Date of Award

Winter 1982

Project Type

Dissertation

Program or Major

Chemistry

Degree Name

Doctor of Philosophy

Abstract

This dissertation evaluates the potential of chemiluminescence analysis for two types of clinical applications: (1) rapid analysis with a flow injection system which consumes small reagent volumes (25 (mu)L) and (2) a new approach to chemiluminescence immunoassay.

The first application studied uses the firefly reaction with a flow system to analyze for adenosine triphosphate (ATP). Two flow configurations called the "valve within a valve" and "merging zones" configurations were evaluated with respect to effect of parameters such as flow and sample volume on precision, sensitivity and sample throughput. With the appropriate choice of parameters, both systems achieve precisions of 1-2% relative standard deviation and throughputs of 5-10 measurements per minute. The merging zones configuration consumes smaller volumes of sample.

The second project demonstrates a new approach to immunoassay based on chemical excitation of fluorophor labelled immunochemicals using the peroxyoxalate reaction. The effects of fluorophors, esters, and solvents, etc. on the chemiluminescence measurement were evaluated. The conditions for achieving the best precision (1-3% R.S.D.) with a high water component in the measurement were rhodamine B in Tris buffer adjusted to pH 8.00, 10('-3) M bis(2,4,6-trichlorophenyl) oxalate (TCPO) in ethyl acetate and 10('-3) M hydrogen peroxide in acetonitrile in the ratio 5:2:3. Under these conditions, the detection limit for rhodamine B is 10('-9) M. The detection limit is established by variations in the background chemiluminescence.

The possibility of both homogeneous and heterogeneous immunoassays was evaluated. It was shown that the chemiluminescence excitation efficiency decreases when fluorophors were bound to albumin but increases when the fluorophor was coupled to folic acid. The feasibility of a homogeneous immunoassay was demonstrated by binding albumin to rhodamine labelled antiserum. It was shown that the chemiluminescence intensity of the fluorophor labelled antibody decreases with increasing concentrations of bound antigen. The heterogeneous chemiluminescence immunoassay was evaluated by comparison with a commercially available fluorescence immunoassay kit by Bio Rad. Both immunoassays are limited by imprecision of mixing of the solvent systems for the chemiluminescence measurement.

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