Development of Monoclonal Antibodies for an Assay of Cardiac Troponin-I and Preliminary Results in Suspected Cases of Myocardial Infarction

Abstract

To improve the specificity of biochemical markers of myocardial infarction (MI), we have developed a double monoclonal "sandwich" enzyme immunoassay to measure cardiac troponin-I (cTnI) in serum. We produced eight IgG monoclonal antibodies against human cardiac troponin-I (cTnI) and tested them against human and animal (canine, bovine, and rabbit) troponins. Five antibodies were cardiac-specific; none of the antibodies were species-specific. Two of the five cTnI-specific monoclonal antibodies were utilized in an immunoassay. Standards were made by adding purified human cTnI to affinity-stripped cTnI-free human sera to cover the range 0-100 micrograms/L for cTnI. The dose-response curve was nonlinear but reproducible. Total assay imprecision (CV) varied between 11% and 21%. The upper limit of the reference range (nonparametric 95% interval) was established as 3.1 micrograms/L by measuring cTnI concentration in sera of 159 hospitalized patients without evidence of cardiac disease. Purified human skeletal TnI up to 10,000 micrograms/L did not affect the assay (calculated cross-reactivity < 0.1%). Diagnostic sensitivities of creatine kinase MB isoenzyme (CK-MB) and cTnI were evaluated retrospectively in 49 consecutive patients with proven MI. In the 30 patients for whom sufficient information was available to establish an accurate time course, CK-MB was more sensitive during the first 4 h after the onset of chest pain, but thereafter the sensitivities were similar up to 48 h. However, cTnI is more cardiac-specific than is CK-MB and remains increased longer than does CK-MB.

Validation of a Commercially Available Immunoassay for the Measurement of Bovine Cardiac Troponin I

“…Cardiac TnI can occur in circulation after myocardial injury as free, binary (complex of cTnI with cTnC), as well as ternary forms (complex of cTnI with cTnC and cTnT). 29,30 The troponin complex released after injury undergoes rapid posttranslational modification. The type of myocardial insult determines which forms of circulating cTnI dominate.…”

“…The rate of degradation of cardiac troponin depends on different factors such as size of the cTnI fragments released and their complex formation with other troponin subunits. 29,30 In humans, free cTnI has a lower stability than the binary or ternary forms. The N-terminal as well as C-terminal regions of troponin I are rapidly cleaved during proteolysis.…”

Cardiac Troponin I in Feline Hypertrophic Cardiomyopathy

“…13 Increased circulating concentrations of both troponins are sensitive and specific markers for myocardial damage. [10][11][12][14][15][16] Measurement of circulating cTnI may be a more sensitive means of detecting myocardial injury in humans than measurement of circulating cTnT. [17][18][19] The molecular structure of troponin proteins is highly conserved across species, and current assays developed for their detection in humans have been validated in several other species.…”

“…[17][18][19] The molecular structure of troponin proteins is highly conserved across species, and current assays developed for their detection in humans have been validated in several other species. 15,16,[20][21][22] In veterinary medicine, reports evaluating cardiac troponins relevant to clinical medicine are limited to a few studies. [23][24][25][26][27][28][29][30] Measurement of circulating cTnI concentration ([cTnI]) is a more sensitive means of detecting myocardial cell injury than measurement of either plasma [cTnT] or serum creatine kinase isoenzyme MB (CK-MB) concentration in dogs and cats after blunt chest trauma.…”

Temperature‐Dependent Postmortem Changes in Human Cardiac Troponin‐T (cTnT): An Approach in Estimation of Time Since Death

“…Cardiac troponin-T is a 37-KD protein that is 10-30% dissimilar from skeletal troponin-T isoforms (4). Different groups have developed monoclonal antibodies specific for cTnT by exploiting residues that are unique to the cardiac isoforms (5,6). Proteases such as calpains, cathepsins, and serine proteases have showed active role in degradation of cTnT (7,8).…”