Sandwich enzyme immunoassay for rat retinol-binding protein using antibody against recombinant antigen and its application.
The development of a sandwich enzyme immunoassay for rat retinol-binding protein using molecular biological techniques was described. Rat retinol-binding protein gene cloned by the PCR method was expressed by a fusion vector pEZZ18 in Escherichia coli strain HB101. A recombinant retinol-binding protein fused with IgG-binding domain ZZ of protein A was purified with IgG-Sepharose. Antibody against the recombinant protein was found to be specific to rat retinol-binding protein in plasma by immunoblot analysis. Affinity-purified anti-recombinant protein IgG was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range is 1.9-60 ng/ml and the coefficients of variation within and between the assay series (assay range: 4-30 ng/ml) are 4.30 +/- 4.33 and 5.32 +/- 1.45%, respectively. Cross-reactivity of the immunoassay was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse serum. In an in vitro experiment, retinol-binding protein produced 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
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D001710:Biotin; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D012177:Retinol-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D054839:Retinol-Binding Proteins, Plasma; D013205:Staphylococcal Protein A
D000906:Antibodies; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma; D013205:Staphylococcal Protein A; D001710:Biotin; D004247:DNA