Doctoral Dissertations

Date of Award

12-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Michael J. Sepaniak

Committee Members

George Schweitzer, E.L. Wehry, Paul Bienkowski

Abstract

Immunoassays are the method of choice for detection and monitoring of many biological markers. This research combines the specificity and sensitivity of fluoroimmunoassay techniques with several fiber-optic chemical sensor protocols. The goal was to design a sensor that could be used for continuous or repetitive in situ analysis of large molecules such as proteins. Immunoglobulin G (IgG) is used as a representative protein molecule.

Several prototype sensors are described. These sensors revealed that immobilization of antibody-containing silica spheres (immunobeads) near the end of an optical fiber gave the largest concentration of antibody in the sensing range of the fiber. Immobilization of the immunobeads was accomplished by entrapment behind a membrane or frit. Protein molecules are too large to be sampled by diffusion, so a method for sampling by aspiration was developed. Reagent delivery into the sensing chamber by way of capillary columns made delivery of reagents in multi-step immunoassays more practical and allowed the sensors to be regenerated between trials. The early sensors revealed the importance of reproducibility of antibody coverage on the immunobeads. Other factors that were studied with these sensors were the density and volume of the bead slurry and the flow rate for reagent and sample delivery.

A sensor molded from acrylic resin was designed. This sensor was similar to the capillary column sensor, but the resin was more resistant to aqueous solvents than the epoxy used in the earlier sensors. It was found that the optimum immunobead conditions for this sensor were 50 μl of a slurry with density of 10 mg silica/ml. Optimum flow rate for this sensor was 136 μl/min. A direct immunoassay for fluorescently-labeled IgG was used to illustrate the function of the sensor and to aid in development of a sandwich immunoassay for the sensor. A calibration curve for the direct assay is exhibited. This sensor gave a limit of detection of less than 0.005 mg/ml of IgG using the direct assay protocol.

There are many more variables in a sandwich assay than in a direct assay. The slurry concentration, flow rates of sample, rinse and second antibody solutions, and the concentration of the antibody solution must all be controlled. Careful selection of the antibody system is crucial for a successful sandwich immunoassay. The epitope of the analyte molecule bound by the immobilized antibody must be well separated from the epitope bound by the free antibody to allow room for binding of both antibodies to the analyte. This may be best accomplished with the use of monoclonal antibodies developed to greatly separated portions of the analyte molecule. Analyte molecules with two distinct portions, such as leutinizing hormone with α and β subunits, may be more amenable than IgG to analysis by sandwich immunoassay using the sensors developed in this work.

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