Immunoenzymometric assay for a small molecule,11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies.
To overcome the sensitivity limit in immunoassays for small molecules (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten molecules. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chemically linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amounts of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse beta-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized alpha-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels determined for healthy subjects were validated as reliable.
Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Kobe, 658-8558, Japan.
Norihiro
N
Kobayashi
Keiichi
K
Iwakami
Shuhei
S
Kotoshiba
Toshifumi
T
Niwa
Yoshinori
Y
Kato
Nariyasu
N
Mano
Junichi
J
Goto
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D003350:Cortodoxone / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000032:analysis* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010913:Pituitary-Adrenal System / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000888:Antibodies, Anti-Idiotypic; D006241:Haptens; D007135:Immunoglobulin Variable Region; D003350:Cortodoxone