Development of a novel diagnostic system based on the use of the compact disc as a platform for biological analysis

White, Elaine M (1999) Development of a novel diagnostic system based on the use of the compact disc as a platform for biological analysis. MSc(R) thesis, University of Glasgow.

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The aim of this project was to develop a novel diagnostic system based on compact discs as a platform for biological analyses - in effect a diagnostic disc. This would allow a blood or serum sample to be tested against a panel of clinically relevant analytes or diseases thus aiding diagnosis and saving time. It would also allow such tests to be decentralised from a large testing laboratory and performed at the point of care such as in field hospitals, clinics and health centres. This has been developed initially for immunoassays with a view to moving away from the standard 96-well plates, where only one analyte can be tested per sample, to multianalyte testing on the same sample. This would then be read using compact disc technology in a specially adapted laser reader, producing optical density (O.D.) values. The initial aim of the project was to show that a panel of immunoassays could be performed in parallel without cross-reaction or cross-contamination. This would then be extended to a more clinically relevant panel for example, parasitic diseases. The system was originally based on the use of sectors, which resembled miniaturised microplates. These contained 44 wells of approximately 3.5mul in volume. The sectors are held within a "spider" structure based on a compact disc. Each spider holds eight sectors giving a total of 352 wells. The ELISAs performed were initially as on a 96-well plate, the main difference being that due to the spinning of the disc while being read, a substrate had to be found that would produce an insoluble end-product. A basic ELISA was developed that could be used while the sectors and spider were being developed. Once the final structure had been determined the sectors were moulded from medical grade polystyrene. Work was then carried out on plain plastic discs while the sectors were being moulded; primarily to determine the sensitivity of insoluble 3,3', 5,5'-Tetramethylbenzidine (TMB) - a substrate not usually used in ELISAs. A novel washing procedure, called "flood and fill" was developed which allowed the wells to be washed simultaneously while the sectors were held in the spider. A multianalyte test was constructed using seven different ELISAs which involved detecting the complementary antibody to the following commercially available antigens. Human Serum Albumin, alpha-1-Antitrypsin, alpha-8-Macroglobulin, Antithrombin III, Catalase, alpha-1-Antichymotrypsin and Plasminogen. There was no cross-reaction or cross-contamination between wells except for a low-level cross-reaction between anti-Antithrombin III and alpha-2-Macroglobulin. There was very low non-specific binding. The incubation times were greatly reduced with a total assay time of one hour, excluding washing. There was inter-sector and inter-disc variation in colour, which was reduced by pre-coating the sector with a silicone spray. Evaluation of colour was mainly by visual examination with the sectors due to problems with the development of the reader There were two main problems associated with the sectors. The first was how to keep them in the spider while the disc was being read and the second was how to prevent the liquid left in the wells at the end of the assay from escaping the disc. There was a requirement for a "snap-up" mechanism to be added to the sector that, at the end of the assay, would both hold the sector in the spider and also create a seal to prevent the leakage of any liquid. This would have been prohibitively expensive and therefore a second prototype platform was developed. Earlier work had shown that the multianalyte assay could be performed on a compact disc without separate wells. A compact disc had a black label applied such that the pattern of wells was the same as the sector. The antigen and blocking agent had to be dried on to the wells to prevent "streaming", an effect whereby there was cross-contamination between wells and the blue colour produced at the end of the assay was not localised in the appropriate wells. It was found that the multianalyte ELISA could be performed in this way and at the end of the assay, the disc was simply allowed to air-dry and then inserted in the reader. The O.D. values were significantly higher with the black disc as compared to the sectors and it was used as the platform for the remainder of the project. There was significant variation in O.D. values between wells and discs and this made it difficult to draw conclusions about assay sensitivity and the effect of changing assay conditions such as incubation times and temperatures. This was primarily due to the antigens being coated on to the disc by hand-pipette (1mul per wells), thus introducing a large degree of error. In addition the insoluble TMB did not produce quantitative results as shown by with poor gradation in colour when the antigen and antibody concentrations were varied. Alternatives under consideration include blue bead conjugated second antibody or changing the signal entirely from colorimetric to fluorescence. Future work will involve comparing results from the disc with those from microtitre plates using clinically defined sera and from there to constructing logical panels of immunoassays for example parasitic immunoassays that can be used in the field.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: Professor John Kusel
Keywords: Information technology, medicine.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Supervisor, not known
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-73267
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 09 Sep 2021 09:35

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