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The invention is directed to entrapped mutated binding proteins, mutated binding proteins containing reporter groups, compositions of mutated binding proteins containing reporter groups in analyte permeable matrixes, and their use as analyte biosensors both in vitro and in vivo.
1. A glucose biosensor for in vivo or in vitro use comprising:
a) at least one mutant glucose/galactose binding protein (GGBP) and at least one reporter group covalently attached thereto wherein said mutant GGBP comprises at least one amino acid substitution of a non-reactive amino acid with a reactive amino acid, wherein said at least one amino acid substitution is selected from the group consisting of a cysteine at position 11, a cysteine at position 14, a cysteine at position 19, a cysteine at position 43, a cysteine at position 74, a cysteine at position 107, a cysteine at position 110, a cysteine at position 112, a cysteine at position 113, a cysteine at position 137, a cysteine at position 149, a cysteine at position 213, a cysteine at position 216, a cysteine at position 238, a cysteine at position 287, a cysteine at position 292, a cysteine at position 112 and a serine at position 238, a cysteine at position 149 and a serine at position 238, a cysteine at position 152 and a cysteine at position 182, a cysteine at position 152 and a serine at position 213, a cysteine at position 213 and a cysteine at position 238, a cysteine at position 149 and an arginine at position 213, a cysteine at position 149 and a serine at position 213 and a serine at position 238, and a cysteine at position 149 and an arginine at position 213 and a serine at position 238 corresponding to amino acid positions of E. coli GGBP, and
b) a first analyte permeable matrix entrapping or encapsulating said at least one mutant GGBP, said matrix comprising a covalently crossliniked hydrogel or a sol-gel comprising organically modified silicates or a combination thereof, wherein the first analyte permeable matrix is formulated to allow three-dimensional conformational movement of the at least one mutant GGBP to bind and release the glucose, and wherein the matrix is adapted to allow glucose to diffuse into and out of the matrix.
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15. A glucose biosensor for in vivo or in vitro use comprising:
a) at least one mutant glucose/galactose binding protein (GGBP) and at least one reporter group covalently attached thereto wherein said mutant GGBP comprises at least one amino acid substitution of a non-reactive amino acid with a reactive amino acid, and
b) a first analyte permeable matrix entrapping or encapsulating said at least one mutant GGBP, said matrix comprising a covalently crossliniked sol-gel comprising organically modified silicates, wherein said sol-gel is selected from at least partially cured hydrolytically condensable siloxanes condensed with at least one water soluble organic polyol component, wherein the first analyte permeable matrix is formulated to allow three-dimensional conformational movement of the at least one mutant GGBP to bind and release the glucose, and wherein the matrix is adapted to allow glucose to diffuse into and out of the matrix.
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This application is a continuation of U.S. Ser. No. 10/039,833, filed Jan. 4, 2002, now abandoned the contents of which are herein incorporated by reference.
1. Field of the Invention
The invention is in the field of biotechnology. Specifically, the invention is directed to entrapped mutated binding proteins, mutated binding proteins containing reporter groups, compositions of mutated binding proteins containing reporter groups in analyte permeable matrixes, and their use as analyte biosensors both in vitro and in vivo.
2. Description of Relevant Art
Monitoring glucose concentrations to facilitate adequate metabolic control in diabetics is a desirable goal and would enhance the lives of many individuals. Currently, most diabetics use the "finger stick" method to monitor their blood glucose levels and patient compliance is problematic due to pain caused by frequent (several times per day) sticks. As a consequence, there have been efforts to develop non-invasive or minimally invasive in vivo and more efficient in vitro methods for frequent and/or continuous monitoring of blood glucose or other glucose-containing biological fluids. Some of the most promising of these methods involve the use of a biosensor. Biosensors are devices capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element which is combined with a transducing (detecting) element.
The biological recognition element of a biosensor determines the selectivity, so that only the compound which has to be measured leads to a signal. The selection may be based on biochemical recognition of the ligand where the chemical structure of the ligand (e.g. glucose) is unchanged, or biocatalysis in which the element catalyzes a biochemical reaction of the analyte.
The transducer translates the recognition of the biological recognition element into a semi-quantitative or quantitative signal. Possible transducer technologies are optical, electrochemical, acoustical/mechanical or colorimetrical. The optical properties that have been exploited include absorbance, fluorescence/phosphorescence, bio/chemiluminescence, reflectance, light scattering and refractive index. Conventional reporter groups such as fluorescent compounds may be used, or alternatively, there is the opportunity for direct optical detection, without the need for a label.
Biosensors specifically designed for glucose detection that use biological elements for signal transduction typically use electrochemical or calorimetric detection of glucose oxidase activity. This method is associated with difficulties including the influence of oxygen levels, inhibitors in the blood and problems with electrodes. In addition, detection results in consumption of the analyte that can cause difficulties when measuring low glucose concentrations.
A rapidly advancing area of biosensor development is the use of fluorescently labeled periplasmic binding proteins (PBP's). As reported by Cass (Anal. Chem. 1994, 66, 3840-3847), a labeled maltose binding protein (MBP) was effectively demonstrated as a useable maltose sensor. In this work MBP, which has no native cysteine residues, was mutated to provide a protein with a single cysteine residue at a position at 337 (S337C). This mutation position was within the binding cleft where maltose binding occurred and therefore experienced a large environmental change upon maltose binding. Numerous fluorophores were studied, some either blocked ligand binding or interfered with the conformational change of the protein. Of those studied IANBD resulted in a substantial increase in fluorescence (160%) intensity upon maltose binding. This result may be consistent with the location of the fluorophore changing from a hydrophilic or solvent exposed environment to a more hydrophobic environment as would have been theoretically predicted for the closing of the hinge upon maltose binding. However this mutant protein and the associated reporter group do not bind diagnostically important sugars in mammalian bodily fluids. Cass also disclosed Analytical Chemistry 1998, 70(23), 5111-5113 association of this protein onto TiO2 surfaces, however, the surface-bound protein suffered from reduced activity with time and required constant hydration.
Hellinga, et al. (U.S. Pat. No. 6,277,627), reports the engineering of a glucose biosensor by introducing a fluorescent transducer into a Galactose/Glucose Binding Protein (GGBP) mutated to contain a cysteine residue, taking advantage of the large conformation changes that occur upon glucose binding. Hellinga et al (U.S. Pat. No. 6,277,627) disclose that the transmission of conformational changes in mutated GGBPs can be exploited to construct integrated signal transduction functions that convert a glucose binding event into a change in fluorescence via an allosteric coupling mechanism. The fluorescent transduction functions are reported to interfere minimally with the intrinsic binding properties of the sugar binding pocket in GGBP.
In order to accurately determine glucose concentration in biological solutions such as blood, interstitial fluids, occular solutions or perspiration, etc., it may be desirable to adjust the binding constant of the sensing molecule of a biosensor so as to match the physiological and/or pathological operating range of the biological solution of interest. Without the appropriate binding constant, a signal may be out of range for a particular physiological and/or pathological concentration. Additionally, biosensors may be configured using more than one protein, each with a different binding constant, to provide accurate measurements over a wide range of glucose concentrations as disclosed by Lakowicz (U.S. Pat. No. 6,197,534).
Despite the usefulness of mutated GGBPs, few of these proteins have been designed and examined, either with or without reporter groups. Specific mutations of sites and/or attachment of certain reporter groups may act to modify a binding constant in an unpredictable way. Additionally, a biosensor containing reporter groups may have a desirable binding constant, but not result in an easily detectable signal upon analyte binding. Some of the overriding factors that determine sensitivity of a particular reporter probe attached to a particular protein for the detection of a specific analyte is the nature of the specific interactions between the selected probe and amino acid residues of the protein. It is not currently possible to predict these interactions within proteins using existing computational methods, nor is it possible to employ rational design methodology to optimize the choice of reporter probes. It is currently not possible to predict the effect on either the binding constant or the selectivity based on the position of any reporter group, or amino acid substitution in the protein (or visa-versa).
To develop reagentless, self-contained, and or implantable and or reusable biosensors using proteins the transduction element must be in communication with a detection device to interrogate the signal to and from the transduction element. Typical methods include placing proteins within or onto the surface of optical fibers or planner waveguides using immobilization strategies. Such immobilization strategies include, but are not limited to, entrapment of the protein within semi-permeable membranes, organic polymer matrixes, or inorganic polymer matrixes. The immobilization strategy ultimately may determine the performance of the working biosensor. Prior art details numerous problems associated with the immobilization of biological molecules. For example, many proteins undergo irreversible conformational changes, denaturing, and loss of biochemical activity. Immobilized proteins can exist in a large number of possible orientations on any particular surface, for example, with some proteins oriented such that their active sites are exposed whereas others may be oriented such that there active sites are not exposed, and thus not able to undergo selective binding reactions with the analyte. Immobilized proteins are also subject to time-dependent denaturing, denaturing during immobilization, and leaching of the entrapped protein subsequent to immobilization. Therefore problems result including an inability to maintain calibration of the sensing device and signal drift. In general, binding proteins require orientational control to enable their use, thus physical absorption and random or bulk covalent surface attachment or immobilization strategies as taught in the literature generally are not successful.
There have been several reports of encapsulating proteins and other biological systems into simple inorganic silicon matrixes formed by a low temperature sol-gel processing methods, for example, as taught by Brennan, J. D. Journal of Fluorescence 1999, 9(4), 295-312, and Flora, K.; Brennan, J. D. Analytical Chemistry 1998, 70(21), 4505-4513. Some sol-gel matrixes are optically transparency, making them useful for the development of chemical and bio-chemical sensors that rely on optical transduction, for example absorption or fluorescence spectroscopic methods. However, entrapped or immobilized binding proteins must remain able to undergo at least some analyte induced conformational change. Conformational motions of binding proteins may be substantially restricted in most sol-gel matrixes as taught in the literature. It has been reported that sol-gel entrapped proteins can exhibit dramatically altered binding constants, or binding constants that change over relatively short time periods or under varying environmental conditions. In addition, a time dependence of the protein function while entrapped in the sol-gel matrix has been reported. This time dependence of protein function in sol-gel entrapped matrixes has limited general applicability of sol-gels in biosensors for in vitro as well as in vivo use.
