Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
2000-06-22
2001-03-13
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C250S459100
Reexamination Certificate
active
06200818
ABSTRACT:
The present invention relates to a method for the detection of association, dissociation, linking or cleaving reactions and conformational changes using coincidence analysis.
Finding molecules having specific properties, such as binding, inhibition or catalytic properties, is a key problem in the development of active substance and in biotechnological applications. Molecules having such properties can be either discovered or designed. In this sense, “discovering” means the isolation and screening of substances, while molecular design relies on rational or evolutive techniques. Rational design requires fundamental insights into molecular biophysics to enable prediction of the structure-function relationship. In contrast, in evolutive design, principles of Darwin's Evolution are employed on a molecular level, and new or modified molecules are generated by a combination of mutation, amplification and selection. The use of evolutive techniques in biotechnology, so-called evolutive biotechnology, was proposed by Eigen and Gardiner at the beginning of the 80es (Pure Appl. Chem. 56, 967-978, 1984) and has meanwhile met with a broad acceptance. However, unfortunately, in many evolutive approaches, the process of selection is not immediately linked to amplification. Therefore, selective processes often have to be introduced deliberately. This may be achieved, for example, in the so-called high throughput screening (HTS) in combination with a suitable assay.
HTS processes are subject to economical restrictions. Therefore, to process a large number of samples, it is required for the time to analyze one individual sample to be extremely short. In recent years, many efforts were therefore made in the miniaturization, parallelization and automation of screening techniques and in the development of homogeneous assays and the integration of highly sensitive and quickly operating detection devices. Among the large number of alternative detection principles, techniques based on fluorescence, such as fluorescence resonance energy transfer (FRET), fluorescence quenching, fluorescence polarization, time-resolved fluorescence techniques and fluorescence correlation spectroscopy (FCS), have found a high interest.
In fluorescence correlation spectroscopy, fluorescence fluctuations of individual molecules within a measuring volume element which is in the femtoliter range, in particular, are measured, and molecular diffusion characteristics are established, for example, by evaluating the autocorrelation function of the one-color fluorescence signals. The basics of FCS and its application, in particular, to biological problems have been described in various articles and patent applications (e.g., Eigen and Rigler, Proc. Natl. Acad. Sci. USA 91, 5740-5747, 1994; WO 94/16313).
Fluorescence cross-correlation spectroscopy, or so-called two-color FCS, is also the subject of some publications. Two-color FCS was proposed at the beginning of the 90es by Eigen and Rigler (Proc. Natl. Acad. Sci. USA 91, 5740-5747) and is also discussed in the International Patent Application published under WO 94/16313. Applications of this technology in the study of hybridization kinetics have been described by Schwille et al. (Biophysical Journal, Vol. 72, 1878-1886, 1997). The applications of fluorescence cross-correlation described in the literature show that analysis times of from 30 to 120 s are required to enable a sufficiently precise determination of the amplitudes of the cross-correlation function and the diffusion times. Such long analysis times are not suitable for high throughput screening, or only so in a limited way.
Further, various signal processing methods for separating signals from the background noise have been described in the literature.
Tellinghuisen et al. (Analytical Chemistry 66, No. 1, 64-72, 1994) describes a method for fluorescence lifetime spectroscopy which is intended to serve for filtering out the photons coming from the light source, in this case a laser, from the overall signal. Thus, the current of the signal counter is compared to the trigger signal, i.e., the excitation pulse of the pulsed laser. If a photon arrives simultaneously with the excitation pulse, allowing for the velocity of light, it is identified as a scattering light pulse and eliminated.
Keller et al. (Applied Spectroscopy 50, No. 7, 12-32A, 1996) describes another method for analyzing fluorescence lifetimes. In this method, the time differences between successive, in time, pulses arriving at the detector are determined by counting the number of trigger pulses generated at 100 kHz between two successive photons. These counts are stored in successive channels of an MCS (multichannel scaler). This MCS signal is then subjected to a time-resolved fast Fourier transformation (FFT) for smoothening. After FFT, if at least 5 time differences of the smoothened signal are below a visually determined threshold value, the signal is considered coherent for the whole period of time during which the time differences were continuously below the visually determined threshold value, and evaluated as a fluorescence signal, called “burst” for some time in scientific language. Subsequently, the thus filtered signal is evaluated for fluorescence lifetime. However, the authors only use those signal fractions in which more than 25 time differences are below the visually determined threshold value.
It has been the object of the present invention to provide a method which enables a reliable and fast detection of association, dissociation, linking or cleaving reactions and conformational changes in minute sample volumes.
The object of the invention is achieved by a method having the features of claim
1
. The further claims relate to preferred embodiments of the method according to the invention.
Thus, the invention relates to a method for the detection of association, dissociation, linking or cleaving reactions and conformational changes of analytes in a sample using coincidence analysis, wherein
the sample contains at least two analytes labeled with different fluorescent dyes, and/or at least one analyte labeled with at least two different fluorescent dyes;
the sample is illuminated by at least one laser for exciting the fluorescence emission of said at least two dyes;
the fluorescent signals emitted by the sample which come from at least one measuring volume element V are detected by at least two detection units;
the signals respectively detected in the detection units or time tracks derived therefrom are cut into arbitrary, but essentially simultaneous, time segments with freely selectable time channel widths;
the number of signals contained in at least one time segment and/or the time intervals between signals within the time segments are established;
for at least one time segment of the first detection unit, a coincidence analysis of the established data with at least one essentially simultaneous time segment of the second detection unit is performed;
at least one statistical analysis of the results of the coincidence analysis is performed, and/or the results are subjected to a threshold analysis;
said statistical analysis or at least one combination of several statistical analyses is evaluated for the presence of properties characteristic of an association, dissociation, linking or cleaving reaction or conformational change.
It may further be preferred that the time channel widths be greater than the longest fluorescence lifetime of said at least two dyes and/or that the time channel widths be smaller than the time required for said at least one sample molecule to pass through the measuring volume.
In a preferred embodiment, the detection units should have different spectral detection sensitivities.
Further, the measuring volume element V should be ≦10
−12
l.
It may further be preferred to illuminate the sample with a laser which emits electromagnetic radiation of at least one wavelength which is capable of exciting said at least two dyes present in the sample. However, it is also possible to use lasers which emit more than one wav
Bieschke Jan
Dörre Klaus
Eigen Manfred
Kettling Ulrich
Koltermann Andre
EVOTEC BioSystems AG
Jacobson Price Holman & Stern PLLC
Snay Jeffrey
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