Simultaneous quadruple-label fluorometric immunoassay of thyroid-stimulating hormone, 17 alpha-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM isoenzyme in dried blood spots.
We describe a quadruple-label fluorometric immunoassay for simultaneously measuring four analytes: thyroid-stimulating hormone (TSH), 17 alpha-hydroxyprogesterone (17 alpha-OHP), immunoreactive trypsin (IRT), and creatine kinase MM (CK-MM). The assay is based on immunoreagents labeled with four different lanthanide ions (Eu3+, Tb3+, Sm3+, and Dy3+), on dissociative fluorescence enhancement applying the principle of co-fluorescence, and on time-resolved fluorometry. The monoclonal anti-alpha-TSH and anti-IRT antibodies and the polyclonal anti-CK-MM antibody were labeled with Eu3+, Sm3+, and Dy3+, respectively; 17 alpha-OHP was labeled with Tb3+. The assay was performed in microtitration strip wells coated with a mixture of monoclonal antibodies against beta-TSH, IRT, and CK-MM and a polyclonal goat anti-rabbit IgG for capture of the rabbit anti-17 alpha-OHP antibodies. After completion of the immunoreactions, the bound fractions of the lanthanides were dissociated into the co-fluorescence enhancement solution, creating highly fluorescent chelates. The four lanthanide-specific signals were subsequently measured in a time-resolved fluorometer. The detection limits of the assay were 0.1 mIU/L for TSH, 2 nmol/L for 17 alpha-OHP, 2 micrograms/L for IRT, and 4 U/L for CK-MM.
Xu
Y Y
YY
Department of Biochemistry, University of Turku, Finland.
Pettersson
K
K
Blomberg
K
K
Hemmilä
I
I
Mikola
H
H
Lövgren
T
T
D019326:17-alpha-Hydroxyprogesterone; D000312:Adrenal Hyperplasia, Congenital / Q000175:diagnosis; D000911:Antibodies, Monoclonal; D003402:Creatine Kinase / Q000097:blood*; D003550:Cystic Fibrosis / Q000175:diagnosis; D004419:Dysprosium; D005063:Europium; D015200:Fluoroimmunoassay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D006801:Humans; D006908:Hydroxyprogesterones / Q000097:blood*; D007037:Hypothyroidism / Q000175:diagnosis; D007202:Indicators and Reagents; D007231:Infant, Newborn; D007527:Isoenzymes; D009136:Muscular Dystrophies / Q000175:diagnosis; D015997:Neonatal Screening / Q000379:methods; D012493:Samarium; D013725:Terbium; D013972:Thyrotropin / Q000097:blood*; D014357:Trypsin / Q000097:blood*
D000911:Antibodies, Monoclonal; D006908:Hydroxyprogesterones; D007202:Indicators and Reagents; D007527:Isoenzymes; D013725:Terbium; D004419:Dysprosium; D012493:Samarium; D005063:Europium; D019326:17-alpha-Hydroxyprogesterone; D013972:Thyrotropin; D003402:Creatine Kinase; D014357:Trypsin