Therefore, there is a need in the art to design additional useful mutated proteins and mutated GGBP proteins generating detectable signals upon analyte binding for use as biosensors, and additionally there is a need for the entrapment of these proteins into analyte-permeable matrixes for interfacing to signal transmitting and receiving elements.
The invention provides entrapped or encapsulated mutated binding proteins and mutated binding proteins having reporter groups attached thereto, for their use as in vivo or in vitro biosensors. Furthermore the invention provides a glucose biosensor including (a) a mutated binding protein and at least one reporter group attached thereto such that said reporter group provides detectable signal when said mutated binding protein is exposed to glucose and (b) a matrix permeable to analyte where the mutated glucose/galactose binding protein and the reporter group are encapsulated in the matrix.
The invention also provides compositions comprising a mixture including (a) at least one mutated glucose/galactose binding protein and at least one reporter group attached thereto and (b) a hydrogel, dialysis membrane, sol-gel, or combinations thereof to provide for a matrix permeable to analyte wherein the mutated glucose/galactose binding protein and the reporter group are encapsulated in the matrix.
The invention also provides a device including (a) a mutated maltose binding protein (MBP) and at least one reporter group attached thereto such that the reporter group provides a detectable signal when the mutated MBP is bound to maltose and wherein the MBP includes a cysteine present at position 337 and (b) a matrix permeable to maltose wherein the mutated MBP and the reporter group are encapsulated in the matrix.
The invention further provides a device and compositions thereof suitable for in vivo use including (a) a mutated glucose/galactose binding protein and at least one reporter group attached thereto such that the reporter group provides a detectable and reversible signal when the mutated glucose/galactose binding protein is exposed to varying glucose concentrations and (b) a matrix permeable to analyte wherein the mutated glucose/galactose binding protein and the reporter group are encapsulated in the matrix.
The term biosensor generally refers to a device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds, usually by electrical, thermal or optical signals. As used herein a "biosensor" refers to a protein capable of binding to analyte which may be used to detect analyte or a change in analyte concentration by a detector means as herein described.
The term "binding proteins" refers to proteins which interact with specific analytes in a manner capable of transducing or providing a detectable and or reversible signal differentiable either from when analyte is not present, analyte is present in varying concentrations over time, or in a concentration-dependent manner, by means of the methods described. The transduction event includes continuous, programmed, and episodic means, including one-time or reusable applications. Reversible signal transduction may be instantaneous or may be time-dependent providing a correlation with the presence or concentration of analyte is established. Binding proteins mutated in such a manner to effect transduction are preferred.
The term "galactose/glucose binding protein" or "GGBP" or "maltose binding protein" or "MBP" as used herein refers to a type of protein naturally found in the periplasmic compartment of bacteria. These proteins are naturally involved in chemotaxis and transport of small molecules (e.g., sugars, amino acids, and small peptides) into the cytoplasm. For example, GGBP is a single chain protein consisting of two globular α/β domains that are connected by three strands to form a hinge. The binding site is located in the cleft between the two domains. When glucose enters the binding site, GGBP undergoes a conformational change, centered at the hinge, which brings the two domains together and entraps glucose in the binding site. X-ray crystallographic structures have been determined for the closed form of GGBP from E. coli (N. K. Vyas, M. N. Vyas, F. A. Quiocho Science 1988, 242, 1290-1295) and S. Typhimurium (S. L. Mowbray, R. D. Smith, L. B. Cole Receptor 1990, 1, 41-54) and are available from the Protein Data Bank (http://www.rcsb.org/pdb/) as 2 GBP and 3 GBP, respectively. The wild type E. coli GGBP DNA and amino acid sequence can be found at www.ncbi.nlm.nih.gov/entrez/ accession number D90885 (genomic clone) and accession number 23052 (amino acid sequence). Preferred GGBP is from E. coli.
"Mutated binding protein" (for example "mutated GGBP") as used herein refers to binding proteins from bacteria containing amino acid(s) which have been substituted for, deleted from, or added to the amino acid(s) present in naturally occurring protein.
Exemplary mutations of binding proteins include the addition or substitution of cysteine groups, non-naturally occurring amino acids (Turcatti, et al. J. Bio. Chem. 1996 271, 33, 19991-19998) and replacement of substantially non-reactive amino acids with reactive amino acids to provide for the covalent attachment of electrochemical or photo-responsive reporter groups.
Exemplary mutations of the GGBP protein include a cysteine substituted for a lysine at position 11 (K11C), a cysteine substituted for aspartic acid at position 14 (D14C), a cysteine substituted for valine at position 19 (V19C), a cysteine substituted for asparagine at position 43 (N43C), a cysteine substituted for a glycine at position 74 (G74C), a cysteine substituted for a tyrosine at position 107 (Y107C), a cysteine substituted for threonine at position 110 (T110C), a cysteine substituted for serine at position 112 (S112C), a double mutant including a cysteine substituted for a serine at position 112 and serine substituted for an leucine at position 238(S112C/L238S), a cysteine substituted for a lysine at position 113 (K113C), a cysteine substituted for a lysine at position 137 (K137C), a cysteine substituted for glutamic acid at position 149 (E149C), a double mutant including a cysteine substituted for an glutamic acid at position 149 and a serine substituted for leucine at position 238 (E149C/L238S), a double mutant comprising a cysteine substituted for histidine at position 152 and a cysteine substituted for methionine at position 182 (H152C/M182C), a double mutant including a serine substituted for an alanine at position 213 and a cysteine substituted for a histidine at position 152 (H152C/A213S), a cysteine substituted for an methionine at position 182 (M182C), a cysteine substituted for an alanine at position 213 (A213C), a double mutant including a cysteine substituted for an alanine at position 213 and a cysteine substituted for an leucine at position 238 (A213C/L238C), a cysteine substituted for an methionine at position 216 (M216C), a cysteine substituted for aspartic acid at position 236 (D236C), a cysteine substituted for an leucine at position 238 (L238C) a cysteine substituted for a aspartic acid at position 287 (D287C), a cysteine substituted for an arginine at position 292 (R292C), a cysteine substituted for a valine at position 296 (V296C), a triple mutant including a cysteine substituted for an glutamic acid at position 149 and a alanine substituted for a serine at position 213 and a serine substituted for leucine at position 238 (E149C/A213S/L238S), a triple mutant including a cysteine substituted for an glutamic acid at position 149 and a alanine substituted for an arginine at position 213 and a serine substituted for leucine at position 238 (E149C/A213R/L238S).
The entrapped or encapsulated mutated binding proteins of the present invention may be used in an in vitro or in vivo analyte assay which, for example, is capable of following the kinetics of biological reactions involving an analyte or glucose, as well as in clinical assays and food or beverage industrial testing. The concentration of the binding protein in the matrix is preferred to be less than the Kd of the protein with its analyte.
The mutation may serve one or more of several purposes. For example, a naturally occurring protein may be mutated in order to change the long-term stability of the protein; to conjugate the protein to a particular encapsulation matrix, polymer; or to provide binding sites for detectable reporter groups, or to adjust its binding constant with respect to a particular analyte, or combinations thereof.
In the instant invention, analyte and mutated protein act as binding partners. The term "associates" or "binds" as used herein refers to binding partners having a relative binding constant (Kd) sufficiently strong to allow detection of binding to the protein by a detection means. The Kd may be calculated as the concentration of free analyte at which half the protein is bound, or vice versa. When the analyte of interest is glucose, the Kd values for the binding partners are preferably between about 0.0001 mM to about 20 mM.
In the present invention, it has been shown that mutated GGBPs may be used to detect glucose binding by attaching thereto a reporter group which provides a detectable signal upon glucose binding. To "provide a detectable signal", as used herein refers to the ability to recognize a change in a property of a reporter group in a manner that enables the detection of ligand-protein binding. For example, in one embodiment, the mutated GGBPs comprise a detectable reporter group whose detectable characteristics alter upon a change in protein conformation which occurs on glucose binding. In a preferred embodiment, the reporter group is a luminescent label which results in a mutated GGBP with an affinity for glucose producing a detectable shift in luminescence characteristics on glucose binding. The change in the detectable characteristics may be due to an alteration in the environment of the label bound to the mutated GGBP.
The luminescent label may be a fluorescent label or a phosphorescent label. The use of fluorescent labels, which may be excited to fluoresce by exposure to certain wavelengths of light is preferred.
In one embodiment, the reporter group is a fluorophore. As used herein, "fluorophore" refers to a molecule that absorbs energy and then emits light. Non-limiting examples of fluorophores useful as reporter groups in this invention include fluorescein, coumarins, rhodamines, 5-TMRIA (tetramethylrhodamine-5-iodoacetamide), Quantum Red™, Texas Red™, Cy3, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenzoxadiazole (IANBD), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), pyrene, Lucifer Yellow, Cy5, Dapoxyl� (2-bromoacetamidoethyl)sulfonamide, (N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl)iodoacetamide (Bodipy507/545 IA), N-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-N′-iodoacetylethylenediamine (BODIPY� 530/550 IA), 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), and carboxy-X-rhodamine, 5/6-iodoacetamide (XRIA 5,6). Preferably, IANBD is used. Many detectable intrinsic properties of a fluorophore reporter group may be monitored to detect glucose binding. Some of these properties which can exhibit changes upon glucose binding include fluorescence lifetime, fluorescence intensity, fluorescence anisotropy or polarization, and spectral shifts of fluorescence emission. Changes in these fluorophore properties may be induced from changes in the fluorophore environment such as those resulting from changes in protein conformation. Environment-sensitive dyes such as IANBD are particularly useful in this respect. Other changes of fluorophore properties may result from interactions with the analyte itself or from interactions with a second reporter group, for example when FRET (fluorescence resonance energy transfer) is used to monitor changes in distance between two fluorophores.
