EP2483666A1 - Procédé pour déterminer des changements in vitro dans un environnement protéique - Google Patents

Procédé pour déterminer des changements in vitro dans un environnement protéique

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Publication number
EP2483666A1
EP2483666A1 EP10762851A EP10762851A EP2483666A1 EP 2483666 A1 EP2483666 A1 EP 2483666A1 EP 10762851 A EP10762851 A EP 10762851A EP 10762851 A EP10762851 A EP 10762851A EP 2483666 A1 EP2483666 A1 EP 2483666A1
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EP
European Patent Office
Prior art keywords
protein
light
changes
frequency
fluorescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10762851A
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German (de)
English (en)
Inventor
David M. Jameson
Dudley J. Williams
Marcella A. Gilmore
Lance E. Steward
Nicholas G. James
Justin A. Ross
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Hawaii
Allergan Inc
Original Assignee
University of Hawaii
Allergan Inc
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Publication date
Application filed by University of Hawaii, Allergan Inc filed Critical University of Hawaii
Publication of EP2483666A1 publication Critical patent/EP2483666A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Definitions

  • Emission heterogeneity could be due to the presence of multiple fluorophores (each giving rise to different exponential decays), excited state processes (such as solvent relaxation or Forster Resonance Energy Transfer (FRET)) or non-exponential decays due to processes such as transient quenching.
  • Models used to fit multiexponential decays are usually based on discrete exponential components or continuous distribution functions.
  • J.A. Ross, and D.M. Jameson Time-resolved methods in biophysics. 8. Frequency domain fluorometry: applications to intrinsic protein fluorescence. Photochem. Photobiol. Sci. 7 (2008) 1301 -1312; B. Valeur, Molecular Fluorescence, Wiley-VCH, Weiheim, Germany, 2002; and, J. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, 2006.)
  • Figure 1 shows a phasor plot of L-tryptophan at various pH values.
  • Figure 2 shows the quenching of NATA and lysozyme using the quencher acrylamide.
  • Figure 3 shows a phasor plot, at 70 MHz and 280 nm excitation, of NATA (circles) and lysozyme (open triangles) as a function of increasing temperature.
  • Figure 4 A shows a phasor plot for dynamin 2 alone (circle), bound with GDP- (closed triangle), and GTPyS -bound (open triangle).
  • FIG. 4 B Monomeric HSA (closed circle) plotted with furosemide bound HSA (open triangle) and D-thyroxine bound HSA (closed triangle).
  • Dimeric HSA (open circle) which can be found up to 10% in lyophilized HSA, has a unique decay/phasor point compared to monomeric HSA.
  • Figure 4 C Phasor plot showing point movements due to protein-protein interaction.
  • Figure 5 shows phasor plot of iron release from hTF N-lobe.
  • Figure 6 shows phasor point trajectory of lysozyme denaturation as a function of increasing GuHCI (solid line) and urea (dashed line) concentration.
  • Figure 7 shows a phasor plot of denaturation of GFP-SNAP25-BFP from OmM (gray star) to 2M (gray circle) with GHCI (Black line).
  • the universal circle (gray line) is illustrated for reference. Number illustrate the GHCI concentration in mM at the respective point.
  • Figure 8 shows a phasor plot of LHn/A and LHn/A-Dynorphin at 50MHz and the affects of addition of GuHCI to 50mM.
  • Figure 9 show LHn/A, Dyn-LHn/A With Cryo-Protectant 5% PEG.
  • any change in a specific protein's environment say due to protein unfolding (denaturation), subunit oligomerization or subunit dissociation, fragmentation due to proteolysis or more subtle allosteric responses or isomeric relationships of protein molecules can be picked up during this type of analysis.
  • subtle movements in the conformation of the protein or changes in its interactions with other molecules induce changes in the immediate environment of the protein's fluorophores which can change the lifetime of their emission.
  • the fluorescence measured can be from the intrinsic fluorescence of a protein, e.g., from the amino acids Tryptophan or Tyrosine, or from extrinsic fluorescence by labeling a protein with a fluorophore, for example amine or sulfhydryl reactive probes.
  • the present application discloses an in vitro solution phase method that tracks conformational changes in a protein and/or changes to the protein milieu.
  • stability of proteins of interest over time periods may be observed.
  • mapping kinetic changes in proteins due to enzymatic turnover or oligomerization may be observed.
  • screening batch to batch protein preparations to rapidly validate the exclusion of potential protein refolding problems may be observed.
