentration of morphine increased. Using a flow-rate of 0.5 ml min-I and a fluorescein derivative dilution of 1: 100, this assay had a sample throughput of 4 samples h-I and a detection limit of 14.1 f,lg ml- I . For a flow-rate of 1.6 ml min-I and a fluorescein derivative dilution of 1: 1 00,the assay had a sample throughput of 6 samples h-I and a detection limit of 10.9 J.!g mri. The origin of the phenomenon was investigated and revealed to be due to the low association rate of the drug tracer with the morphine antibody used and the near equivalence of the monoclonal antibody affinity for its respective tracer and drug. It was found that when these values are exceeded, the "split peak" phenomenon was not observed but the reagents could be used in conventional displacement flow injection fluoroimmunoassays as was demonstrated for benzoylecgonine and methadone.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:251355 |
| Date | January 2002 |
| Creators | Taylor, Carolyn |
| Publisher | University of Sunderland |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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