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Title: Immunoassays by flow injection analysis
Author: Hughes, Arwel
ISNI:       0000 0001 3583 664X
Awarding Body: Sheffield City Polytechnic
Current Institution: Sheffield Hallam University
Date of Award: 1986
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A home-built flow injection analysis (FIA) system was fully automated using an Apple IIe microcomputer, and subsequently used as a versatile system for the study of immunoassay procedures. The performance of the automated system was monitored by using the turbidimetric detection of barium sulphate as a model. A model immunoassay between concanavalin A (antibody) and yeast mannan (antigen) was investigated using the computer controlled FIA system. The automated injection procedure gave acceptable precision %RSD < 6 for a turbidimetric method and the stop-flow merging zones technique used gave an acceptable sample throughput (40 samples hour-1) with minimal consumption of both sample and reagent (30 mul per analysis). An immunological reaction between human serum immunoglobulin G (IgG) and goat antihuman IgG was investigated using a similar system to the above. Turbidimetric detection at 340 nm was used to monitor the rate of reaction. A sampling rate of 40 samples hour-1 and a precision of 2.0-6.8 %RSD was obtained for a range of human standards. Serum samples and a human reference serum were analysed and their IgG concentrations interpolated from the calibration data. Satisfactory correlation (r=0.9881) was observed with the existing technique of radial immunodiffusion, as used at Doncaster Royal Infirmary, over the range 0-2844 mg dl-1 IgG.Efforts to increase the sensitivity of the method for IgG determinations were based on enzyme immunoassay procedures, using urease as the enzyme label on the antibody. A homogeneous EMIT type assay for IgG was studied using the computer controlled FIA system. However the expected increase in sensitivity was not achieved. A pseudo heterogeneous ELISA type assay for IgG was also studied, a method which allowed for further reduction in consumption of expensive reagents by immobilising the enzyme conjugate on controlled pore glass or polystyrene. The results presented demonstrate that both EMIT and ELISA type assays are possible using FIA, however on this occasion problems of sensitivity did not allow for determinations using real samples. The determination of ABO blood grouping is the first qualitative application of FIA that has been reported. Results for 20 human red cell samples using the automated FIA system correlated well with results obtained by an automated agglutination technique used at the Blood Transfusion Service, Sheffield.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry