The study of disease processes requires a number of tools for detection of proteins and biomarkers in cell and animal
based assays. Near infrared (NIR) technologies offer the advantage of high signal without interference from
background producing factors such as tissues, blood, or plastics. NIR fluorescence quenching biochemical assays
employing a novel NIR quencher are homogeneous and sensitive. NIR-based immunocytochemical assays offer a
means of quantitatively evaluating cell signaling pathways. The technology can be extended to the development of
targeted molecular imaging agents for disease analysis in animal models. We describe here model assays for each of
these categories. A fluorescence quenching caspase-3 assay was developed employing a novel, broadly applicable
quencher dye suitable for use with both visible and NIR dye chemistries. An NIR cell based assay is described for
assessment of phosphorylation of p53 in response to a cellular stimulus. Finally, we describe the development and
application of a targeted NIR optical imaging agent for monitoring tumor growth in whole animals. The NIR
biochemical and cell based assays are robust with Z' factors greater than 0.7. The use of an IRDye (R)800CW-labeled
cyclic RGD peptide is presented as a model for development and application of targeted imaging agents. NIR
technologies are compatible with the complete spectrum of assay needs for disease analysis and therapeutic
development.
KEYWORDS: Luminescence, Fluorescence resonance energy transfer, Light scattering, Absorption, Control systems, Quenching (fluorescence), Signal to noise ratio, Tolerancing, Proteins, Imaging systems
The HIV-1 protease enzyme is an excellent target for drug therapy of HIV infection/AIDS. To measure the
protease activity and screen for potent protease inhibitors, homogeneous protease assays based on quenched fluorescent
peptide substrates have been widely used as a high-throughput screening methods. The major problem in these assays is
the compound interference or assay artifacts from colored or insoluble materials in the assay, e.g. assay components,
screening library compounds, etc. We report in this paper a near-infrared fluorescence resonance energy transfer (NIRFRET)
based HIV-1 protease assay that can dramatically reduce or completely eliminate these assay artifacts by using a
novel near-IR donor-quencher pair and long wavelength excitation (780 nm) and detection (820±10 nm). In this assay, a
HIV-1 protease peptide substrate is conjugated with a near-IR fluorescent donor (IRDyeTM 800CW), and a novel near-IR
non-fluorescent quencher (QC1) on opposite sides of the proteolytic cleavage site. The quencher, QC1, has extremely
good spectral overlap of its absorption spectrum with the donor emission spectrum to ensure the efficient quenching of
the donor's fluorescence. In the HIV-1 protease assay, this NIR-FRET system shows a large dynamic range, high signal
to noise ratio, excellent Z'-factors, a wide range of DMSO tolerance, and no compound interference. This system
provides a sensitive, robust assay for high-throughput screening (HTS) and can be readily adapted to other
therapeutically significant protease targets.
Current organic fluorophores used as labeling reagents for biomolecule conjugation have significant limitations
in photostability. This compromises their performance in applications that require a photostable fluorescent reporting
group. For example, in molecular imaging and single molecule microscopy, photostable fluorescent labels are important
for observing and tracking individual molecular events over extended period of time. We report in this paper an
extremely photostable and highly fluorescent phthalocyanine dye, IRDyeTM 700DX, as a near-infrared fluorescence
labeling reagent to conjugate with biomolecules. This novel water-soluble silicon phthalocyanine dye has an isomericly
pure chemical structure. The dye is about 45 to 128 times more photostable than current near-IR fluorophores, e.g.
Alexa Fluor"R"680, CyTM 5.5, CyTM 7 and IRDyeTM 800CW dyes; and about 27 times more photostable than
tetramethylrhodamine (TMR), one of the most photostable organic dyes. This dye also meets all the other stringent
requirements as an ideal fluorophore for biomolecules labeling such as excellent water solubility, no aggregation in high
ionic strength buffer, large extinction coefficient and high fluorescent quantum yield. Antibodies conjugated with
IRDyeTM 700DX at high D/P ratio exist as monomeric species in high ionic buffer and have bright fluorescence. The
IRDyeTM 700DX conjugated antibodies generate sensitive, highly specific detection with very low background in
Western blot and cytoblot assays.
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