ABSTRACT
We describe a new one-step enzyme immunoassay of troponin T that uses two specific monoclonal antibodies and streptavidin-coated tubes as the solid phase. The monoclonal antibodies were obtained by conventional hybridoma technology, with human troponin T as antigen. The identity of the cardiac troponin T antigen was confirmed by analysis of the amino acid composition and by partial sequence analysis. The specificity of the monoclonal antibodies for cardiac troponin T was proved by immunoblot analysis and displacement curves. The capture antibody is labeled with biotin. The second antibody is conjugated to horseradish peroxidase (EC 1.11.1.7). The assay [Enzymun-TestR system (Boehringer Mannheim GmbH)] is performed in only 90 min at room temperature; the measuring range for troponin T is 0.1 to 15 micrograms/L. The assay shows excellent between-run precision (CV = 3.3-4.9%).
Subject(s)
Immunoenzyme Techniques , Muscles/chemistry , Myocardium/chemistry , Troponin/analysis , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Bacterial Proteins , Cattle , Humans , Immunoenzyme Techniques/standards , Immunoenzyme Techniques/statistics & numerical data , Molecular Sequence Data , Streptavidin , Troponin/blood , Troponin/chemistry , Troponin TABSTRACT
For the diagnosis of acute myocardial infarction (AMI) in patients circulating constituents of the contractile apparatus may be measured instead of cytosolic cardiac enzymes. The potential advantages of the use of myofibrillar cardiac proteins as marker proteins for AMI results from their expression as cardio-specific isoforms, their high intracellular concentration, and their continuous release from infarcting myocardium. While analyzing the specificity of polyclonal goat anti-human cardiac myosin light chains antisera a cardio-specific antibody fraction was identified which is directed against cardiac troponin T contaminations of the myosin light chains antigen. Using this antibody fraction a standardized enzyme immuno-assay for circulating troponin T was developed to detect AMI in patients. In this assay troponin T is bound on different epitopes by affinity purified goat anti-cardiac troponin T antibodies immobilized on polyvinyl chloride test tubes as well as horse raddish peroxidase labeled monoclonal anti-troponin T antibody in liquid phase. The assay procedure can be completed semiautomatically in 90 min with a detection limit of the assay of 0.5 ng/ml human or bovine cardiac troponin T. There is 1% crossreactivity with skeletal troponin T. In 26 healthy volunteers no cardiac troponin T was detectable in serum of 25 persons, while in 1 further volunteer 1 ng/ml troponin T was found. In the sera of all 50 patients with transmural AMI troponin T was elevated ranging from 7.2 to 110 ng/ml. In the mean troponin T remained elevated from three until 300 hours after onset of ischemic pain showing a biphasic serum concentration curve.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Enzyme-Linked Immunosorbent Assay , Myocardial Infarction/diagnosis , Myocardium/metabolism , Troponin/blood , Humans , Middle Aged , Time Factors , Troponin TABSTRACT
We report the results obtained with a modification of Hillmann's method (1) for determination of the catalytic concentration of acid phosphatase in serum using alpha-naphthylphosphate as the substrate. In a group of 158 males aged 16-85 years, the upper limit of the reference range (95th percentile) for the total acid phosphatase was established as 4.7 U/l (37 degrees C) and 4.2 U/l (30 degrees C), respectively; the corresponding values for the tartrate-inhibited acid phosphatase were 1.6 U/l (37 degrees C) and 1.5 U/l (30 degrees C). The upper limit of the reference range (95th percentile) for the total acid phosphatase determined in 60 females aged 18-80 years was 3.7 U/l (37 degrees C) and 3.0 U/l (30 degrees C). The catalytic concentrations in men and women did not show any age-related differences. The catalytic concentration of the tartrate-inhibited acid phosphatase was determined with the substrates alpha-naphthylphosphate and p-nitrophenylphosphate in a group of 89 patients with prostatic carcinoma (stages C and D). In 74 of these patients, the concentration of the prostatic specific acid phosphatase was assayed by enzyme-immunoassay and radioimmunoassay. The sensitivity of the method with p-nitrophenylphosphate was found to be unsatisfactory (66%), while that obtained with the other methods was superior and intercomparable (approx. 90%). The results obtained with the two substrates (p-nitrophenylphosphate vs. alpha-naphthylphosphate) differed significantly.(ABSTRACT TRUNCATED AT 250 WORDS)
