A sensitive sandwich enzyme immunoassay for human myoglobin using Fab'-horseradish peroxidase conjugate: Methods and results in normal subjects and patients with various diseases

Abstract

A sensitive sandwich enzyme immunoassay (EIA) for human myoglobin (Mb) was developed. Serum Mb was assayed by incubation with an anti-Mb rabbit IgG-coated polystyrene ball and then with affinity-purified anti-Mb Fab'-horseradish peroxidase (HRP) conjugate. The HRP activity bound to the polystyrene ball was assayed fluorimetrically. The sensitivity was 3.1 pg/tube and serum Mb levels of 0.4–1000 μg/l could be determined. The recoveries of Mb added to human sera at 3 concentrations were 92.8–99.8%. The within- and between-assay coefficients of variations (CV values) were both below 10%. The regression equation of values determined by EIA and radioimmunoassay (RIA) was y( EIA) = 0.964 x( RIA) − 2.30 and the correlation coefficient was 0.984. The mean normal serum concentration of Mb was 21.5 ± 6.0 μg/l (mean ± sd) in males and 16.9 ± 5.8 μg/l in females. The significance of serum Mb determination by the EIA in various diseases was confirmed. The present EIA for Mb is more sensitive and economical than RIA and should be useful for determining Mb in biological materials.