Although the use of fluorescent labels is desired, it is contemplated that other reporter groups may be used. For example, electrochemical reporter groups could be used wherein an alteration in the environment of the reporter will give rise to a change in the redox state thereof. Such a change may be detected using an electrode.
Furthermore, it is envisaged that other spectroscopically detectable labels, for example labels detectable by NMR (nuclear magnetic resonance), may be used
The reporter group may be attached to the mutated protein or GGBPs by any conventional means known in the art. For example, the reporter group may be attached via amines or carboxyl residues on the protein. However, especially preferred is covalent coupling via thiol groups on cysteine residues. For example, for mutated GGBP, cysteines located at position 11, position 14, position 19, position 43, position 74, position 107, position 110, position 112, position 113, position 137, position 149, position 152, position 213, position 216, position 238, position 287, and position 292 are preferred in the present invention.
Any thiol-reactive group known in the art may be used for attaching reporter groups such as fluorophores to an engineered or mutated protein's cysteine. For example, an iodoacetamide, bromoacetamide, or maleimide are well known thiol-reactive moieties that may be used for this purpose.
Fluorophores that operate at long excitation and emission wavelengths (for example, about 600 nm or greater excitation or emission wavelengths) are preferred when the molecular sensor is to be used in vivo, for example, incorporated into an implantable biosensor device (the skin being opaque below 600 nm). Presently, there are few environmentally sensitive probes available in this region of the spectrum and perhaps none with thiol-reactive functional groups. However, thiol-reactive derivatives of Cy-5 can be prepared for example as taught by H. J. Gruber, et al, Bioconjugate Chem., (2000), 11, 161-166. Conjugates containing these fluorophores, for example, attached at various cysteine mutants constructed in mutated GGBPs, can be screened to identify which results in the largest change in fluorescence upon glucose binding.
Mutated GGBPs useful in the present invention may be engineered or mutated to have a histidine tag on the proteins N-terminus, C-terminus, or both termini. Histidine fusion proteins are widely used in the molecular biology field to aid in the purification of proteins. Exemplary tagging systems produce proteins with a tag containing about six histidines and preferably such tagging does not compromise the binding activity of the mutated GGBP.
As used herein, "matrix" refers to essentially a three-dimensional environment which has at least one binding protein immobilized for the purpose of measuring a detectable signal from ligand-protein interaction. The relationship between the constituents of the matrix and the binding protein include, but are not limited to covalent, ionic, and Van derWals interactions and combinations thereof. The spatial relationship between the matrix and binding protein includes heterogeneous and homogeneous distribution within and or upon any or all of the matrix volume. The matrix may be comprised of organic, inorganic, glass, metal, plastic, or combinations thereof. The matrix provides for the binding protein transducing element configuration which may be for example, be incorporated at the distal end of a fiber or other small minimally invasive probe to be inserted within the tissue of a patient, to enable an episodic, continuous, or programmed reading to the patient. Information from the transducing element to the patient may be provided, for example, by telemetry, visual, audio, or other means known in the art, for example, as taught in U.S. Pat. Nos. 5,517,313, 5,910,661, 5,894,351, and 5,342,789 as well as in Beach, R. D., et al. IEEE Transactions on Instrumentation and Measurement (1999) 48, 6, p. 1239-1245. Information includes electrical, mechanical, and actinic radiation suitable for deriving analyte concentration or change in concentration, as is suitable.
In one aspect of the present invention, the biosensor is used for analyte sensing in vivo. In this aspect, the biosensor is encapsulated into a matrix which may then be used as an implantable device. The "matrix" can be in any desirable form or shape including one or more of disk, cylinder, patch, nanoparticle, microsphere, porous polymer, open cell foam, and combinations thereof, providing it permits permeability to analyte. The matrix additionally prevents leaching of the biosensor. The matrix permits light from optical sources or any other interrogating light to or from the reporter group to pass through the biosensor. When used in an in vivo application, the biosensor will be exposed to a substantially physiological range of analyte and determination or detection of a change in analyte concentration would be desired whereas the determination or detection includes continuous, programmed, and episodic detection means. Thus, the envisaged in vivo biosensor of the present invention comprises at least one mutated binding protein in an analyte permeable entrapping or encapsulating matrix such that the mutated binding protein provides a detectable and reversible signal when the mutated binding protein is exposed to varying analyte concentrations, and the detectable and reversible signal can be related to the concentration of the analyte. The implantable biosensors may, in some embodiments, be implanted into or below the skin of a mammal's epidermal-dermal junction to interact with the interstitial fluid, tissue, or other biological fluids. Information from the implant to the patient may be provided, for example, by telemetry, visual, audio, or other means known in the art, as previously stated.
Preferably, the matrix is prepared from biocompatible materials or incorporates materials capable of minimizing adverse reactions with the body. Adverse reactions for implants include, inflammation, protein fouling, tissue necrosis, immune response and leaching of toxic materials. Such materials or treatments are well known and practiced in the art, for example as taught by Quinn, C. P.; Pathak, C. P.; Heller, A.; Hubbell, J. A. Biomaterials 1995, 16(5), 389-396, and Quinn, C. A. P.; Connor, R. E.; Heller, A. Biomaterials 1997, 18(24), 1665-1670.
The biosensor may be encapsulated into a matrix derived substantially from a hydrogel. The polymer portion of the hydrogel may contain functionality which is suitable for hydrogen bonding or covalent coupling (e.g. hydroxyl groups, amino groups, ether linkages, carboxylic acids and esters and the like) to either the protein or reporter group.
Numerous hydrogels may be used in the present invention. The hydrogels may be, for example, polysaccharides such as agarose, dextran, carrageenan, alginic acid, starch, cellulose, or derivatives of these such as, e.g., carboxymethyl derivatives, or a water-swellable organic polymer such as, e.g., polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyethylene glycol, copolymers of styrene and maleic anhydride, copolymers of vinyl ether and maleic anhydride and derivates thereof. Derivatives providing for covalently crosslinked networks are preferred. Synthesis and biomedical and pharmaceutical applications of hydrogels based on, comprising polypeptides, have been described by a number of researchers. (See, e.g. "Biosensors Fundamentals and Applications", edited by A. D. F. Turner, I. Karube and G. S. Wilson; published from Oxford University Press, in 1988). An exemplary hydrogel matrix derived from a water-soluble, UV crosslinkable polymer comprises poly(vinyl alcohol),N-methyl-4(4′-formylstyryl)pyridinium methosulphate acetal (CAS Reg. No. [107845-59-0]) available from PolyScience Warrington, Pa.
In one embodiment of the encapsulation process, one or more hydrogels in water is added the mutated binding protein in an aqueous buffer solution having a pH in the range of about 4 to about 10 depending on the protein. Subsequent curing of the matrix, for example crosslinking, provides physical form. Using this technique and a conventional fabrication process (e.g. block casting, reverse emulsion polymerization, screen or contact printing, fluid-bed coating and dip or spin coating) one can obtain matrixes in various configurations (e.g. granulates, nanoparticles, microparticles, monoliths, and thick and thin films) suitable for in vitro and in vivo use.
The matrix may, in one embodiment, be comprised of modified sol-gels. Modified sol-gels includes at least partial cured (or gelled) preparations comprised of permeable metal oxide glass structures containing in addition to the sol-gel precursor materials, preferably one or more organic components which hydrolytically condense along with the sol-gel precursor such that the resultant sol-gel matrix imparts properties suitable for, by example, implantation. Suitable properties include low volume shrinkage over time, resistance to cracking and other physical defects, maintenance of protein function, and compatibility with the protein and or reporter group, and compatibility with the animal or subject to which it may be implanted. Suitable organic materials include polyols such as glycerol, ethylene glycol, propylene glycol, polyethylene glycol, and the like for example, as taught by Gill and Ballesteros Journal of the American Chemical Society 1998, 120(34), 8587-8598. It is understood that those skilled in the art can appreciate the attributes described are generally not predictable for a given protein/sol-gel/reporter group combination, thus optimization of sol-gel precursor, organic component and protein solution materials may be expected for any given binding protein-reporter pair. It has been found by the applicants that such optimization may provide for unexpected enhanced signal, shifted binding constants, improved physical performance attributes of the matrix, and combinations thereof relative to that of other matrixes or aqueous solutions thereof. Optimization of performance attributes of the protein-reporter pair and functional performance attributes of the matrix encapsulating same may be achieved, for example, by way of combinatorial methods or other statistical based design methods known in the art.
Sol-gel matrixes useful for the present invention include material prepared by conventional, well-known sol-gel methods and include inorganic material, organic material or mixed organic/inorganic material. The materials used to produce the sol-gel can include, but are not limited to, aluminates, aluminosilicates and titanates. These materials may be augmented with the organically modified silicates (Ormosils) and functionalized siloxanes, to provide an avenue for imparting and manipulating hydrophilicity and hydrophobicity, ionic charge, covalent attachment of protein, and the like. As used herein the term "hydrolytically condensable siloxane" refers to sol-gel precursors having a total of four substituents, at least one, preferably two, and most preferably three or four of the substituents being alkoxy substituents covalently bound to silicone through oxygen and mixtures thereof. In the case of three, two, and one alkoxy substituent precursors, at least one of the remaining substituents preferably is covalently bound to silicone through carbon, and whereas the remaining substitutent contains organic functionality from alkyl, aryl, amine, amide, thiol, cyano, carboxyl, ester, olefinic, epoxy, silyl, nitro, and halogen.
