Potentiometric enzyme immunoassay using miniaturized anion-selective electrodes for detection.
An enzyme-linked immunosorbent assay (ELISA) for prostate specific antigen (PSA) detection in human serum was developed based on the potentiometric detection of 6,8-difluoro-4-methylumbelliferone (DiFMU). The assays were carried out in anti-human PSA capture antibody modified microtiter plates (150 microL volume). After incubation in the PSA-containing serum samples, beta-galactosidase-labeled PSA tracer antibody was added. The beta-galactosidase label catalyzed the hydrolysis of 6,8-difluoro-4-methylumbelliferyl-beta-D-galactopyranoside (DiFMUG) and the resulting DiFMU(-) anion was detected by potentiometric microelectrodes with anion-exchanger membrane. The selectivity of the anion-exchanger electrode is governed by the lipophilicity of the anions in the sample. Since DiFMU(-) is much more lipophilic (log P = 1.83) than any of the inorganic anions normally present in the working buffers and occurs in its anionic form at the physiological pH (pK(a) = 4.19), it was chosen as the species to be detected. The potentiometric ELISA-based method detects PSA in serum with a linear concentration range of 0.1-50 ng/mL. These results confirm the applicability of potentiometric detection in diagnostic PSA assays. Owing to simple methodology and low cost, potentiometric immunoassays seem to offer a feasible alternative to the development of in vitro diagnostic platforms.
Szucs
Júlia
J
Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szt. Gellért tér 4, H-1111 Budapest, Hungary.
Pretsch
Ernö
E
Gyurcsányi
Róbert E
RE
D000838:Anions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007118:Immunoassay / Q000379:methods; D008297:Male; D011199:Potentiometry / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000032:analysis*; D001616:beta-Galactosidase / Q000378:metabolism
C504401:6,8-difluoro-4-methylumbelliferyl sulfate; D000838:Anions; D005697:Galactosides; D006923:Hymecromone; C026845:4-methylumbelliferyl-galactopyranoside; D001616:beta-Galactosidase; D017430:Prostate-Specific Antigen
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