Chemistry: analytical and immunological testing – Optical result – Including reagent preparation
Reexamination Certificate
1999-07-30
2001-08-07
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
Including reagent preparation
C436S172000
Reexamination Certificate
active
06271039
ABSTRACT:
PRIOR FOREIGN APPLICATIONS
This application is a 35 USC §371 filing of PCT/GB98/03750, filed Dec. 15, 1998, and claims priority from GB patent application number 9727355.1, filed Dec. 24, 1997.
1. Field of Invention
The present invention relates to a composition for use in fluorescence detection. The invention further pertains to a fluorescence assay method and uses of compositions in fluorescence assay methods.
2. Background of the Invention
Fluorescence measurements have been used increasingly for determination and analysis, especially in the biological field. For example, enzyme immunoassays (EIA) based on the determination of the enzyme as a marker has been extensively applied in the bioanalytical field [T. Portmann and S. T. Kiessig, J. Immunol. Methods (1992), 150, 5 and M. Oellerich, J. Clin. Chem. Clin. Biochem., (1989), 22, 895] owing to the high sensitivity and specificity of EIA, availability of many enzyme markers and long term stability of the label reagents and their operational safety. The most commonly used enzyme labels for EIA are alkaline phosphatase, horse radish peroxidase and galactosidase. Most enzyme labels are detected by spectrophotometry [H. Labrousse, J. L. Guesdon, J. Ragimbeau, S. Avrameas, J. Immunol. Methods (1982), 48, 133], spectrofluorimetry [E. Ishikawa, Clin. Biochem., (1987), 20, 375], chemiluminescence [H. Sasamoto, M. Maeda and A. Tsuji Anal. Chim. Acta (1995), 306, 161] and electrochemistry [K. P. Wehemeyer, H. B. Halsall and W. R. Halsall, Anal. Chem., (1995), 31, 1546].
Alkaline phosphatase has been widely used as a label in EIA and has been assayed spectrophotometrically using p-nitrophenyl phosphate (PNPP) [C. S. Chiang, T. Grove, M. Cooper et al., Clin. Chem. (1989), 35, 946], spectrofluorimetrically using 4-methyl umbelliferyl phosphate (4MUP), by chemiluminescence using adamantyl 1,2,-dioxetane aryl phosphate (AMPPD) and its derivatives [I. Bronstein, B. Edwards and J. C. Voyta, J. Biol. Chem., (1989), 4, 99], and electrochemically using phenyl phosphate [K. R. Wehemeyer, H. B. Halsall, W. R. Volle, and I. W. Chen, Anal. Chem., (1986), 58, 135] and p-aminophenyl phosphate (PAPP) [D. A. Palmer, T. E. Edmonds and N. J. Seare, Analyst, (1992), 117, 1679].
The known fluorimetric substrates of alkaline phosphatase are 4-methyl umbelliferyl phosphate and fluorescein diphosphate. These substrates (4-methyl umbelliferyl phosphate and fluorescein diphosphate) are hydrolysed by alkaline phosphatase to 4-methyl umbelliferone and fluorescein which fluoresce at circa 450 nm and 525 nm respectively. Naphthofluorescein phosphate (NFP) is an alternative to the above mentioned alkaline phosphatase substrates with numerous benefits. The product of NFP hydrolysis by alkaline phosphatase is naphthofluorescein which has spectroscopic characteristics of excitation 595 nm and emission 660 nm which clearly fall in the long wavelength
ear infrared region of the electromagnetic spectrum. The benefits of working in the long wavelength region have been reviewed recently [J. N. Miller, Fluorescence spectroscopy, (1993), 5/2, 34]. These include
a) lower background scattering—Raman and Rayleigh
b) less photodecomposition
c) fewer bright fluorophores, hence less background fluorescence
d) availability of compact bright light sources
e) good solid state detectors.
It has recently been demonstrated by our research group that assays for or using alkaline phosphatase can be determined using a combination of naphthofluorescein phosphate, cyclodextrins or surfactant reagents and a solid state laser diode detector. Coinciding with the advances made in the fluorophores, has been corresponding advances in the detectors.