In one embodiment of the encapsulation process, one or more of hydrolytically condensable siloxane is hydrolyzed in water, either spontaneously or under acid or base catalysis to form derivatives with an organic polyol component present in a molar amount relative to the hydrolytically condensable siloxane up to about 10:1 to 1:10, preferably to about 5:1 to 1:5, and most preferably to about 1:1. To this mixture, prior to final gellation, is added the mutated binding protein in an aqueous buffer solution having a pH in the range of about 4 to about 10 depending on the protein. At least partial condensation reactions give rise to the final matrices.
In another embodiment, the hydrolytically condensable siloxane hydrolyzed in water, either spontaneously or under acid or base catalysis to form derivatives with the organic polyol, is mixed with a water soluble polymer component. Suitable water soluble polymers include polyvinyl alcohol (PVA), poly-(maleic acid co-olefin) sodium salt (PMSA), poly-(vinylsulfonic acid) sodium salt (PVSA), and polyvinyl pyrollidone (PVP). Poly-(maleic acid co-olefin) includes copolymers of maleic anhydride with styrene, vinyl ether, and C1-C8 olefins and salts thereof, for example, sodium, potassium, ammonium, tetraakylammonium, and the like. Preferably, the water soluble polymer component is from 0 to about 30% by weight of the sol-gel composition.
In another embodiment the hydrolytically condensable siloxane hydrolyzed in water, either spontaneously or under acid or base catalysis to form derivatives with the organic polyol, is mixed with one or more functionalized silicone additives (FSA) in amounts from 0 to about 0.6% mole ratios to hydrolytically condensable siloxane. Exemplary FSA's include alkyl derivatives: for example, methyltrimethoxysilane (MTMOS): amine derivatives: for example, 3-aminopropyl triethoxysilane (ATEOS); and bis silane derivatives: for example, (bis(3-methyldimethoxysilil)propyl) polypropylene oxide (BIS).
In another embodiment, both the water soluble polymer component and the functionalized silicone additive are mixed together with the hydrolytically condensable siloxane hydrolyzed in water, either spontaneously or under acid or base catalysis to form derivatives with the organic polyol, to provide for a matrix suitable for entrapment or encapsulation of the binding protein. Using the afore-mentioned sol-gel technique and a conventional fabrication process (e.g. block casting, reverse emulsion polymerization, screen or contact printing, fluid-bed coating and dip or spin coating) one can obtain aerogel- or xerogel-matrixes in various configurations (e.g. granulates, nanoparticles, microparticles, monoliths, and thick and thin films) suitable for use in vitro and in vivo.
In another embodiment the matrix may be formed from dialysis membranes. The dialysis membranes can be constructed to physically encapsulate or entrap the protein. Covalent attachment to the membrane is considered within the scope of the as described embodiment. The membrane should be chosen based on its molecular weight cut-off such that analytes of interest can readily permeate the membrane whilst high molecular weight materials would be restricted from entering, or in the case of the mutated binding proteins, leaving the membrane matrix. The molecular weight cut-off required would be such as to meet the afore-mentioned requirement and is within the skill of one familiar with this art. Typically, membranes having molecular weight cut-off between about 1000 to about 25,000 Daltons are suitable. Using this technique, matrixes in various configurations and shapes suitable for use in vitro and in vivo can be prepared.
It is also contemplated that matrixes containing the binding protein and reporter group be combinations of one or more hydrogel, sol-gel, and dialysis membranes. For example, a protein entrapped or encapsulated within a hydrogel or sol gel can be placed within a dialysis membrane of a suitable shape and size as would provide for implantation within a subject, or to manipulate mass-transport properties or permeability of the analytes with respect to the matrix.
The matrix entrapped or encapsulated binding protein biosensors of this invention are capable of measuring or detecting micromolar (10−6 molar) to molar analyte concentrations without reagent consumption. In some embodiments, their sensitivity to analyte may enable the biosensors to be used to measure the low analyte concentrations known to be present in low volume samples of interstitial fluid. The implantable biosensors may, in some embodiments, be implanted into or below the skin of a mammal's epidermal-dermal junction to interact with the interstitial fluid, tissue, or other biological fluids. The binding protein biosensors of the present invention provide for the means to monitor analyte continuously, episodically, or "on-demand" as would be appropriate to the user or to the treatment of a condition.
In other embodiments, the biosensors sensitivity to analyte (for example glucose) is such that they may be used to test blood analyte levels or the concentration of analyte in a biological solution or other solution may be determined. As used herein, a "biological solution" includes but is not limited to blood, perspiration, and or ocular or interstitial fluid including combinations thereof.
The following examples illustrate certain preferred embodiments of the instant invention, but are not intended to be illustrative of all embodiments. Labeled mutated maltose binding protein S337C MBP with fluorophore reporter probe NBD used herein in accordance with the procedure set forth by Cass, A. et al. (Anal. Chem. 1994, 66, 3840-3847). Fluorescence emission spectra of mutated, labeled protein was measured using an SLM Aminco fluorimeter (Ontario, Canada) with slit settings of 8 and 4 for excitation and settings of 5 and 5 on the MC250 emission monochromator to compare the ligand-binding performance of the entrapped fluorophore-labeled proteins in various matrixes to the performance of the same proteins in solution. The initial fluorescence emission intensity is defined as Io. The relative ratio of the emission intensity maxima in the presence of the protein's respective ligand (If) to the ligand's absence (Io) is defined as ΔF.
Binding constants were determined by titration of increasing concentrations of glucose into a protein solution with mixing following each addition of glucose. Slit settings were the same as listed above. The Kd was determined from the following relationships as adapted from Pisarchick and Thompson (1990):
where F is fluorescence intensity, Finf is fluorescence at infinity, F0 is fluorescence at zero glucose, and x is the free concentration of glucose ([Glc]free) as determined by the relationship:
where [Glc]tot and [Prot]tot are the total concentrations of glucose and protein, respectively.
This example describes the method for the expression and purification of mutant Proteins Without Histidine Tags. GGBP is coded by the Mg1B-1 gene in E. coli. This protein was altered by introducing the amino acid cysteine at various positions through site-directed mutagenesis of the Mg1B-1 gene. These proteins were then expressed in E. coli and purified.
Cassette mutagenesis of Mg1B-1 was accomplished as follows. The wild-type Mg1B-1 gene was cloned into a pTZ18R vector (Dr. Anthony Cass, Imperial College, London, England). Mutant plasmids were generated from this parent plasmid using cassette mutatgenesis producing randomized amino acid sequences, essentially as described by Kunkel (1991) and cloned in E. coli JM109 (Promega Life Science, Madison, Wis.). Mutant plasmids were identified by sequencing. The mutant protein was induced in JM109 and purified as described below. An E. coli JM109 colony containing the mutant plasmid was grown overnight at 37� C. with shaking (220 rpm) in LB broth containing 50 μg/mL ampicillin (LB/Amp). The overnight growth was diluted 1:100 in 1 L fresh LB/Amp and was incubated at 37� C. with shaking until the OD600 of the culture was 0.3-0.5. Expression of the mutant was induced by the addition of 1 mM IPTG (Life Technologies, Gaithersburg, Md.) final concentration with continued incubation and shaking at 37� C. for 4-6 hours. The cells were harvested by centrifugation (10,000�g, 10 min, 4� C.).
The mutant protein was harvested by osmotic shock and was purified by column chromatography. The cell pellet was resuspended in a sucrose buffer (30 mM Tris-HCL pH 8.0, 20% sucrose, 1 mM EDTA), incubated at room temperature for 10 min, and then centrifuged (4000�g, 15 min, 4� C.). The supernatant was poured off and kept on ice. The cell pellet was resuspended, and 10 mL ice cold, sterile deionized H2O was repeated, and the suspension was incubated on ice and centrifuged. The remaining supernatant was pooled with the other collected supernatants and was centrifuged once again (12,000�g, 10 min, 4� C.). The pooled shockate was filtered through a 0.8 μm and then a 0.45 μm filter. Streptomycin sulfate (Sigma Chemical Co., St. Louis, Mo.), 5% w/v, was added to the shockate and was stirred once for 30 min followed by centrifugation (12,000�g, 10 min, 4� C.). The shockate was then concentrated using the Amicon Centriprep 10 (10,000 MWCO) filters (Charlotte, N.C.) and dialyzed overnight against 5 mM Tris-HCl pH 8.0, 1 mM MgCl2. The dialyzed shockate was centrifuged (12,000�g, 30 min, 4� C.). The resulting supernatant was added to a pre-equilibrated DEAE Fast Flow Sepharose column (Amersham Pharmacia Biotech, Piscataway, N.J.) at 0.5 mL/min. The column was washed with 5-10 column volumes. A linear gradient from 0-0.2 M NaCl was applied to the column and fractions were collected. The mutant protein containing fractions were identified by SDS-PAGE with Coomassie Brilliant Blue staining (mw. Approx. 32 kDa). The fractions were pooled and dialyzed overnight (4� C.) against phosphate buffered saline (PBS) or 10 mM ammonium bicarbonate (pH 7.4) concentrated using Amicon Centriprep 10 filters, and stored at 4� C. or −20� C. with glycerol. The ammonium bicarbonate dialyzed protein was lyophilized.
