Cardiac troponin T immunoassay on biotin-streptavidin-coated microplates: preliminary performance in acute myocardial infarction

Abstract

We have adapted the cardiac troponin T (TnT) immunoassay system (ELISA troponin T, Boehringer Mannheim, Germany), which is based upon streptavidinbiotin immunoassay technology, to a sensitive microplate system. A coating of microplates with biotinylated bovine serum albumin (biotin-LC-BSA) remained stable for months. A secondary streptavidin coating was prepared as the first step of the assay. By using o-phenylenediamine (o-PD) as a substrate for the peroxidase-anti-TnT conjugate, the system allowed rapid kinetic measurement of TnT levels. The upper limit of a reference population (97.5th percentile) was found to be 0.04 μg 1⁻¹. Intra-assay imprecision at 0.08 μg 1⁻¹ was 8%, and 3–4% between 0.28 and 4 μg 1⁻¹. Between-assay imprecision was 6.2% at 0.28 μg 1⁻¹. Studies of TnT and CK-MB mass concentration in acute myocardial infarction patients, treated with streptokinase, demonstrated a clinical sensitivity of the TnT micro-plate system similar to that of the CK-MB mass concentration test, during the first 8 h after initiation of thrombolytic therapy, at discriminator levels of 0.1 μg 1⁻¹ (TnT) and 8 μg 1⁻¹ (CK-MB mass concentration). The early CK-MB/TnT ratio was lower in patients with signs of successful reperfusion (early peak CK-MB) than in the remaining patients (p<0.001). Serum samples from two patients with renal failure and one patient with rhabdomyolysis demonstrated strong non-linear behaviour with dilution, indicating the presence of an interfering factor. Kinetic measurement compared favourably with end-point analysis with respect to sensitivity and total analysis time. The system described greatly reduces the costs of TnT measurements compared to the ES systems. The total assay time is 70–90 min.

Key Words:

• CK-MB mass
• ELISA
• reperfusion
• serum cardiac marker
• thrombolytic therapy
• unstable angina pectoris