CHROMATOGRAPHIC AUTOMATION OF IMMUNOASSAYS

In clinical samples the free, or non-protein bound, fraction of a drug or
hormone is of great interest since it represents the compound's
biologically-active form. This makes the measurement of this fraction
ideal for patient diagnosis and treatment. But current analytical methods
for these measurements (typically based on immunoassays) are slow, labor-
intensive, and cannot be performed by existing automated systems. This is
due to the low concentrations which must be determined and the need to
separate the bound and free fractions of the analyte prior to analysis. In
addition, the effects of bound analyte dissociation during the separation
process are not well understood. The aim of this study is to obtain a
better understanding of this dissociation and to develop new, automated
systems for free drug/hormone measurements. Dissociation effects will be
studied by examining the protein binding of two model compounds, thyroxine
and cortisol. Systems for automating free drug/hormone assays for these
compounds will be developed based on high-performance immunoaffinity
chromatography and chemiluminescent detection (HPIAC/CL). In this method,
an immobilized antibody column will be used to rapidly extract free analyte
from a sample, followed by analyte detection using chemiluminescent
acridinium ester labels. The first section of this work will examine the interaction kinetics of
thyroxine and cortisol with their binding proteins. This will be done by
studying the retention and band-broadening of their binding proteins as
they pass through columns containing immobilized thyroxine or cortisol.
The goal is to obtain a better understanding of dissociation effects in
free drug/hormone assays and to provide data for minimizing these effects
in HPIAC/CL systems. The second section will study the processes involved
in the adsorption of analytes to immobilized antibody columns. The goal is
to determine what optimum chromatographic conditions are needed for
automating free drug/hormone immunoassays using HPIAC/CL. This will be
studied using computer simulations and work with a model system, the
binding of concanavalin A to fluorescent and nonfluorescent sugars. The
last section will use data from the previous studies to design systems
based on HPIAC/CL for free thyroxine and cortisol measurements. A
comparison of this approach to current methods will be made to determine
its potential advantages and limitations. The use of this system in
measuring other drugs/hormones will then also be considered.