This example describes the expression and purification of mutant GGBPs containing Histidine Tags. GGBP mutants were engineered by either site-directed mutagenesis or the cassette mutagenesis. Site-directed mutagenesis (QuikChange, Stratagene, La Jolla, Calif.) was performed to alter individual amino acids in the pQE70 vector by replacing one amino acid with another, specifically chosen amino acid. The cassette mutagenesis method (Kunkel 1991) was performed to randomize amino acids in a specified region of the GGBP gene. The mutated cassettes were then subcloned into the pQE70 expression vector. The pGGBP-His plasmid contained the GGBP gene cloned into the pQE70 expression vector (Qiagen, Valencia, Calif.). This construct places six histidine residues on the C-terminus of the GGBP gene. E. coli strain SG13009 was used to over express mutant GGBP-His following standard procedures (Qiagen). After over expression of a 250 mL culture, the cells were collected by centrifugation (6000 rpm) and resuspended in 25 mL bugbuster (Novagen, Madison, Wis.). Lysozyme (25 mg was added to the lysate and the mixture was gently mixed at room temperature (RT) for 30 min. Clear lysate was produced by centrifugation (6000 rpm) and to this, 0.5 ml imidizole (1 M) and 3 ml of Ni-NTA beads (Qiagen) was added. After 30 minutes of gently mixing at RT, the mixture was centrifuged (6000 rpm) and the lysate removed. The beads were washed with 25 ml of solution (1M NaCl, 10 mM tris, pH 8.0) and recentrifuged. The mutant GGBP-His was eluted from the beads by adding 5 mL solution (160 mM imidazole, 1 M NaCl, 10 mM Tris, pH 8.0) and mixing for 15 min. The protein solution was immediately filtered through a Centriplus YM-100 filter (Amicon, Charlotte, N.C.) and then concentrated to 1-3 mg/ml using a Centriplus YM-10 filter. The protein was dialyzed overnight against 2 L of storage solution (1 M NaCl, 10 mM Tris, 50 mM NaPO4, pH 8.0).
This example describes generically the labeling of binding protein with reporter probe. An aliquot of mutant GGBP containing cysteine (4.0 nmol) in PBS was treated with 2 mM dithiothreitol (5 μL, 10 nmol) for 30 min. A stock solution of N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD amide, 0.5 mg) was prepared in DMSO (100 μL, 11.9 mM) and 3.36 μL (40 nmol) was added to the protein. The reaction proceeded at room temperature for 4 h on a Dynal rotamix in the dark. The labeled protein was purified by gel filtration on a NAP-5 column (Amersham Pharmacia). The labeling rations were determined using an estimated extinction coefficient (50 mM−1 cm−1) for GGBP that was calculated in GeneWorks 2.45 (IntelliGenetics), ε478 (IANBD amide)=25 mM−1cm−1), and a measurement of O.D. for a standard solution of IANBD amide at 280 nm and 478 nm. The dye concentration in the protein was calculated as Cdye=γ478/A478. The absorbance of protein at 280 nm was calculated as Aprot(280)=Atotal(280)−Adye(280), where Adye(280)=A478�(A280/A478)standard. The concentration of protein was then Cprot(280)=ε280/Aprot(280).
This example describes the immobilization of a biosensor of the instant invention using glycerol modified silicate condensate (GMSC). The additions of glycerol directly followed the initial tetraethoxyorthosilicate (TEOS) or tetramethoxyorthosilicate (TMOS) acid hydrolysis. A range of hydrolysis times, pH levels, reagent addition order, and TEOS:glycerol ratios were evaluated to determine the optimal conditions for beginning the glyceration reaction. Preferred conditions were found using an interval of 10 to 30 minutes between hydrolysis and glycerol addition, a pH range of between 0.5 and 1, and a 1:1 mole ratio of TEOS to glycerol. The following describes a modified procedure of Gill and Ballesteros for a TEOS-based glycerol modified silicate condensate (GMSC) preparation using the following ratios of reagents: TEOS or TMOS:1; H2O:1, Methanol:4, Glycerol:1. TEOS or TMOS in methanol was added to a flask and cooled to 0� C. over ice. Next 0.6M HCl was added drop-wise to the solution. After 20 minutes of stirring, glycerol was added dropwise. The reaction was warmed slowly over 1-2 hours to 20-25� C. Following this the reaction vessel was heated further and maintained at a temperature range of 60-70� C. under nitrogen for between 36 and 42 hours. The optimal time was 40 hours. Incomplete glyceration was indicated by an observable phase separation for reactions stopped before 36 hours. Reactions maintained beyond 42 hours produced GMSC sol-gel monoliths with greatly reduced physical properties, for example, increased brittleness. Following the 40 hour reaction at 60-70� C., the solution volume was reduced by rotary evaporation until it was viscous and transparent, at which point methanol was added to the solution in a 4:1 ratio by weight. This GMSC solution proved to be stable and provided consistent results for several months when stored at freezer temperature. When the GMSC solution was to be used, methanol was removed by rotary evaporation and distilled water was added in a 1:1 ratio by weight to the GMSC reagent to catalyze the final hydroylsis/gelation. Monoliths, thin films, and powders were created with this procedure using an appropriate container to function as a mold. The GMSC sol-gel monoliths were not brittle and had shrinkage of about 8% after curing at 4� C. at 50% relative humidity for 2 weeks (% shrinkage was the average of changes in diameter and length measured with a microcaliper and compared to original mold dimensions). Electron microscopy (SEM) further illustrated the significant improvements in surface fracturing between monoliths created with TEOS hydrolysis and the monoliths created through the GMSC procedure described above. This set of experiments demonstrates how sol-gels with improved physical characteristics can be produced in accordance with the methods taught in the instant invention.
This example describes further optimization of physical properties by GMSC sol-gels in which glycerol has been partly substituted with either ethylene glycol (EG) or polyethylene glycol (PEG). Ethylene glycol (EG) was evaluated as a substitute for glycerol in mixtures where the ratio of glycerol and EG was varied but the mole ratio of total glycerol and EG was maintained constant relative to other reagents. Sol-gel monoliths were prepared by the procedure described in the preceding example, cured for two weeks at 4� C. and 50% relative humidity and their % shrinkage was determined and stated in Table 3. % Shrinkage is defined as the average of the decrease in length and diameter versus original dimensions. Monoliths used for determination of shrinkage had no protein/fluorophore present. For ΔF measurements, the samples listed in Table 1 were prepared containing H152 GGBP-H6 NBD (from Example 3) as will be described shortly.
The 1% and 5% EG/GMSC sol-gels (entries 5 and 6 respectively in Table 3) were found to have significantly less % shrinkage than either the plain TEOS sol-gels or GMSC modified TEOS sol-gels (entries 2 and 3 respectively in above Table 3). Polyethylene Glycol (PEG) was also evaluated qualitatively as a partial substitute for glycerol in similar proportions in GMSC sol-gels and produced monoliths with favorable surface properties and rubber-like flexibility. In summary, partial substitution of either ethylene glycol (EG) or polyethylene glycol (PEG) for glycerol in GMSC sol-gels provides improvements in physical properties, for example, minimized shrinkage and reduced surface fracturing. These sol-gel matrixes containing binding protein were found to possess performance equal to or better than that of protein in solution.
Entrapment Of Binding Proteins In GMSC Sol-Gels Containing Functionalized Silicone Additives (FSA) and Polymers. This example describes the addition of polymer and organic polyol additives to optimize the GMSC sol-gels for entrapping binding proteins to both maintain and enhance their spectral properties upon ligand binding. The binding proteins were labeled with a fluorophore (as described in example 3). The protein solutions were added during the final hydrolysis/gelling step described previously to produce final concentrations of 2-4 μM protein within the sol-gel. The polymer additives and FSA's were obtained from Sigma-Aldrich Chemicals (St. Louis, Mo.). Polymer additives were evaluated in amounts between 0 to about 30 wt. Functionalized silicone additives (FSA) were evaluated as additives to the GMSC sol-gels in amounts from 0 to about 0.6% mole ratio. Thus, rotary evaporation of the GMSC reagent to remove methanol from its storage solution was followed by reconstitution in water in a 1:1 ratio by weight. To a 400 μL aliquot of this mixture, 800 μL of buffer (HEPES, PBS or Tris) with a premixed water soluble polymer additive was added along with any FSA-modified GMSC. A mutated binding protein in solution was then, and after thorough mixing, 100 μL of the mixture was dispensed into a 96 well microplate (Falcon white flat bottom plates, product # 35-3941, BD Labware, N.J.). The sol-gel containing microplates were cured 12-18 hours at 4� C. and 50% relative humidity. GMSC-BIS was prepared by the same procedure as the TEOS-based GMSC, but with substitution of (Bis(3-methyldimethoxysilyl)propyl) polypropylene oxide for TEOS. GMSC-MTMOS and GMSC-ATEOS were prepared similarly except that the hydrolysis was carried out with either 10% of the amount of acid, or no acid in the hydrolysis step, respectively compared to the TEOS-based GMSC procedure. Fluorescence emission was measured with a Varian Cary Eclipse scanning fluorometer with microwell plate adapter (Varian Instruments, Victoria, Australia). Excitation was at 475 nm and emission recorded from 500 to 600 nm, typically monitoring emission maximum peak fluorescence. Slit widths were 5 nm for excitation and 10 nm for emission. Individual Io determinations were made for each well and 100 μL of a ligand solution (1 mM maltose in the case of S337C MBP, and 10 mM glucose in the case of H152C GGBP, or 100 mM glucose in the case of A213C/L238C) was added and If readings were obtained, from which ΔF values were calculated. The modified sol-gel entrapped proteins exhibited greater initial fluorescence (Io) in the absence of ligand when compared to equivalent concentrations of the same protein in solution.
The formulation optimization experiments described above used Design-Expert 6.0.5 (Stat-Ease, Inc., Minneapolis, Minn.) to design several Design of Experiments (DOE's). Among other variables in formulation which were optimized in each DOE were buffer type (HEPES, PBS and Tris) and pH (from 6.6 to 7.8). Surprisingly, the optimal formulation constituents and concentration ranges were quite different for each protein. In all cases, however, substantial performance improvements were obtained for the optimized formulations in comparison to either solution performance or performance in unmodified sol-gels.
