Construction of Sandwich Enzyme Immunoassay for Rat Transthyretin Using Recombinant Antigen Approach, and Its Application

Abstract

A sandwich enzyme immunoassay for rat transthyretin using a recombinant antigen approach is described. Rat transthyretin cDNA was cloned by PCR. Rat transthyretin fused with the IgG-binding ZZ domain of protein A was expressed in E. coli using the fusion vector pEZZ18. Antibody against the fusion protein was specific to rat transthyretin in plasma as shown by immunoblotting. Affinity-purified anti-rat transthyretin-ZZ was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range was 1.2–50 ng/ml and the coefficients of variation within and between the assay series (assay range: 2.5–20 ng/ml) were 1.31 ± 0.08% and 3.50 ± 1.46%, respectively. Cross-reactivity was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse transthyretin. In an in vitro experiment, transthyretin secreted by rat hepatocytes could be measured by the sandwich enzyme immunoassay.