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Non-competitive immunochemiluminometric assay for cardiotrophin-1 detects elevated plasma levels in human heart
Leong L Ng1, Russell J O'Brien, Bettina Demme
1Department of Medicine & Therapeutics, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, U.K. lln1@le.ac.uk
Insights
A new non-competitive assay for Cardiotrophin-1 (CT-1) allows direct measurement in plasma, simplifying detection of elevated CT-1 levels in heart failure patients.
Area of Science:
- Biochemistry
- Immunology
- Cardiology
Background:
- Cardiotrophin-1 (CT-1) is implicated in ventricular hypertrophy and elevated in heart failure.
- Existing competitive assays for CT-1 are complex, requiring plasma extraction and lengthy incubations.
Purpose of the Study:
- To develop and validate a novel non-competitive assay for measuring plasma Cardiotrophin-1 (CT-1) levels.
- To assess the utility of this assay in distinguishing heart failure patients from normal controls.
Main Methods:
- Development of two rabbit-generated antibodies targeting distinct CT-1 regions (mid-section and C-terminal).
- Establishment of a non-competitive immunochemiluminometric assay using immobilized capture antibody and biotinylated detection antibody.
- Assay validation using recombinant CT-1 and measurement in plasma from heart failure patients and healthy controls.
Main Results:
- The assay demonstrated linear chemiluminescence with increasing recombinant CT-1 concentrations, with a low limit of detection (2.9 fmol/ml).
- High assay precision was confirmed with low intra-assay (3.1-4.2%) and inter-assay (3.5-4.5%) coefficients of variation.
- Significantly elevated plasma CT-1 levels were observed in heart failure patients (median 166.5 fmol/ml) compared to controls (median 43.5 fmol/ml; P<0.0001).
- The assay achieved 95% sensitivity and 82.5% specificity for heart failure detection at 68 fmol/ml CT-1.
Conclusions:
- A novel non-competitive immunochemiluminometric assay effectively measures full-length CT-1 in unextracted human plasma.
- This assay confirms significantly elevated plasma CT-1 levels in patients with heart failure.
- The developed assay offers a simplified and sensitive method for assessing CT-1 in clinical settings.
Abstract:
Cardiotrophin-1 (CT-1) leads to a specific form of ventricular hypertrophy characterized by sarcomeres added in series, and has been reported to be elevated in heart failure. Previous competitive assays for CT-1 necessitate the extraction of plasma and involve prolonged incubations. We describe the development of a non-competitive assay for CT-1 that can measure plasma levels without the need for extraction. Two antibodies specific for the mid-section (amino acids 105-120) and C-terminal (amino acids 186-199) portions of CT-1 were developed in rabbits. One antibody was immobilized and used as the capture antibody. The other antibody was affinity purified and biotinylated. Unextracted plasma was incubated with these antibodies, and detection was with methylacridinium ester-labelled streptavidin. Plasma was obtained from 40 patients with heart failure and 40 normal control subjects. The non-competitive assay demonstrated a linear increase in chemiluminescence (measured as relative light units) with increasing amounts of full-length recombinant CT-1, with no evidence of a hook effect at high concentrations. The lower limit of detection was 2.9 fmol/ml. Intra-assay coefficients of variation ranged from 3.1% to 4.2% in the 10-40 fmol/well concentration range, and interassay coefficients of variation ranged from 3.5% to 4.5% in the 550-950 fmol/ml range. Measurements of CT-1 levels in patients with heart failure (median 166.5 fmol/ml; range 49.5-2788 fmol/ml) revealed very significantly elevated levels compared with those in normal controls (median 43.5 fmol/ml; range 11.2-258.6 fmol/ml; P<0.0001 by Mann-Whitney test). At a CT-1 concentration of 68 fmol/ml, sensitivity and specificity were 95% and 82.5% respectively. Thus this new non-competitive immunochemiluminometric assay for CT-1 could successfully detect full-length recombinant CT-1 in unextracted plasma, and demonstrated that plasma levels of CT-1 were significantly elevated in patients with heart failure.