This example describes the entrapment of GGBP H152C in UV cross-linked hydrogel matrix and the effect of the matrix on the fluorescence change and binding affinity. In this experiment SbQ-PVA from Polysciences Inc. was added 100 ul of PBS buffer and mixed for one hour to mix in a rotary mixer. 80 ul of this solution was then mixed with 20 ul of labeled protein. Final protein concentration was spectroscopically determined to be 0.15 mg/ml. After mixing, aliquiots were dispensed into 96-well plates and dried in a chamber maintained at 20% humidity for 12 h followed by curing with UV light. Wells containing protein encapsulated in matrix were challenge with 2 ul of 10 mM glucose and compared to protein solution without matrix having equivalent protein loading.
This example describes the immobilization of a biosensor of the instant invention into a dialysis membrane matrix and the ability of the matrix to provide reversible and continuous readings. Using a Varian Eclipse fluorimeter with a fiber optic attachment, GGBP L238C/A213C protein (2 μM in PBS buffer) entrapped within a dialysis membrane having a molecular cut-off of 3500 Daltons affixed to the distal end of the fiber. Solutions were prepared containing PBS buffer, 2 mM, and 20 mM glucose in PBS buffer. With the probe in PBS solution, readings were recorded at 0.02 seconds intervals of the emission wavelength 521 nm, followed by insertion of the fiber into the glucose solutions. Replacement of the fiber into buffer-only solution resulted in the return of initial signal.
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text:
An anonymous hacker offers some tips on how to protect yourself online:
An anonymous hacker offers some tips on how to protect yourself online:
I was really hoping this was going to be a "Get a Mac" joke…
Can't think about this too long…or I get freaked out…
I'm on Facebook "timeout" right now, but I've always squirmed a bit when people post status updates like, "leaving for a week vacation to Greece and no one is home but the cat".
Seems like that also is just asking for trouble.
Thanks. Very useful. It's interesting why a hacker would want to help others.
Yeah….it does make you wonder. Consider me "Puzzled Christian"
Hacker does not mean what you think it might mean.
Some Sysadmin people call themselves white hat hackers. (referencing old westerns)
So there are hackers who use there ability for good and hackers that use their ability for illegitimate game.
And then their are hackers that use their ability illegally thinking they are helping people. (AKA people who took down the scientology website and servers a while back)
Comments are closed.
Justin Taylor is senior vice president and publisher for books at Crossway and blogs at Between Two Worlds. You can follow him on Twitter.

subset: commoncrawl


text:
The UALR Student Philanthropy Council, Trojans Supporting Trojans, aims to create a culture of tradition of giving FOR students BY students.
Nov. 26, 2013Â New student philanthropy council supports peers
June 10, 2014Â Student philanthropists donate to scholarship honoring Boccarossa
January 8, 2015 Student philanthropists raise $1,700 for memorial scholarship
The Purpose
- Educate students on the need for philanthropy on campus and begin a culture and tradition of giving back to UALR.
- Encourage a culture of giving back, both as students and alumni.
- Instill in students the importance of alumni support and involvement.
- Raise money for The Annual Fund for UALR.
The Need
- UALR budget last year: $170 million
- State appropriations covered 38%
- Tuition and fees covered 44%
- Remaining 18% had to be covered by private philanthropy. That amount was just over 30 million dollars.
- This year, UALR awarded more than 1,000 privately funded scholarships totaling over $1 million.
 How Can I Get Involved?
- The Trojans Supporting Trojans Council will distribute banks and information on the need for philanthropy and The Annual Fund for UALR.
- Students may check out a logoed piggy bank from the Office of Alumni or a member of the Trojans Supporting Trojans Council Member.
- Students are encouraged to fill their banks and let their "change make change".
- All banks returned at the end of the semester will be receipted to those who checked out a bank as a donation to UALR.
How Can I Join the UALR Student Philanthropy Council?
- Apply Now for Fall 2014- Spring 2015
- For more information contact Derek Boyce at 683-7355.


text:
Dancing With The Stars
DWTS All-Stars Pro Speculation: Who Will Dance With Whom?
Twelve celebrities have been named for Dancing With the Stars All-Stars Season 15, with one more to come when we're done voting. But what about their pro partners? The official pairings will be revealed on Monday, August 13, but everyone is already busy speculating. Will some original couples stay together? Will everyone get a new partner?
ABC posted 12 pro photos on its "professionals" link — Anna Trebunskaya, Chelsie Hightower, Cheryl Burke, Derek Hough, Karina Smirnoff, Kym Johnson, Maksim Chmerkovskiy, Mark Ballas, Peta Murgatroyd, Tony Dovolani, Tristan MacManus, and Val Chmerkovskiy.
Those are the same pros from Season 14, so either they just left the photos up, or we can expect these exact pros to return for Season 15 — plus a final male or female pro, depending on whether Carson Kressley, Kyle Massey, or Sabrina Bryan is voted in as the 13th contestant.
It sounds like even the pros themselves still don't know with whom they will be paired. Cheryl noted that she has three past partners in the mix and Mark has two (potentially three if Sabrina returns). Anna doesn't have anyone from her past unless Carson is voted in. But she's not worried about the celeb she gets. Anna tweeted on July 28, "I appreciate all the speculations but will know who my partner is soon enough + few weeks. So whoever I get It'll be great! #dwts."
So with absolutely nothing to go on, we're jumping into the water with pro/celeb partner speculation, assuming — perhaps falsely — that at least a few original couples will stay together.
Gilles Marini & Cheryl Burke
Gilles & Cheryl have unfinished business from Season 8. Cheryl won with her other two partners on All-Stars (Drew Lachey and Emmitt Smith), so she needs to at least try to get Gilles that Mirror Ball trophy. We think it wouldn't be fair for past champions to re-team, but runners-up or third-place contestants are fine to try again. If anyone ends up re-paired, it has to be Gilles and Cheryl — especially since they both said they would only return to dance with each other.
Kirstie Alley & Maksim Chmerkovskiy
Maks was very much against the idea of re-pairing couples — and since Season 12 runner-up Kirstie is the only one of his past partners returning, chances are he'll get her again. If not, what are they going to do, give him to Bristol Palin just to see if they fight? Kirstie and Maks brought out the best in each other and it would be nice to see that Maks return and enjoy himself, whether they win or not.
Melissa Rycroft & Tony Dovolani
All we want for Tony is for him to have fighting chance with a really talented partner — and he could have a shot at winning with his Season 8 partner, Melissa. Melissa is young, fit, determined, and a good fit with Tony. They would be Team Smile with those insanely bright white teeth. Melissa and Tony took third place on Season 8, and this would be their chance to take out the two competitive stars who beat them — Shawn Johnson and Gilles Marini.
Joey Fatone & Kym Johnson
Maybe they won't re-team Season 4's Joey and Kym, but since they weren't champions, why not? Joey has more recent experience from DWTS: Live in Las Vegas and by now Kym already has two Mirror ball trophies under her belt so she knows what it takes to win. If Joey isn't re-paired with Kym, we'd guess he gets Peta — since Kym and Peta are pretty tall and Joey is one of the taller male All-Stars.
Emmitt Smith & Karina Smirnoff
Karina has never danced with a football player, so maybe it's her turn. Kym and Peta have already won with football players so they shouldn't get another instant advantage. Cheryl already won with this particular football player so she definitely shouldn't get another chance with Twinkle Toes. If Karina doesn't get Emmitt, we'd guess either Anna or Chelsie will.
Pamela Anderson & Val Chmerkovskiy
Pam is going to need help — extreme dancing assistance (since she didn't even come close to the finals and she's competing against six champions) and someone to flirt back with her so she doesn't come off as cougar creepy, the way she kind of did back on Season 10 with Damian Whitewood. Derek would be great with giving Pamela dancing instruction and patience, and you can't argue with his success rate. Tristan would bring his adorable attitude, patience, and fan base. Val, on the other hand, would bring his dance skills and Chmerkovskiy fan base and — and this is key — he would probably flirt back and give as good as he got. Would they make it to the end? Who knows. But it would be fun to see their chemistry together. Val would enjoy himself for as long as they were around.
Helio Castroneves & Peta Murgatroyd
Frankly, we have no idea where to put the lovely Peta so we're putting her here. She's already danced with two athletes — and some people consider race-car drivers athletes — but she's also tall and some of these guys are really short. No idea what to do with the reigning champion but whoever gets her will be lucky. Helio is adorable and had such a great energy with Julianne. If he doesn't get Peta, maybe Karina or Chelsie?
Shawn Johnson & Tristan MacManus
Originally we had Mark Ballas here before remembering — oh yeah, he and Shawn won together. It's possible DWTS will re-pair some champs, but that seems like a way unfair advantage. Shawn is a 20-year-old Olympian with boundless energy, a huge fan base and a smile for days. Our favorite lucky charm has yet to have a young partner — and he and Shawn would be so cute together just imagining their Jives and Cha-Chas makes rainbows appear in the sky.
Drew Lachey & Anna Trebunskaya
Drew has mad skills, a likable personality, and a competitive attitude. He knows what it takes to win. As he showed with Cheryl back on Season 2, if you give him an ambitious routine — like their famous Freestyle — he can pull it off in spades. The last time Anna had a partner with as much natural skill as Drew was back on Season 10 with Evan Lysacek. They made it to second place and it's possible Drew and Anna would able to climb the list just as well. Then Anna would just need to bring it out in the Freestyle.
Kelly Monaco & Mark Ballas
Kelly will probably be the rustiest of all the stars since she was the original winner, back in 2005 on Season 1, when there were only six couples. Mark is patient and creative and has a good success rate. Plus, Kelly is still fit and gorgeous, so once she gets up-to-speed they could be a dark horse couple to watch.
Apolo Anton Ohno & Chelsie Hightower
Olympian Apolo is a frontrunner all on his own, so it would be nice to see him with a young, hungry pro like Chelsie, who has yet to make it to the finals. She has a great chill energy — just like she did with another athlete, Louie Vito — and they would be cute together.
