TY - JOUR
T1 - Liquid chromatography coupled on-line to flow cytometry for postcolumn homogeneous biochemical detection
AU - Schenk, T.
AU - Molendijk, A.
AU - Irth, H.
AU - Tjaden, U.R.
AU - de Greef, J
PY - 2003
Y1 - 2003
N2 - The feasibility of flow cytometry as read-out principle for homogeneous cell- or bead-based assays coupled on-line to LC is demonstrated using digoxin-coated beads (Dig-Beads) and fluorescent-labeled anti-digoxin (AD-FITC) as model system. The assay is carried out in a postcolumn continuous-flow reaction detection system where the AD-FITC and Dig-Beads are simultaneously added to the eluate of an LC separation column. Binding of AD-FITC to Dig-Beads results in a constant amount of fluorescence associated with the beads, which is detected by the flow cytometer. The presence of active compounds, such as digoxin and its analogues, in the sample will results in a decrease of the AD-FITC-Dig-Bead complex and, consequently, in the bead-associated fluorescence. Hence, the bead-associated fluorescence detected is inversely related to the digoxin concentration. A data-handling algorithm was developed in-house for adequate analysis of raw data output from the flow cytometer. Various conditions that influence the performance of this novel LC-biochemical detection (LC-BCD) system were investigated to determine the optimal settings of the bead-based biochemical interaction. The optimized flow injection bead-based assay was capable of detecting very low concentrations of digoxigenin (0.5 nmol/L), digoxin (0.1 nmol/L), and gitoxigenin (50 nmol/L). The applicability of LC coupled on-line to flow cytometry was demonstrated by the individual detection of digoxin, digoxigenin, and gitoxigenin in a single LC analysis. The successful coupling of LC on-line to flow cytometry principally enables the use of a wide range of new homogeneous assay formats in LC-BCD, such as membrane-bound receptor assays, cell-binding assays, and functional cell-based assays. Next to the ability to use insoluble targets, and also multiplexing assays, i.e., performing a number of assays simultaneously, using color- or size-coded beads becomes at hand in LC-BCD.
AB - The feasibility of flow cytometry as read-out principle for homogeneous cell- or bead-based assays coupled on-line to LC is demonstrated using digoxin-coated beads (Dig-Beads) and fluorescent-labeled anti-digoxin (AD-FITC) as model system. The assay is carried out in a postcolumn continuous-flow reaction detection system where the AD-FITC and Dig-Beads are simultaneously added to the eluate of an LC separation column. Binding of AD-FITC to Dig-Beads results in a constant amount of fluorescence associated with the beads, which is detected by the flow cytometer. The presence of active compounds, such as digoxin and its analogues, in the sample will results in a decrease of the AD-FITC-Dig-Bead complex and, consequently, in the bead-associated fluorescence. Hence, the bead-associated fluorescence detected is inversely related to the digoxin concentration. A data-handling algorithm was developed in-house for adequate analysis of raw data output from the flow cytometer. Various conditions that influence the performance of this novel LC-biochemical detection (LC-BCD) system were investigated to determine the optimal settings of the bead-based biochemical interaction. The optimized flow injection bead-based assay was capable of detecting very low concentrations of digoxigenin (0.5 nmol/L), digoxin (0.1 nmol/L), and gitoxigenin (50 nmol/L). The applicability of LC coupled on-line to flow cytometry was demonstrated by the individual detection of digoxin, digoxigenin, and gitoxigenin in a single LC analysis. The successful coupling of LC on-line to flow cytometry principally enables the use of a wide range of new homogeneous assay formats in LC-BCD, such as membrane-bound receptor assays, cell-binding assays, and functional cell-based assays. Next to the ability to use insoluble targets, and also multiplexing assays, i.e., performing a number of assays simultaneously, using color- or size-coded beads becomes at hand in LC-BCD.
UR - https://www.scopus.com/pages/publications/0042476371
UR - https://www.scopus.com/inward/citedby.url?scp=0042476371&partnerID=8YFLogxK
U2 - 10.1021/ac0341822
DO - 10.1021/ac0341822
M3 - Article
SN - 0003-2700
VL - 75
SP - 4272
EP - 4278
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 16
ER -
