text:
|Publication number
|Publication type
|Application number
|Publication date
|30 May 1989
|Filing date
|20 Nov 1986
|Priority date
|20 Nov 1986
|Fee status
|Also published as
|Publication number
|Patrick D. Mize, James P. O'Connell
|Original Assignee
|Becton, Dickinson And Company
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (11), Non-Patent Citations (3), Referenced by (64), Classifications (59), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for immunoassay in which enhancement of a detectable signal is achieved by modulation of enzymatic catalysis of an indicator reaction.
Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous, requiring a separation of bound tracer from free (unbounded) tracer or homogeneous in which a separation step is not required.
Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed.
Fluoroimmunoassay (FIA) uses fluorochromes as labels and provides direct detection of the label. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium.
Enzymes have also been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. When the signal is a color change, detection with the naked eye is limited because the average individual can detect the presence of chromophores only down to about 10-5 or 10-6 M.
EIA sensitivity can often be increased by spectrophotometric techniques; however, these procedures require expensive equipment. In another approach, the sensitivity may be increased by various amplification methods. Single enzyme amplification methods have been disclosed in which ligands present at concentrations of 10-6 to 10-10 M have been detected. These methods however, have been generally unsatisfactory at ligand concentrations above 10-11M. In cascade amplification procedures, the number of detectable (generally colored) molecules is increased by use of two or more enzymes or enzyme derivatives. U.S. Pat. No. 4,463,090 to Harris discloses a cascade amplification immunoassay in which a large molecule activator, such as an enzyme or a proenzyme coupled to a ligand, activates a second enzyme which reacts with a substrate to produce a detectable signal or in turn activates a third enzyme.
U.S. Pat. No. 4,446,231 to Self discloses a cycling amplification enzyme immunoassay which includes primary and secondary enzyme systems and a modulator for the second enzyme system. The primary system includes a first enzyme coupled to a ligand. In a first embodiment of the Self invention, the first enzyme system acts on a modulator precursor to liberate a modulator. The modulator is a cofactor of the secondary enzyme which activates the second enzyme system to catalyze the reaction of a substrate to a detectable product. During the reaction, the modulator is converted to an inactive form, and cycling is accomplished by a third enzyme which reactivates the modulator. In a second embodiment the modulator is an inhibitor of the secondary system, and is removed by the primary enzyme system whereby the secondary system is activated to act on the substrate and thereby produce the detectable product.
Boguslaski et al., U.S. Pat. No. 4,492,751 teaches a cycling system in which an enzyme substrate or coenzyme is conjugated to one member of the specifically binding pair.
A variety of molecules has been shown to cause specific inactivation of a target enzyme. A subset of inhibitors, termed mechanism-based inhibitors, are substrates for enzymes which react with an enzyme to form a covalent bond. Mechanism-based inhibitors have been reviewed by Walsh (Tetrahedron 38, 871 (1982). Another subset of inhibitors includes molecules which act as stable transition-stable analogs. Gelb et al. have disclosed some fluoroketones as transition-state inhibitors of hydrolytic enzymes in Biochemistry 24, 1813 (1985).
One aspect of the present invention is a method for detection of a ligand in a liquid sample, hereinafter referred to as the unknown sample. The unknown sample suspected of containing the ligand is combined with a specific antiligand and a tracer for the ligand. The tracer includes a first enzyme conjugated to either the ligand or to a second antiligand, also specific for the ligand. Conditions conducive to binding between ligand, antiligand and tracer are provided to give a bound phase. After separation of the bound phase from the fluid, the bound phase is contacted in a liquid with a blocked modulator and a second enzyme. The first enzyme removes the blocking group to provide a modulator for the second enzyme. A substrate for the second enzyme is then added. The substrate is converted by the second enzyme to a product which provides a detectable signal, the conversion of the substance to the product by the second enzyme being modulated by the modulator.
The ligand may be an antigen, a hapten or an antibody. The preferred ligand is an antigen, most preferably a viral antigen. The method may be carried out by a competitive immunoassay technique, in which case the tracer is the ligand conjugated to the first enzyme. Preferably, a sandwich immunoassay technique may be used, in which case the tracer is a second antiligand conjugated to the first enzyme.
The preferred blocked modulator of the present invention is a blocked inhibitor which is converted by the first enzyme component of the tracer to an inhibitor. The preferred substrate is a chromogen which is convertible by the second enzyme to a product of a different color. Most preferably the chromogen is colorless and is converted to a colored product by the second enzyme, the conversion of chromogen to product being inhibited by the inhibitor.
In the most preferred assay format, an antibody may be affixed to a solid support and contacted under binding conditions with a viral antigen and a second antibody, specific for the antigen, labeled with alkaline phosphatase (first enzyme). After binding and separation, the solid support having an antibody:antigen:alkaline phosphatase-labeled antibody sandwich affixed thereto is contacted with the blocked inhibitor and the second enzyme in a liquid. If antigen is present in the fluid, alkaline phosphatase captured on the solid support removes the blocking group to give the inhibitor. A colorless chromogen is added. Inhibitor generated in the liquid inhibits the second enzyme from converting the chromogen to the colored product. Thus the development of color indicates the absence of antigen in the sample and failure of color development indicates the presence of antigen in the sample.