Bristol Palin & Derek Hough
Derek has won DWTS three times and he's never been voted out early. Every time he succeeds, someone sniffs that it's because he always gets the best partners. Well, bless her heart, Bristol has never been confused with the best partner. She definitely has her own fan base, and they helped her take third place with Mark back on Season 11, but she's going to be competing against six past winners and at least three runner-ups. She'll need to bring more than she did before and that's where Derek comes in. If anyone can make her great, it's him. Plus, Mark is his best friend, so he can give Derek tips on what works with her and what doesn't. If Derek doesn't get Bristol, we'll guess Tristan does.
Sabrina Bryan & Mark Ballas
If Sabrina is chosen as the "Viewers Choice," she'll probably be re-paired with her Season 5 partner, Mark. That would change our list above, perhaps giving Dmitry Chaplin or Louis van Amstel an opportunity to compete.
Kyle Massey & Lacey Schwimmer
Dying for Kyle and Lacey to come back. They were so much fun on Season 11 and Lacey is nowhere to be found on this list yet. It would be great to see them both again.
Carson Kressley & Anna or Kym
Carson and Anna already had their fun back on Season 13 and they could dance together again — or maybe Carson could dance with his DWTS BFF, Kym, and Anna could take Kym's old partner, Joey. Unless Peta gets Joey, since Peta's pretty tall and so is Joey. Then maybe Anna with Helio? This whole thing is so confusing. Maybe Carson should just dance with Tom Bergeron. He's usually just standing there — ask him to dance!
What partnerships are you dying to see? Let us know in the comments below.
Dancing With the Stars Season 15: All-Stars will premiere on Monday, September 24 at 8 p.m. ET/PT on ABC.
Gina Carbone is a contributing writer at Wetpaint Entertainment. Follow her on Twitter @ginacarbone.
For more news about Dancing With the Stars, Like us on Facebook and Follow us on Twitter.


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Jonathan Feinberg
I haven't really seen any thread on Jonathan in here; I first heard him on Lisa Loeb's cut Stay (I Missed You) off the Reality Bites soundtrack back in the early 90s. Her album, Tails, featured Jonathan too if I'm not mistaken. I always wondered what drums he played on, and later went on to find that he played Brady. His exact specs and that poppin' snare, though, remains a mystery. Fine pop-rock playing, beautiful feel, deep pocket.
Re: Jonathan Feinberg
Re: Jonathan Feinberg
Jonathan Feinberg here, and happy to answer any questions about my dim, dim memories of making Tails, or anything else. :)
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UNDP's support in the regional-level fight against this epidemic is led by the HIV/AIDS Regional Programme in the Arab States (HARPAS). Launched to heighten awareness and build commitment to scale up prevention measures to the spread of HIV/AIDS in the region, HARPAS works with a broad range of partners including religious leaders, NGOs, parliamentarians, the media, the private sector, and people living with HIV/AIDS.
HIV/AIDS Reports
Featured Publications
Exploring the Dynamics and Vulnerabilities of HIV Transmission Amongst Sex Workers in the Palestinian Context 31 Jul 2013
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North Charleston police have released photos of a suspect in three jewelry store burglaries.
Investigators believe the same man pulled off all three burglaries, and possibly other burglaries in other jurisdictions, North Charleston police spokesman Spencer Pryor said.
The most recent incident was Monday at Polly's Jewelry on Rivers Avenue. An alarm went off about 1:30 a.m., and an officer found a window shattered, according to an incident report.
Video from the store showed a white man with something black covering the lower half of his face, wearing a long-sleeved camouflage shirt, gray pants, white hat with brown writing and black shoes, according to the report. The owner was doing an inventory to find out what was missing.
A window was broken out of the same jewelry store about 3:40 a.m. on Aug. 30. Store video showed a light-skinned man with a white cloth covering the lower half of his face entering the store. Again, an inventory was being conducted to discover what was taken.
Zales at Tanger Outlets was burglarized about 6 a.m. on Aug. 11. A security guard chased a man carrying a trash can from the store after seeing a window broken, according to a police report. The guard described the man as white, between 25 and 35 years old, between 5 feet 5 and 8 inches tall and weighing about 150 pounds.
A video showed the man was wearing a light gray hoodie with USC on the chest. He broke into several cases and took earrings, watches and rings, according to the report. Most of the items spilled out of the trash can when he tripped while running away, the guard told police.
Investigators don't see a connection between the North Charleston burglaries and the one in Summerville this month in which three men from Charlotte was arrested, he said.
Anyone with information about the North Charleston burglaries is asked to call police at 554-5700 or leave an anonymous tip with Crime Stoppers at 554-1111.
Reach Dave Munday at 937-5553 or follow him on twitter @dmunday.
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The New Twinkies: Actually the Sweetest Comeback in the History of Ever?
Like all good Americans, I have a fondness for Twinkies, though I suspect that's more informed by their hallowed status in pop culture than an actual appreciation for them as a dessert. With the new version on the market (spoiler alert: yes, they have been tweaked), it was time to revisit the king of snack cakes. More


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Fit in S.A.
Jessica Belasco on the latest on local fitness news, upcoming races, gym openings and how San Antonio residents are getting in shape.
Monthly Archive for November, 2009
Aging with a shimmy and a shake
Think growing older means think growing less active? Then you should check out the Move Free Dancers performing at the Senior Lifestyles Show next week. [Read More]
Latino health Web site launches
In an effort to promote healthier lifestyles among Latinos, the Institute for Health Promotion Research at the University of Texas Health Science Center at San Antonio has launched a Web site, www.SaludToday.com, focusing on health news, resources and events. You can learn about the latest health research on the blog, watch public service announcements, read […] [Read More]
Review: Valerie Bertinelli's Losing It and Keeping Fit!
(Courtesy photo) Celebrity fitness DVDs are a mixed bag. I've seen some great ones. I've seen some not-so-great ones. Valerie Bertinelli (of "One Day at a Time" fame) is the latest celebrity to come out with an exercise DVD, on the heels of her much-publicized weight loss and new book "Finding It: And Satisfying My […] [Read More]
Harvey Walden IV returns to pump S.A. up
(Courtesy photo) Retired drill Sgt. Harvey Walden IV (left) and trainer Bay Bay McClinton team up on Saturday for another round of Boot Camp: The Fitness Challenge. Think you're fit? Why don't you let the experts be the judge? San Antonio trainer Bay Bay McClinton is again teaming up with Harvey Walden IV, the tough […] [Read More]
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By Army Staff Sgt. Jim GreenhillNational Guard Bureau
WASHINGTON, June 24, 2010 About 1,200 National Guard troops will support the Homeland Security Department in enhancing the security of the nation's Southwest border, Homeland Security Secretary Janet Napolitano said here this week.Video
The Guardsmen will assist in ongoing efforts to secure the border and combat cartels, Napolitano said during remarks June 22 at the Center for Strategic and International Studies about ongoing Homeland Security initiatives and new strategies to bolster border security and enforce immigration laws.
The National Guard deployments have worked over the last 20 years in a coordinated fashion that frees up border patrol agents for other aspects of their duties, David Aguilar, deputy commissioner of U.S. Customs and Border Protection, said during a panel discussion following Napolitano's remarks.
"Support from the National Guard - but yet a clear division: them not arresting, not engaging in enforcement activities directly attributed to any illegal crossings of either aliens, narcotics or things of that nature …
has worked out very well," Aguilar said. "The National Guard … will bring us a tremendous amount of capability in securing our borders."
Homeland Security officials work with federal, state, local, tribal and Mexican partners to crack down on border-related crime and smuggling while facilitating legitimate travel and commerce.
President Barack Obama has requested $500 million in supplemental funds for enhanced border security and law enforcement activities in addition to the deployment of National Guard troops.
Napolitano announced several additional initiatives that include creating new partnerships with state and local law enforcement, building information-sharing capabilities, enhancing technology, prioritizing the arrest and removal of dangerous criminal aliens and expanding unprecedented law enforcement partnerships with Mexico.
The National Guard supported the border security mission during Operation Jump Start from 2006 to 2008. The latest mission, employing 1,200 volunteers drawn from the four border states, will provide criminal investigative analysis and entry identification teams that will monitor the border from strategic observation points with state-of-the-art surveillance and detection tactics and technology for one year.
The deployment of National Guard troops is part of a multi-layered effort targeting illicit networks trafficking in people, drugs, illegal weapons and money, Guard officials said.
Related Sites:Department of Homeland Security National Guard Bureau
Article is closed to new comments.
The opinions expressed in the following comments do not necessarily reflect those of the U.S. Department of Defense.
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Just before midnight, NYPD officers moved in on the southwest corner of Liberty Park, the site of Occupy Wall Street for the past month, to take down and confiscate a medical tent that had been erected during the day. A commotion immediately erupted in this section of the park. Occupiers rushed over and a human chain around the tent was formed. And, Rev. Jesse Jackson suddenly appeared to help the occupiers defend the medical tent from being forcibly removed.
Rev. Jackson told the occupiers, "I am not visiting, I'm participating." When asked to link arms and help the occupiers defend the tent, he linked arms with them. They stood their ground and were able to convince the NYPD to back down. [Here is video of the human chain. And, here are photos. ]
I suspected a raid of some kind would happen. Last night, around 6:30 pm ET, I watched a suit and an officer, who both appeared to be high-ranking, stand by the sanitation work station and discuss something in the park that needed to be addressed. I couldn't hear the conversation, but I sensed they were planning something. [cont'd.]
The medical tent is the only tent up in the camp. It would seem, given the peaceful nature of the occupation, an agreement could be worked out to keep the tent erected. But, that has not stopped Mayor Michael Bloomberg from lying about Occupy Wall Street by labeling the camp a "tent city."
