Date of Award

Summer 1989

Document Type


Degree Name

Master of Science (MS)


Chemistry & Biochemistry



Committee Director

James H. Yuan

Committee Member

Patricia A. Pleban

Committee Member

Mark S. Elliott

Call Number for Print

Special Collections LD4331.C45Y36


A microwell based competitive enzyme immunoassay for determination of total thyroxine and triiodothyronine in human serum was developed with use of horseradish peroxidase conjugate. The assay, which can be performed in only 1.5 hours at room temperature, involves simultaneous incubation of T3- or T4- HRPO conjugate and serum sample in anti-T3 or anti-T4 gamma globulin fraction coated polystyrene microwells.

Isolation of anti-T3 and anti-T4 gamma globulin fractions were accomplished from whole antiserum by using DEAE-cellulose chromatograph. A carbodiimide coupling method was described to prepare the T4- and T3-HRPO conjugates. Binding of hormones to serum protein was minimised using 8-anilino-1-naphthalene sulfonic acid.

The detection limit of the T3 assay was 15.0 ng/dL and that of T4 was 0.8 ug/dL. Precision studies involving high and low levels of T3 and T4 were performed. Intra-assay coefficients of variation were 12.6% and 5.78% for T3 concentrations at 80.82 ng/dL and 207.9 ng/dL, and 8.51% and 11.1% for T4 concentrations at 14.4 ug/dL and 6.90 ug/dL, respectively. The inter-assay coefficients of variation of 15.3% and 5.91% were obtained for T3 concentrations at 85.87 ng/dL and 366.9 ng/dL, and 9.44% and 8.39% were obtained for T4 concentrations at 6.86 ug/dL and 15.66 ug/dL. Values for total T3 and total T4 so determined correlated well with those determined by radioimmunoassay with correlation coefficient of 0.976 and 0.971 respectively.


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