Enzyme-Linked Immunosorbent Assay (ELISA) Determination of Skeletal Troponin I (TNI) in Human Serum as Evidence of Muscle Damage

• Other Authors: Fredericks, William (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Food
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-4401

Skeletal muscle damage is a common occurrence and can lead to severe complications. Early diagnosis of skeletal muscle damage is a leading factor in successful treatment and recovery. The ability to rapidly, accurately, and non-invasively diagnose muscle damage is predicated on the ability to detect various biomarkers in blood. Creatine kinase (CK) is a biomarker commonly used for this purpose, but limitations exist such as tissue specificity. Troponin I (TnI), a contractile protein in muscle, is a potential alternative. Enzyme-linked immunosorbent assay (ELISA), a technique used to determine the presence of a specific protein based on an antibody (Ab)-antigen (Ag) interaction, has been successfully applied in the detection of cardiac TnI (cTnI) to diagnose myocardial infarction (AMI). Attempts by other investigators to develop an ELISA assay for detecting skeletal muscle damage using skeletal troponin I (sTnI) have been unsuccessful because of cross-reactivity of the antibodies used with cTnI. The goal of this study was to initiate development of an ELISA for sTnI by testing four mammalian monoclonal antibodies (MAbs - 2G3, 8A12, 1F9, and 8F10) previously used for food science purposes and not for detecting skeletal muscle damage. These four MAbs were first screened for the ability to detect human sTnI by testing them against a protein extract from human skeletal muscle; three of the MAbs (2G3, 8A12, 1F9) reacted positively. Secondly, these three MAbs were then tested for cross-reactivity to cTnI with a beef heart protein extract, and only one of the three MAbs (1F9) was eliminated. Thirdly, limit of detection (LOD) was approximated for the MAbs 2G3 and 8A12; the approximate LODs were calculated to be 14 ng/ml and 35 ng/ml, respectively. Finally, the MAbs 2G3 and 8A12 were tested to see if sTnI could be detected in serum samples from human subjects with evidence of skeletal muscle damage based on CK values, but the results were inconclusive. In summary, the MAbs 2G3 and 8A12 are specific for human sTnI, but experimental conditions need to be further optimized to determine whether these two MAbs can detect sTnI in human serum and therefore be used to develop an ELISA-based assay for diagnosing skeletal muscle damage. === A Thesis submitted to the Department of Nutrition, Food, and Exercise Sciences in partial fulfillment of the requirements for the degree of Master of Science. === Spring Semester, 2009. === January 28, 2009. === Muscle damage, Troponin I === Includes bibliographical references. === Jodee Dorsey, Professor Directing Dissertation; Susan Lynn, Committee Member; Cathy Levenson, Committee Member; Yun-Hwa Hsieh, Committee Member.