"The Constitution doesn't protect tents," he said at a news conference in Queens. "It protects speech and assembly."
The mayor expressed concern that those exercising a "right to be silent" might be getting drowned out amid the din of the protests.
So, in addition to the mendacious lie about the camp being a "tent city," the mayor is saying people need to be concerned about the New Yorkers, who do not wish to exercise their First Amendment rights? Seems absurd. And, basically, underlying this "concern" is the belief that the elites know best (i.e. Bloomberg, Big Bank CEOs, NYPD Commissioner, etc) and the people, who are not a part of the elite, are out-of-bounds as they vociferously and persistently share their grievances with the media and the public. The people in the park aren't meant to be taken seriously because they are not a kind of people that should be allowed to make decisions and run the country.
I have been in New York for about a week now (except for the two days when I was traveling to and from Occupy Boston). There is a narrative being constructed by the police and city. The cost of babysitting the occupiers is now millions of dollars. The police and city are going to repeatedly remind New York taxpayers that they are footing the tab for the peaceable assembly in Liberty Park. The occupiers need to expose this and find a creative way to neutralize this attempt to decrease the support New Yorkers have for the protest (currently, around 87% say they should be able to stay if they follow the law).
Firedoglake's premier live blog continues. I'll be down at Occupy Wall Street for another day of reporting on whatever happens. I will be conducting more interviews with occupiers and supporters.
Here's the Twitter list to follow for the latest updates on all things "Occupy." (cont'd)
1:48 PM How banks became too big to fail: Mother Jones charts bank merger history
1:44 PM Occupy Wall Streets month of success: has gone global, flourished with diversity, gained support in the heartland and changed the conversation.
1:42 PM Adbusters calls for October 29 to be a day for a Robin Hood Global March
1:17 PM Al Jazeera English's "Inside Story" does panel on Occupy movement. Features FDL friend Dean Baker.
12:46 PM The Guardian covers the push for an investigation into Deputy Inspector Johnny Cardona's assault on a protester
Marine Sgt. Shamar Thomas, who confronted NYPD in Times Square on Saturday, appeared on "Countdown w/ Keith Olbermann":
I've been to Iraq twice. I was in a riot in Rubai, Iraq in 2004, where we had rocks thrown at us. After the rocks were thrown, we didn't go beating up people and arresting people. You know what I mean? We kind of treated it with a level of humility. You know what I mean? And, to have the cop punching a woman in the face, to see that in my own country—You know my family fought for people in this country for people to have a right and these people are peaceful. I haven't seen a video yet of people trying to hurt the cops or why they are using riot gear when there's nobody starting a riot…
…There's a time and place for things. When a situation arises that is when it is time to say, "You know what? The crowd is getting frantic." When people are just shouting, "We are the 99 percent. Come join us." These are not chants that incite violence. You know what I mean? The riot police were in form just walking and marching up and down the street like there was a battle going on. I've been in battle. There was not a battle going on.
12:36 PM "March Against Police Brutality" at 5 pm ET today in New York City to District Attorney Cy Vance's office at 1 Hogan Place near Foley Square. Facebook event has further details.
On Friday morning, a senior NYPD officer sucker-punched Felix Rivera-Pitre in an unprovoked attack during an #OccupyWallStreet march. Now the police say they are looking for him and threatening multiple charges to cover up the assault.
Felix is a grassroots leader in the community group VOCAL-NY and has participated in #OWS solidarity actions during the past several weeks, including arriving early on Friday morning at Zuccotti Park to help resist the eviction.
Demand District Attorney Cy Vance hold the NYPD and Deputy Inspector Johnny "Fists" Cardona accountable for violence against Occupy Wall Street
I will be at the scene and intend to broadcast live on the FDL stream.
12:30 PM The Atlantic with 50 high-resolution photos of the Occupy movement.


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The Solitaire 1-Cell AAA Flashlight with Presentation Box (Black) from Maglite has a very compact, lightweight, and durable design made in the USA. The Solitaire features a bright and adjustable spot to flood beam, and is powered by a single AAA battery that provides 3.75 hours of operation. Constructed out of an anodized aluminum alloy, the Solitaire is corrosion resistant, impact resistant, and weather resistant. Weighing only 0.8 oz it is the perfect size to carry with you and comes with a key lead and presentation box. With the dependability of the Solitaire on your keys, you never have to be without light again.
- Bright 2 Lumen lamp with an adjustable spot to flood beam, and a maximum spot range of 65' / 20 m
- Compact, lightweight design
- Rugged anodized aluminum alloy construction that is corrosion resistant, impact resistant, and weather resistant
- Powered by 1 AAA battery
- Key lead to secure the Solitaire on keys or a bag
- Presentation box makes a great gift box and provides protection for the flashlight
- Candle mode for general illumination
- Designed and made in the USA


text: The Sheriff's Office is continuing to investigate a car crash reported early yesterday morning near Toutle. A 911 call came in a few minutes after 3 yesterday morning, when a passer-by reported finding a car in the ditch in the 600 block of Sightly Road. The caller said that there was no one around the car; when Deputies arrived, they reported that there was still steam coming from underneath the hood. After an hour after the first 911 call, someone else called and gave the name of the man that was supposedly driving, also claiming that he'd been driving under the influence. That second caller also reported that the woman that owned the car had been injured. The effort turned to locating that woman; Deputies called hospitals in Longview, Centralia and Vancouver, without success. A little before 11 am, the daughter of the car's owner called the Sheriff's Office saying that they were in the Emergency Department at St. John Medical Center. An interview was conducted, but no action by the Sheriff's Office has been reported.


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Vegan: Cheesy Baked Potatoes With Broccoli
I'm not all that fond of baked potatoes on their own, but baked potatoes with cheesy sauce and tiny broccoli florets is another story entirely. I love smooshing the sauce into the fluffy potato below, turning what was once dry and bland into something creamy and exciting. During my vegan month, it's not quite so easy a goal to accomplish. At least it wasn't, until I developed my Vegan Nacho Cheese recipe. Now, it's a piece of cake. More


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Rabbit monoclonal antibodies are one of the latest advances in antibody development. Many rabbit monoclonal antibodies bind with greater affinity to their target antigen, thereby offering superior performance and higher working titers in tissue staining applications compared to monoclonal antibodies derived from mice or rats. In most instances, these improved tissue staining and dilution characteristics result in substantial cost savings.
All of these rabbit monoclonal antibodies provide excellent results on formalin-fixed, paraffin embedded tissue sections using standard immunohistochemistry methods. These antibodies are applied to sections just like mouse monoclonals, and then detected using anti-rabbit secondary detection reagents.
Our rabbit monoclonal antibodies are supplied as liquid concentrates and are stable for 2 years when stored at 4 degrees C.


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Printed Cut Out Crop Top
Style # 226198
Sexy meets sweet. This feminine floral crop top gets taken over the edge with super sexy cut outs along the shoulders. It's irresistible with a high waist legging and stacked platform booties.
- 92% polyester, 8% spandex
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Rated 5 out of 5�by 1 reviewer.
Rated 5 out of 5�by elegantgrace Love the print! This floral print top is super cute and versatile. fit true to size and is very well made. i hope bebe will make this same top in regular length too, not just cropped, as it would make a great top for work. :) November 13, 2013
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Though we do get various accounts of what happens inside the compound, there are two key areas of interest for Amélie. The first is the continuation of World War II and the second is her first love. The children have little to do in the compound so they decide to continue World War II. It may be over for the adults but not for the children. There are several compounds. The British, Americans and Russians live in another compound, so they are not involved. However, the remaining nationalities represent the Allies. The remaining nationalities include the francophones - Belgians, French and the former French colonies - and a few other assorted nationalities, including a few East Europeans and the Italians (no-one seems to be aware that they were with the German in World War II). The Germans are represented exclusively by the East Germans. The adults, in one of their rare appearances in this book, decide that the West Germans are on our side now and must be excluded. The children of the Allies refuse to accept them so that they have to remain neutral. The East Germans - all male - are fairly numerous. The War, which is continuous, involves various acts of sabotage - urinating in the yoghourt left on the steps of the residences is a favourite - as well as capturing enemy troops and punishing them. The Germans punish their prisoners by knocking them around. The Allies, after having furious discussions in French about what to with their prisoner, which often has him very worried, usually try humiliation, with immersion in a large container of urine kept especially for this purpose being the one finally selected.
Amélie is the youngest of the Allies, so she is selected as the scout, to determine where the enemy is hiding. She performs this job well but likes to get mixed in and her final triumph is when she urinates - standing up - on the face of the captured German leader. This, of course, brings her great accolades from all of the Allies. However, the one she hopes to most impress is her first love. Soon after the arrival of the Nothombs, an Italian family arrives and Amélie is immediately in love with Elena, the very arrogant daughter of the family. (Elena's brother is universally despised, particularly by Amélie and even more so when he succumbs to torture and reveals he secret hiding-place of the Allies' hospital.) To Amélie's surprise Elena does not reciprocate her love. Amélie is even more shocked when Elena shows interest in a boy, Fabrice. Amélie has already decided that there are three types of people in the world - girls, women and ridiculous people. Girls are, of course, the best, while women serve some purpose, namely bringing up children, cooking, etc. Men and boys - the ridiculous people - serve no useful purpose in Amélie's world whatsoever. So when Elena is interested in a boy Amélie is astounded. She proceeds to do what any girl would do in her situation - sabotage the relationship, which she does. She briefly wins Elena back, first with her urination on the enemy and then, at the instigation of her mother, by playing hard to get.
Nothomb tells a very funny story from a child's perspective. Indeed, she comes close (but not too close) to Lord of Flies in her sweet viciousness and her absolute self-centeredness. (She states that the universe only exists to serve her.) Her ambition was to become either a Nobel prize-winner in medicine or a martyr or both. Thankfully, she has become neither.
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