FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor
necrosis factor alpha (TNF-α) and two of its protein antagonists Humira(TM) (adalimumab), a fully humanized monoclonal
antibody, and Enbrel(TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have
the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be
lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the
protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the
trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry.
Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our
FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even
larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition,
distinct differences between Humira and Enbrel were observed in their association with TNF-α.
Fluorescence Correlation Spectroscopy (FCS) may be used to assay the binding of drug-like ligands to signaling proteins and other bio-particles. For High Throughput Screening (HTS), a competitive format is typically used in which binding of an unlabeled compound results in displacement of a fluorescently labeled ligand. Unweighted curve-fitting of the normalized autocorrelation function (ACF) to a two-diffusion-component model can resolve the fractions of free and bound ligand if the diffusion rates differ sufficiently and if the experimentally estimated ACF has adequate precision. However, for HTS (and also for intracellular FCS studies) it is desirable to minimize the experimental data collection time. In this case, the precision of the ACF is limited and it becomes important to account for the statistical features of the ACF estimate when designing an assay. The errors at different points in the estimated ACF are correlated and hence least-squares fitting methods are not strictly statistically rigorous. We compare different methods for estimating and curve-fitting the ACF from the raw data of short duration FCS measurements. The methods are applied to data from experiments to assay binding of Alexa-488-labeled Bak peptide with Bcl-xL, which is an intracellular protein that acts to protect against programmed cell death. We present results from a detailed Monte Carlo simulation of the experiment, which is useful for validating short-duration assay capabilities. We also discuss the measurement of changes in steady state fluorescence anisotropy due to restricted rotational diffusion upon binding, which provides a complementary assay method.
In this work we characterized seven dyes that are one-photon excited and fluoresce in the far-red region of the visible spectrum, five of which became available only recently, as to their suitability as labels on the red emission channel in a two-color FSC method. Spectroscopic properties and binding to albumin were studied. In addition, when one of these was used as the label in a first-pass high-throughput screen with single-color fluorescence polarization detection, its foreseen advantage of avoiding the excitation of interfering background autofluorescence from compounds in libraries was deemed valuable after a comparison of two complete high-throughput screens of a chemical compound library at Abbott.
The possibility and potential benefits of using an extreme red fluorescent dye such as Cy5.5TM to label drug discovery target proteins was studied experimentally. Cy5.5 labeled BSA, GFP, and CRP were used as examples of protein-dye conjugates whose binding to corresponding antibody was detected by changes in either rotational or transnational diffusion properties, that is, by either fluorescence polarization or confocal fluorescence correlation spectroscopy (FCS). In addition, FCS was used to quantitate excitation of Cy5.5 to its triplet state. Fluorescence polarization and lifetime were measured as a function of excitation wavelength or glycerol concentration and solvent viscosity.
Multifrequency phase-modulation data for fluorescence lifetime and anisotropy decay standards have been obtained for liquid samples in 96-well microtiter plates. We coupled the bifurcated fiber optic probe of a fluorescence plate reader to a phase and modulation time- resolving fluorometer. Laser excitation was used and multifrequency data could be acquired quickly over a range of modulation frequencies from 15 to greater than 150 MHz, at each well position, through the use of multiharmonic Fourier methods. We evaluated the system's performance for UV-excited fluorescence, with the goal of screening quality or properties of proteins or peptides by steady-state or dynamic intrinsic fluorescence intensity or anisotropy using the high throughput 96-well plate format.
We report here a method for measuring and eliminating artifacts caused by background in fluorescence lifetime or rotational correlation data using multiharmonic Fourier transform frequency domain fluorometry. A single measurement on a blank yields phase and amplitude values for multiple modulation frequencies simultaneously, and these may then be used to correct the sample's data. Experimental results demonstrate the applicability of the method over a wide range of background contributions.
The technical simplicity and speed of cross-correlation frequency domain fluorometry has been compromised recently by the need to measure and analyse the phase and modulation responses of a sample over a wide range of excitation frequencies. Phase fluorometers currently use single frequency cross-correlation detection combined with either single frequency excitation or harmonic excitation from a pulsed source. Phase and modulation response curves are constructed by taking a series of point-by-point measurements over the frequency region of interest. This can be a time consuming process which requires the attention of an experienced operator. The Fourier transform spectrofluorometer described here combines multi-harmonic excitation with multi-harmonic cross-correlation detection and digital signal processing techniques to implement a frequency domain fluorometer which can acquire phase and modulation responses simultaneously at 40 to 50frequencies. A particularly dramatic capability of the Fourier transform fluorometer is its ability to collect multi-frequency data at millisecond rates thus allowing complex lifetime analysis of rapidly evolving fluorescence systems. Further, multi-harmonic excitation and detection is the equivalent of measuring the time course of a sample's response to impulse excitation. Therefore, time domain data is inherently available from the instrument and may be fitted to models of fluorescence decay by iterative reconvolution techniques as an alternative non-linear least squares fitting of the frequency domain data.
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