  • protein milieu means the setting or surrounding of a protein, and includes, for example, pH, temperature, ionic strength, excipient, formulation, etc.
  • therapeutic protein refers to a protein that are often extracted from animal cells or engineered in the laboratory for pharmaceutical use.
  • the large number of therapeutic proteins are recombinant human proteins manufactured using non-human mammalian cell lines that are engineered to express certain human genetic sequences to produce specific proteins.
  • Therapeutic proteins are used to relieve patients' suffering from many conditions, including various cancers (treated by monoclonal antibodies and interferons), heart attacks, strokes, cystic fibrosis and Gaucher's disease (treated by Enzymes and blood factors), diabetes (treated by insulin), anemeia (treated by erythropoietins), hemophilia (treated by blood clotting factors), and botulinum toxin for a variety of disorders.
  • the term "industrial protein” refers to a protein from plant, animal or microbial origin that can be produced and/or processed on an industrial scale. Industrial proteins are increasingly used in food products because of their functional properties - both techno-functional properties (such as gelling and emulsifying behavior and taste) and bio-functional properties (such as nutritional value and physiological activity).
  • the functionality is determined by (bio)chemical characteristics and is affected by the kind of protein, by the isolation procedure and by processing. Furthermore, the functionality may deliberately be altered by modification procedures.
  • ISS Chronos spectroflurometer ISS Inc., Champaign IL
  • time resolved capabilities ISS Inc., Champaign IL
  • Sample in the ⁇ - ⁇ concentration range is used in an 800 ⁇ quartz cuvette with a 1 cm optical path length.
  • Frequency modulated exciting light from a 280nm LED at 280nm is passed through a 280-20 band pass filter (Melles Griot, Voisins Le Bretonneaux ) to the sample chamber. 300 nm LEDs as the light source may also be used.
  • the time domain is utilized wherein the decay of the fluorescence is recorded with time after excitation of the fluorescence with a brief pulse of light and the S and G functions are calculated from the time domain by Equations (3) and (4) below:
  • ⁇ can be chosen as the repetition frequency of the pulsed excitation source or another value which depends on the kinetics of the excited state process under investigation and l(t) is the observed fluorescence intensity at time t.
  • the bandpass filter FF01-280/20-25 or FF01 -295/15-25 was used where appropriate with the excitation light and the emission collected through longpass filters (WG315 or UK330) or a 357/50 nm bandpass filter.
  • Polarizers were set at magic angles to eliminate polarization effects. (G.D. Reinhart, P. Marzola, D.M. Jameson, and E. Gratton, A method for on-line background subtraction in frequency domain fluorometry. J. Fluoresc.
  • Raw data are plotted to generate a phasor plot using a routine written in Matlab software.
  • Example 1 Tryptophan lifetime/phasor as a function of pH.
  • Figure 1 shows a phasor plot of L-tryptophan at various pH values.
  • the black points represent pH 6.0 (circle), pH 9.5 (open triangle), and pH 1 1 .0 (square) data at 25 MHz.
  • the lifetime data were collected with 280 nm excitation at 25°C.
  • pH 9.5 there should be a -1 : 1 anion to zwitterion molecular ratio (based on the pK a values of tryptophan), however, one notes that the phasor point corresponding to pH 9.5 is not midway between the high and low pH points.
  • the exact distance of the phasor point along the line joining the starting and ending points on the universal circle depends not only on the relative concentration but also the quantum yields of the species in question (in the case of L-tryptophan, the ratio of the lifetimes and quantum yields of the anion to zwitterion forms is ⁇ 3).
  • D.M. Jameson, and G. Weber Resolution of the pH-dependent heterogeneous fluorescence decay of tryptophan by phase and modulation measurements. The Journal of Physical Chemistry 85 (1981 ) 953-958.
  • the frequencies utilized will also weight the fractional contributions of the components differently, i.e., the lower frequency phasor points will weight the longer lifetime component while higher frequencies will favor the shorter component.
  • R.D. Spencer, and G. Weber Measurement of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer. Ann. N. Y. Acad. Sci. 158 (1969) 361-376.
  • Chemical quenchers may provide information on the exposure of a tryptophan residue to the solvent as a consequence of collision of the excited fluorophore with the quencher molecule (dynamic quenching) or, in some cases, by formation of a ground state dark complex (static quenching) (B. Valeur, Molecular Fluorescence, Wiley-VCH, Weiheim, Germany, 2002). Both steady-state and time-resolved methodologies can be used to derive quenching information.
  • FIG. 2 shows the quenching of NATA and lysozyme using the quencher acrylamide.
