Date of Award

Summer 1996

Document Type

Thesis

Degree Name

Master of Arts (MA)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

James H. Yuan

Committee Member

Roy L. Williams

Committee Member

Mark S. Elliot

Abstract

Nondiagnostic ECGs and unknown characteristic chest pains increase the diIIiculty of Acute Myocardial Infarction (AMI) diagnosis. Biochemical markers, such as creatine kinase MB isoenzymes (CK-MB) and myoglobin, are employed for the early detection of AMI. Compared to myoglobin, CK-MB reveals a higher specific indicator for AMI detection owing to its abundance in cardiac tissue. Hence, a successful CK-MB detection is expected to improve AMI diagnosis and therapy.

A CK-MB detecting ELISA system was set up based on a sandwich assay principle by using specific CK-B and CK-M monoclonal antibodies. The dimeric nature of the CK-MB isoenzyme provides two epitopes on CK-B and CK-M subunits for antibody binding in the system. One anti-CK-BB hybridoma clone (B IDIO) and three anti-CK-MM hybridoma clones (M1D6, Ml1G9, and M2B7) were obtained by the hybridoma technique.

Using Immulon I microwells, optimal performance of the CK-MB detecting ELISA system was achieved by using CK-B antibody as the solid phase immobilized antibody, CK-M antibody as the second antibody conjugated with horseradish peroxidase, glutathione and BSA containing PBS buffer as the antigen incubation buffer, and BSA containing PBS buffer as the enzyme conjugate incubation buffer. A 12.3o/o CV for within-batch operation was determined for the system. A narrow CKMB detection range, 7.3-15.0 ng.mL, was found in the current system, and this may be due to the deterioration of the purified CK-MB or the low affinity of the antibody. Furthermore, because 4.8o/o of cross-reactivity was observed with CK-MM, clinical validation was not performed in this study. Thus, this CK-MB detecting ELISA is successful in CK-MB detection but is not useful for AMI diagnostic detection.

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DOI

10.25777/fxdr-n381

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