Non-competitive immunochemiluminometric assay for cardiotrophin-1 detects elevated plasma levels in human heart failure.
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.
Ng
Leong L
LL
Department of Medicine & Therapeutics, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, U.K. lln1@le.ac.uk
O'Brien
Russell J
RJ
Demme
Bettina
B
Jennings
Sonja
S
D015153:Blotting, Western; D016207:Cytokines / Q000097:blood* / Q000276:immunology; D005260:Female; D006333:Heart Failure / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000097:blood; D012680:Sensitivity and Specificity
D016207:Cytokines; D011994:Recombinant Proteins; C092073:cardiotrophin 1