  • NATA circles
  • lysozyme open triangles
  • phasor points at 89 MHz, with 300 nm excitation at 20°C, with the addition of various concentrations of acrylamide (0 - 0.2 M with NATA and 0- 0.5M with lysozyme).
  • the data was recorded at 89 MHz and 20°C using a 300 nm LED as the excitation source; emission was observed through a WG315 longpass filter.
  • the position of the phasor points is dependent on the quencher concentration.
  • lysozyme one notes that the phasor points are all within the universal circle indicating the heterogeneous nature of the lifetime data.
  • Addition of acrylamide results in shorter lifetimes, indicating one or more of the tryptophan residues in lysozyme are sensitive to dynamic quenching, and the subsequent shift in the phasor points in a clockwise direction.
  • Addition of the quencher iodide (data not shown) also shifted the phasor point, following a similar trajectory to shorter lifetimes, albeit to a lesser extent, as with the acrylamide quenching.
  • Dynamin 2 comprises 5 domains (an N-terminal GTPase domain, a middle domain, a pleckstrin homology (PH) domain, a GTPase Effector Region (GED) and a proline/arginine rich domain (PRD)) and contains 5 tryptophan residues, 4 of which are in the PH domain, while 1 tryptophan is in the C-terminal PRD domain.
  • PH pleckstrin homology
  • GED GTPase Effector Region
  • PRD proline/arginine rich domain
  • HSA which contains a single tryptophan residue (Trp 214)
  • Trp 214 has been the subject of numerous fluorescence studies, including time-resolved studies, see for example (S. Kasai, T. Horie, T. Mizuma, and S. Awazu, Fluorescence energy transfer study of the relationship between the lone tryptophan residue and drug binding sites in human serum albumin. Journal of Pharmaceutical Sciences 76 (1987) 387-392; G. Hazan, E. Haas, and I .Z. Steinberg, The fluorescence decay of human serum albumin and its subfractions. Biochim. Biophys. Acta 434 (1976) 144-153; P. Marzola, and E.
  • HSA is commonly targeted for drug uptake studies and often these drug interactions are studied via changes in tryptophan fluorescence.
  • Figure 4B we show the phasor point, collected using 300 nm excitation at 84 MHz and 25°C, of the intrinsic fluorescence from monomeric HSA.
  • furosemide or D-Thyroxine two drugs known to bind to HSA and to quench the tryptophan fluorescence, the phasor point shifts clockwise, indicating a shortening of the average lifetime.
  • Figure 4C shows a set of data (acquired with 280 nm excitation at 43 MHz and 20°C) for thrombin, anti-thrombin, lysozyme, and mixtures therein. Antithrombin and lysozyme are not predicted to interact and therefore a solution containing the two proteins should produce a phasor point that falls directly on a line between their individual points. This outcome is clearly observed (Figure 4C) for the phasor plot of a 1 : 1 (1 ⁇ ) mixture of the two proteins.
  • thrombin/antithrombin is known to form a tight complex (D. Beeler, R. Rosenberg, and R. Jordan, Fractionation of low molecular weight heparin species and their interaction with antithrombin. J. Biol. Chem. 254 (1979) 2902-2913) and may be expected to produce a distinct phasor point away from the linear combination.
  • Figure 4 A shows a phasor plot for dynamin 2 alone (circle), bound with GDP- (closed triangle), and GTPyS-bound (open triangle).
  • Figure 4 B shows monomeric HSA (closed circle) plotted with furosemide bound HSA (open triangle) and D-thyroxine bound HSA (closed triangle).
  • Dimeric HSA open circle
  • Figure 4 C shows a phasor plot showing point movements due to protein-protein interaction.
  • Solid lines between antithrombin (circle), lysozyme (closed square) and thrombin (closed triangle) are the projected linear movement of phasor point for non-interacting species. Mixtures of antithrombin/lysozyme (both at 1 ⁇ , open square) and antithrombin/thrombin (gray triangle 0.5:1 , open triangle 1 : 1 ) are shown on the plot. The dashed line represents the projected phasor movement for increasing concentrations of antithrombin in the presence of thrombin. Cross-hairs seen in the upper part of each figure represent the statistical error for each phasor point under the expanded phasor plot scale.
  • the phasor plot method recorded at a single frequency, is, however, well-suited for rapidly tracking changes in the phase and modulation data.
  • the phase and modulation of the intrinsic fluorescence of human serum transferrin were recorded over -300 sec at 80 MHz in pH 6.0 buffer and the presence of a chelator.
