Date of Award

Fall 1995

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

James H. Yuan

Committee Member

Roy L. Williams

Committee Member

Mark S. Elliot

Call Number for Print

Special Collections LD4331.C45 F43

Abstract

( +)-Catechin is a type of polyphenolic compound, and it is enriched in fruits, plants, and various beverages. Polyphenolic compounds also play an important role in the human diet. Many scientists, especially wine researchers, have proposed that(+)catechin and its polymers could have many positive health effects. Thus, it is essential to learn more about (+)-catechin and other polyphenolics. Presently, high performance liquid chromatography is the only method to detect ( + )-catechin levels in human blood serum, but this method has the high cost of instrumentation and does not provide good sensitivity. The objective of this project is to develop a high sensitivity, low cost, method to detect ( + )-catechin in serum.

A diazonium coupling reaction and a water soluble carbodiimide reaction were performed to prepare an antigen, (+)-catechin-bovine serum albumin. Approximately 12 mL of gamma globulin was isolated from whole antiserum, and the final concentration of gamma globulin was determined to be 2. 81 mg/mL. From the electrophoresis densitometry, the isolated gamma globulin was found to have the purity of98.9%. Polystyrene microtubes were used as the solid phase for immobilization of antibodies, and the concentrated antibody was diluted to I ng/mL for coating onto the solid phase. The nonlabeled-antigen and the labeled-antigen were allowed to compete for the binding of the immobilized antibodies for I hour, then the excess labeled and nonlabeled antigens were washed away from the solid phase with IO mM phosphate buffer saline and deionizd water.

The detection limit of the ( +)-catechin enzyme immunoassay was determined to be 7 ± 1.4 pM, and the coefficient of variation for the intra-assay precision study performed at (+)-catechin concentrations of 10 pM and 100 pM were found to be 13 .1 % and 11. 4 %, respectively.

The recovery of (+)-catechin from serum was determined to be 71.3% ± 3%, by the recommendation of the International Federation of Clinical Chemistry. The developed EIA method exhibits a linear range of 10 to 100 pM for the catechin standard solutions.

The developed EIA method exhibits a sensitivity that is 5 orders of magnitude higher than the conventional HPLC method. Furthermore, the cost of the instrumentation for the EIA method is one tenth that of HPLC method. Finally, the developed method is capable of analyzing multiple samples within 60 minutes whereas HPLC method can only analyze 4 samples within 60 minutes.

This newly developed EIA method can now be applied to enhance the study of the health effects of (+)-catechin on human body, especially wine drinkers. In addition, other EIA methods to detect flavanoids can also be developed based on the concept of the current EIA method.

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DOI

10.25777/yf2q-dn26

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