We have recently developed a detector apparatus which, inter alia, uses a fluorescence detector to analyse a sample in a fluid stream. One of the particular advantages of our new detector is its capability to analyse for more than one fluorophore in a single sample. This requires each fluorophore in the sample to be matched to a particular laser in the detector, i.e. the absorbance maximum substantially at the maximum wavelength of the excitation of a narrow bandwidth laser. There are a number of fluorophores which can be matched with commercially available lasers and the number of such fluorophores is constantly increasing.
SUMMARY OF THE INVENTION
The present invention seeks to provide new compositions for fluorescent detection.
According to a first aspect of the present invention there is provided a composition comprising an enzyme substrate which is a non-fluorescent derivative of a fluorophore, and a shifting reagent which shifts the absorbance wavelength maximum of the fluorophore which maximum is naturally above 450 nm, the shifting reagent being present in an amount predetermined to shift the maximum to a preset value.
The ability to control the absorbance maximum greatly increases the potential substrates when matching existing fluorophores to available light sources. The amount by which the maximum is shifted can be varied, normally by varying the amount of shifting reagent in the composition. In this way the analysis for samples containing a matched fluorophore is simplified compared to analysis with unmatched fluorophores as the spectrum is cleaner for the matched fluorophores.
Preferably, the fluorophore is selected from the group consisting of: xanthene dyes; polymethine cyanine dyes; phenoxazine dyes; thiazine dyes; phycobiliproteins; and mixtures thereof.
In many such cases the fluorophore is:
a) a xanthene dye selected from the group consisting of fluorescein, naphthofluorescein, and fluorescent derivatives of fluorescein and naphthofluorescein, other xanthene dyes and anthracene-based dyes;
b) a polymethine cyanine dye selected from the group consisting of Cy3, Cy5, Cy7 and indocyanine green;
c) a phenoxazine dye selected from the group consisting of nile blue, nile red and oxazine 750;
d) a thazine dye being methylene blue; and
e) mixtures thereof.
For example some preferred xanthene dyes such as Vita Blue and other fluorescent derivatives of fluorescein are described by Lee, L. et al. in Cytometry 10:151-164 (1989) and by Menchen, S. et al. in U.S. Pat. No. 5,188,934.
Normally, the amount of shifting reagent is less than 10% w/v in the appropriate solvent, e.g. a buffer and preferably the amount of shifting reagent is less than 5% w/v of the appropriate solvent.
Advantageously, the shifting reagent is selected from the group consisting of: cyclodextrins; substituted cyclodextrins; surfactants; detergents; -(3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulphonate (CHAPS); -(3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulphonate (CHAPSO); octyl &bgr;-glucoside; octyl &bgr;-thioglucopyranoside; sodium dodecyl sulphate (SDS); derivatives thereof; and mixtures thereof.
It is particularly preferred if the substrate is a phosphate derivative, and ester derivative, an imide derivative, an amide derivative or another derivative which is cleavable to the fluorophore.
For assaying for two unknowns in a sample, the composition further includes a second substrate comprising a non-fluorescent derivative of a second fluorophore, wherein the absorbance wavelength maximum of the second fluorophore in the presence of the shifting reagent is different from the maximum of the first fluorophore. The maximum of the second fluorophore may be shifted by the shifting reagent.
For assaying for a plurality of unknowns in a sample (multi-analyte determination), the composition comprises a plurality of substrates comprising a non-fluorescent derivative of a plurality of fluorophores, wherein each absorbance wavelength maximum of each respective fluorophore in the presence of the shifting reagent is different from the maximum of the other fluorophores. In which case the maximum of the some or each of respective fluorescent molecules may be shifted by the shifting reagent.
The composition will normally be used in an assay sys
French Martin Thomas
Palmer Derek Adeyemi
Heslin & Rothenberg, P.C.
Kalibrant Limited
Snay Jeffrey
LandOfFree
Composition for use in fluorescence assay systems does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Composition for use in fluorescence assay systems, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Composition for use in fluorescence assay systems will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2516558