  • Human serum transferrin (hTF) is a bilobal glycoprotein that serves as the major transporter of iron in humans (A.B. Mason, and S.J. Everse, Iron Transport by Transferrin, in: H. Fuchs, (Ed.), Iron Metabolism and Disease, Research Signpost, Huawei, India, 2008, pp. 83-123.)
  • Both lobes termed the N- and C-lobes, coordinate ferric iron via four amino acid ligands and a synergistic carbonate anion.
  • Numerous studies on hTF have shown that binding of iron quenches the intrinsic tryptophan fluorescence, through radiative and non-radiative means. Rate constants for iron removal are thus determined by tracking the enhancement in fluorescence emission over time under endosomal like conditions (pH ⁇ 5.5, -150-200 mM salt and the presence of a chelator), which takes place within seconds to minutes.
  • Figure 5 which shows the phasor plot (taken at 80 MHz and 300 nm excitation) during iron release (black line) and the points for iron-bound (circle) and apo (triangle), clearly demonstrates the predicted linear progression in the phasor point during iron release from the iron-bound point towards the apo phasor point.
  • Sample heterogeneity which was expected based on the fitting to a discrete exponential model, is seen in the phasor vectors of hTF N-lobe as each point is inside the universal circle. Calculation of the distance at each point over time can provide information regarding excited state changes during iron removal (i.e. one can recover the fractional contribution of each emitting state using standard linear methods.
  • Chaotropic agents such as GuHCI and urea
  • Unfolding experiments using such chemical denaturants have one major underlying assumption: that the overall, thermodynamic unfolding of the protein is independent of denaturing agent although the structural changes associated with the change are dependent.
  • W. Pfeil, and P.L. Privalov Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride. Biophys. Chem. 4 (1976) 33-40; G.I. Makhatadze, and P.L. Privalov, Protein interactions with urea and guanidinium chloride. A calorimetric study. J. Mol. Biol. 226 (1992) 491 -505.
  • Figure 6 shows phasor point trajectory of lysozyme denaturation as a function of increasing GuHCI (solid line) and urea (dashed line) concentration. Concentrations of 0 M (closed circle), 1 M (open circle), 2 M (open triangle) and 6/8 M (closed triangle) GuHCI and urea, respectively, are highlighted. Data was excited with 280 nm and data plotted were at 70 MHz and 25°C.
  • the mapping of a Clostridial toxin substrate using the Phasor Plot method was accomplished as follows.
  • the protein substrate used is GFP-SNAP25-BFP an approximately 80kDa protein.
  • the unfolding process of the protein is studied using the Phasor Plot mapping as shown in Figure 7.
  • the universal circle (gray line) is illustrated for reference. Number illustrate the GuHCI concentration in mM at the respective point.
  • Example 8 Conformation/dissociation of Botulinum Neurotoxin in denaturant.
  • Example 9 Conformation/dissociation of Botulinum Neurotoxin in denaturant with PEG added.
  • Clostridial toxin activity assay can be evaluated by phasor plot analysis.
  • Clostridial toxin activity assays include those disclosed in U.S. Patent 7, 183,066; U.S. Patent 7,208,285; U.S. Patent 7,332,567; and U.S. Patent 7,399,607; each of which is hereby incorporated by reference in its entirety.
  • the activity of any toxin comprising a Clostridial toxin enzymatic domain derived from a light chain can be assessed by phasor plot analysis, including chimeric toxins are retargeted toxins, such as, e.g., those disclosed in U.S.
  • a Clostridial toxin activity assay is performed and a phasor plot analysis is conducted.
  • the instrumental conditions reagents and general experimental conditions are very similar to other Examples listed herein.
  • the phasor plot indicates a conformational- aggregation change indicative of substrate cleavage by the toxin.
  • Example 1 1 - Analysis of Formulation Stability of Pharmaceutical Composition Stored Frozen Using Phasor Plot Analysis
  • This example illustrates that the stability of a formulated pharmaceutical composition can be assessed using phasor plot analysis without any disruption to the packaged composition.
  • Packaged vials comprising a dried pharmaceutical composition are assessed by phasor plot analysis using similar instrumental conditions, reagents and general experimental conditions as disclosed in Examples above.
  • the quality and quantity of the composition are initially assessed at the time of initial packaging.
  • the vials are then stored at the desired temperature, e.g., room temperature, -20 °C, or -70 °C.
