Rapid Homogeneous Immunoassay for Human Ferritin in the Cobas Mira Using Colloidal Gold as the Reporter Reagent

Abstract

Serum ferritin concentrations, with some exceptions (1)(2)(3), reflect iron stores (4)(5). Ferritin assays must have a broad dynamic range because the serum concentrations can be 1 mg/L in some types of malignancies (7). Radio- and enzyme immunoassays have been used routinely (8), but rapid, automated latex agglutination immunoassays have been developed and validated (9)(10)(11). These methods have drawbacks (12), of which the disturbance of colloidal stability by nonspecific bridging processes particularly should be avoided (13). In our quest for new reporter reagents (14)(15), our attention was drawn to colloidal gold as a potential substitute for latex in particle-enhanced agglutination immunoassays. Colloidal gold was used by Leuvering et al. (16) in sol particle immunoassays (SPIAs) for several serum or urine analytes. Unfortunately, the technique was prone to interference when undiluted serum samples were used (17). Recently, we showed (18)(19) that the change in visible absorbance at 600 nm ( A 600) observed when colloidal gold particles coated with an antibody interact with the antigen results not only from agglutination but also from subtle changes in the refractive index at the particle surface [surface plasmon resonance effect (SPR)]. Thus, the unidentified random interferences noted by Leuvering et al. (16) could result from interactions between nonspecific reacting sites on the antibody molecule (distinct from the binding site) and several serum components (distinct from the analyte). According to this model, these interactions were transduced as a photometric signal by the SPR effect of gold (19)(20), which adds to the signal produced by the agglutination. With this in mind, we optimized the buffer to be used in the SPIA with colloidal gold and carefully selected the antibodies to be used for their lack of SPR …