CHROMATOGRAPHIC AUTOMATION OF IMMUNOASSAYS

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DESCRIPTION: (applicant's abstract) The free, or non-protein bound, form of many drugs and hormones is of great clinical interest since this form is believed to represent the biologically-active fraction of these compounds. This means that the free concentrations of such compounds is what should ideally be measured for patient diagnosis or treatment. However, there are several problems associated with the current methods used for this, including their speed and/or need for separate steps for the isolation and detection of the free drug or hormone fraction. The goal of this work is to develop improved analytical methods for free drugs and hormones by using chromatographic-based immunoassays. These systems will be designed based on information obtained for model drugs and hormones regarding their rates of binding and release in clinical samples, and their rate of extraction on immobilized antibody columns. Previous work has provided us with initial estimates of rate constants for the protein-binding of L-thyroxine and R/S-warfarin. A portion of our future studies will be aimed at obtaining more specific information on the kinetics of these interactions by using stopped-flow analysis and work with immobilized protein columns. A second part of this study will use computer simulations and the kinetic information to design immobilized antibody columns for the rapid extraction of free drugs and hormones. The last portion of this study will explore the use of these extraction columns in the development of automated systems for the analysis of free drugs and hormones. In some of these methods, the extracted free analyte will be determined directly, while in others a second immunoaffinity system and a post-column detection scheme based on chemiluminescent labels will be used for quantitation. Later work will examine ways of modifying these methods for the specific determination of free drugs and hormones that occur in the presence of structurally-similar compounds or that exist in different chiral forms. As these assay systems are developed and evaluated, their extension to the determination of other free drugs or hormones, plus the simultaneous measurement of free and bound solute fractions, will also be considered.