Measurement of serum ferritin by a 2-site immunoradiometric assay

Abstract

A 2-site immunoradiometric assay (2-site IRMA) for human serum ferritin is carried out by reaction of the ferritin solution with a solid-phase anti(human ferritin), followed by a second reaction in which the insoluble product is incubated with purified, radioactively labeled anti(human ferritin). Unreacted labeled antibodies remain in solution and are washed away. As the amount of ferritin increases, the radioactivity in the solid-phase increases. Factors affecting the assay were evaluated including (a) concentration and stability of solid-phase antibody, (b) variation in temperature, reaction times, and reagent concentrations, (c) stability and storage of antibody coated tubes, (d) effect of tube washing cycles, (e) effect of serum proteins and anticoagulants, (f) organ specificity of ferritin. A paradoxical fall in dose-response was seen at high dose. Statistical analysis, dose interpolation, and automatic data processing were carried out by a generalization of the logit/log method and computer programs used in conventional radioimmunoassay. The dependence of the variance on the position on the dose-response curve is different than that seen in radioimmunoassay systems and is reflected in a greater effective assay range. 2-Site IRMA may also have advantages in reagent stability, specificity, antigen protection, and suitability for automation. The properties of 2-site IRMA are closely related to the usual IRMA assay system, but 2-site IRMA is more economical in antigen, is unlikely to be subject to deleterious allosteric reactions, and has a lower zero dose-response (0.5–2% of the total radioactivity in the system).