  • the vials are periodically assessed over time to monitor both quality and quantity of the pharmaceutical composition, e.g., once every month, once every three months, once every six months, once every year. In this case assessment every three months over a period of three years indicated that the pharmaceutical composition remained stable in that the quality and quantity of the composition after three years was substantially the same as the initial assessment made after packaging.
  • Example 12 Analysis of Formulation Stability of Pharmaceutical Composition Stored at Room Temperature Using Phasor Plot Analysis.
  • This example illustrates that the stability of a formulated pharmaceutical composition can be assessed using phasor plot analysis without any disruption to the packaged composition.
  • Packaged vials comprising a liquid pharmaceutical composition are assessed by phasor plot analysis using similar instrumental conditions, reagents and general experimental conditions as disclosed in other Examples herein.
  • the quality and quantity of the composition are initially assessed at the time of initial packaging.
  • the vials are then stored at the desired temperature, e.g., room temperature, -20 °C, or -70 °C.
  • the vials are periodically assessed over time to monitor both quality and quantity of the pharmaceutical composition, e.g., once every month, once every three months, once every six months, once every year. In this case assessment every three months over a period of three years indicated that the pharmaceutical composition remained stable in that the quality and quantity of the composition after three years was substantially the same as the initial assessment made after packaging.

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Abstract

L'invention concerne un procédé pour déterminer des changements in vitro dans un environnement protéique. Le procédé consiste à : sélectionner une protéine d'intérêt, et placer un premier échantillon de la protéine dans un spectrofluorimètre, de telle sorte que l'échantillon de protéine peut être éclairé par une lumière d'excitation modulée en fréquence, et la lumière émise est détectée ; obtenir un ensemble de références de mesures de lumière à une fréquence sélectionnée par mesure du changement d'angle de phase (Φ) entre la lumière émise et la lumière excitée et enregistrer simultanément un changement de modulation de signal (m) ; appliquer des équations de domaine temporel ou des équations de domaine de fréquence de l'ensemble de référence de mesures de lumière pour obtenir un ensemble de données de référence ; obtenir au moins un second ensemble de mesures de lumière à la fréquence sélectionnée pour la protéine ; appliquer les équations de domaine temporel ou les équations de domaine de fréquence du second ensemble de mesures de lumière pour obtenir un second ensemble de données ; reporter S par rapport à G pour l'ensemble de données de référence et le second ensemble de données ; et, déterminer si l'environnement protéique a subi des changements entre la mesure de référence et une seconde mesure par observation du changement de position des points reportés générés. Le spectrofluorimètre nécessite une source de lumière qui peut provoquer la fluorescence d'une protéine et un dispositif pour détecter et mesurer la lumière émise par la protéine. La source de lumière est modulée dans le domaine de fréquence et dans le domaine temporel. Les changements entre la mesure de référence et la seconde mesure sont sélectionnés dans le groupe constitué par : a) le même échantillon de protéine à un moment différent ; et b) la protéine dans un milieu différent. Les changements dans l'environnement protéique sont des changements de conformation d'une protéine unique, des changes dans les interactions protéine-protéine et/ou des changements dans les interactions protéine-excipient.
EP10762851A 2009-10-02 2010-10-04 Procédé pour déterminer des changements in vitro dans un environnement protéique Withdrawn EP2483666A1 (fr)

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Family Cites Families (9)

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Publication number Priority date Publication date Assignee Title
WO2001014570A1 (fr) 1999-08-25 2001-03-01 Allergan Sales, Inc. Neurotoxines de recombinaison activables
US7138127B1 (en) 2000-01-19 2006-11-21 Allergan, Inc. Clostridial toxin derivatives and methods for treating pain
US6903187B1 (en) 2000-07-21 2005-06-07 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US7273722B2 (en) 2000-11-29 2007-09-25 Allergan, Inc. Neurotoxins with enhanced target specificity
US7332567B2 (en) 2001-08-28 2008-02-19 Allergan, Inc. Fret protease assays for clostridial toxins
US7208285B2 (en) 2001-08-28 2007-04-24 Allergan, Inc. Fret protease assays for botulinum serotype A/E toxins
US7183066B2 (en) 2002-09-27 2007-02-27 Allergan, Inc. Cell-based fluorescence resonance energy transfer (FRET) assays for clostridial toxins
JP4235440B2 (ja) 2002-12-13 2009-03-11 キヤノン株式会社 半導体デバイスアレイ及びその製造方法
US7399607B2 (en) 2004-09-22 2008-07-15 Allergan, Inc. Fluorescence polarization assays for determining clostridial toxin activity

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