Construction of sandwich enzyme immunoassay for rat transthyretin using recombinant antigen approach, and its application.
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.
Matsumoto
T
T
Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Osaka, Japan.
Morita
M
M
Shirai
H
H
Kishi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003429:Cross Reactions; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007124:Immunoenzyme Techniques; D008099:Liver / Q000166:cytology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011228:Prealbumin / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D004277:DNA, Single-Stranded; D011228:Prealbumin; D011993:Recombinant Fusion Proteins