Dual-label time-resolved fluoroimmunoassay for simultaneous detection of myoglobin and carbonic anhydrase III in serum

Abstract

We developed a dual-labeled time-resolved fluoroimmunoassay for simultaneous quantification of myoglobin (Mb) and carbonic anhydrase III (CA III) in serum involving polyclonal antibodies and the fluorescent lanthanides europium (Eu3+) and samarium (Sm3+). This solid-phase immunoassay is based on competition between Eu(3+)- or Sm(3+)-labeled antigen and the sample antigen for polyclonal rabbit antibodies. Standards and patients' samples containing antigen inhibit binding of the lanthanide-labeled antigen to the antibody. A second antibody directed against rabbit IgG is coated on a solid phase and binds the IgG-antigen-lanthanide complex, giving rapid and complete separation of antibody-bound and free antigen. The assay requires only one incubation step. An enhancement solution dissociates Eu3+ and Sm3+ ions from the labeled CA III and Mb, respectively, into a solution where they form highly fluorescent chelates. Spectra of the fluorescent chelates in the microtitration-strip wells were run on a time-resolved fluorometer equipped with filters for Eu3+ (613 nm) and Sm3+ (643 nm), the fluorescence from each sample being inversely proportional to the concentration of antigens. The measurement range for both analytes is from 5 to 1500 micrograms/L. The mean within- and between-assay precisions (CV) were 4.6% and 6.2% for CA III and 5.9% and 7.3% for Mb, respectively. Good correlations were obtained with the results of CA III RIA and a commercial myoglobin RIA kit.