for HTS can read a 96-well plate in 10 min. The schematic diagram of Origen Flow system is given in Fig. 16C.
Four derivatives of ruthenium chelate, TAG-amine, TAG-hydrazide, TAG-malei-mide, and TAG-phosphoramidite, are available for convenient covalent linking to biomolecules. ECL can be used for large- and small-molecule immunoassays such as for hormones, second messengers, drugs ; for enzyme-substrate interaction; for receptor-ligand interactions; and for DNA-DNA, DNA-protein, and protein-protein interactions , quantification of nucleic acids , and PCR products .
PTK Assay. In ECL PTK assay, the peptide substrate is end labeled with biotin, and the phosphotyrosine (PY) antibody is labeled with ruthenium (II) tris-(bipyridine) NHS ester (Fig. 17). The biotinylated peptide is incubated with ATP, Mg2+, PTK, ruthenyl PY antibody, and streptavidin coated magnetic beads (M280 Dynabeads) in 100 |L for 30 min; a 200 |L quench buffer is added and read in the ORIGEN analyzer. An automatic sipper aspirates a specified volume (1251000 |L) of the reaction mixture and pumps into a flow cell; the magnet moves close to the platinum electrode, which captures the streptavidin magnetic beads (due to the magnetic field) with bound tyrosine phosphorylated biotin-peptide complexed to Ru2+-anti-phosphotyrosine antibody. Voltage (< 2 V) is applied to the electrode to begin the oxidation reaction to produce light and is measured in a PMT in less than 1 sec. The flow cell is then washed with buffer and the next sample is read.
The ECL is a promising technology that can be used in a wide spectrum of assay formats. Screening can be performed using whole blood, culture medium, crude extracts, and membrane preparations. The dynamic range is very broad. The ECL method utilizes nonisotopic small-molecule labels. In this assay the sample has to pass through the flow cell and is washed to remove free Ru2+ labeled molecules, resulting in a high signal-to-noise ratio (the background is low because the beads are concentrated on the electrode and washed with TPA). However, ECL cannot be used for measuring real-time kinetics. It is suitable for end point determinations. As the signal produced 30-50 nm above the electrode is measured, ECL can not be used efficiently for whole cell assays. At least four derivatives of Ru2+ chelates are available that can be coupled to a variety of bio-molecules, and also a custom labeling service is available from IGEN. ECL instruments are one- to eight-module instruments able to read 1-8 samples at a time. The ECL homogeneous assays can be adapted to HTS with the eight-module instrument, which reads each plate in 10 min.
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