In another aspect of the invention, there is provided a new class of enzyme modulators and blocked modulators. The preferred modulators are enzyme inhibitors, most preferably fluoroketones having a functional group to which there is chemically bonded a blocking group which may be cleaved by the first enzyme.
Another aspect of the invention includes a kit of materials for performing the method of the invention substantially as described above.
In any EIA system which includes unblocking of an enzyme inhibitor, severe constraints are placed on both the inhibitor and the blocked inhibitor if the assay is to achieve maximum sensitivity. In accordance with the present invention, the blocked inhibitor is an excellent substrate for the first (unblocking) enzyme, but is essentially unreactive toward the second (color forming) enzyme. Likewise, the unblocked inhibitor is a potent inhibitor of the second enzyme, but has essentially no effect on the first enzyme. Because the reactions of the blocked inhibitor and inhibitor with the first and second enzymes respectively are highly selective, "short circuits" characteristic of prior art cycling EIA systems due to cross reactivities are substantially eliminated.
Thus, the invention provides a versatile method for assay for ligands present in very low concentrations in a fluid. The method makes possible naked eye detection and measurement of an assay signal even though the ligand is present in concentrations as low as 10-12 M and greatly extends the range of ligands which can be detected or determined without expensive or cumbersome equipment. Further, the assay sensitivity, i.e., time required to detect the presence or absence of the ligand, may be reduced by up to 100 fold compared to conventional EIA. Significant savings in cost and space are thereby achieved, enabling assays in accordance with the invention to be carried out in small clinical laboratories or even in a physician's office.
The FIGURE depicts the result of a typical assay in accordance with the method and materials of the invention.
While this invention is satisfied by embodiments in many different forms, there is described in detail preferred embodiments of the invention, with the understanding that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the embodiments described. The scope of the invention will be measured by the appended claims and their equivalents.
In accordance with the method of the invention, a substance, hereinafter referred to as the ligand, present in the unknown sample, may be detected visually, i.e., by naked eye observation, even when present in very low concentrations. The method includes at least two amplification stages. A first amplification stage is enzymatic unblocking of a modulator. A second amplification stage is enzymatic catalysis of an indicator reaction. These amplification steps take place sequentially to provide signal amplification of 106 fold or higher whereby a ligand present in the sample at a level of 10-12 M or lower may be detected with the naked eye. If additional amplification is desired, a first enzyme may be provided which initiates a cascade of sequential reactions involving a plurality of enzymes, wherein any one or all of the reactions may provide further signal amplification.
An immunological reaction is used in the method of the invention for detection of the ligand in the unknown sample. By the term "immunological reaction," as used herein, is meant a specific binding reaction of an antigen and an antibody, a hapten and an antibody, or any appropriate analogue of an antigen, an antibody, or a hapten which also binds specifically.
The immunological reaction may be carried out in any suitable liquid. For example, the liquid may be a body fluid suspected of containing the ligand, such as serum, urine, cerebrospinal fluid, pleural fluid or the like. Alternatively, the liquid may be water, saline or any appropriate buffer, or a mixture of body fluids and other liquids to which has been added a sample suspected of containing ligand.
The preferred method for sandwich assay of the invention will first be described with reference to the assay flow sheet below to provide a general understanding of the assay components and their interaction, after which each component will be discussed in detail. ##STR1## In the above flow sheet, the following definitions apply, wherein a colon indicates an immunological binding, a hyphen indicates a chemical bond or a physical attachment, such as absorption, a solid arrow indicates a chemical conversation and a dotted arrow indicates modulation of a reaction or an assay component.
E1 --first enzyme
E2 --second enzyme
B--M--blocked modulator
M--modulator for enzyme
B--blocking group for modulator
It is seen from the flow sheet that conditions conducive to binding of ligand to antiligand and tracer are provided, followed by a separation step and appropriate wash steps. A second enzyme and a blocked modulator consisting of a modulator (activator or inhibitor) and a blocking group are added, and the blocking group is removed by the first enzyme component of the tracer. A substrate for the second enzyme is added, and the conversion of the substrate to a detectable product by the second enzyme is modulated by the modulator liberated from the blocked modulator by the first enzyme. The level of product is either directly or inversely proportional to the level of modulator, and thus to the ligand, depending on whether the modulator is an activator or inhibitor respectively. The actual signal measured may be a color associated with the indicator reaction, as, for example, the color of the product or rate of formation thereof, or the color of the substrate or the rate of disappearance thereof.
In a preferred embodiment of the invention, one or more assay components may be attached to the surface of a solid support. As known in the art, the solid support may be any support which does not substantially interfere with the assay. Exemplary of solid supports which may be used are glass and polymeric materials, such as polyethylene, polyvinylidene fluoride, polystyrene and the like. Such supports may be fabricated into any suitable shape, such as sheets, tubes, wells, or preferably, plates such as microtiter plates. For example, an assay component may be attached to the inside walls and bottom of a tube, preferably a plastic tube with one closed end, or most preferably, to the wells of a microtiter plate. Preferably, after the desired quantity of antiligand is attached to the solid support, any remaining binding sites on the support may be filled by an inert protein. The inert protein may be any protein, as, for example, ovalbumin, which can be attached to the support and which does not interfere in any way with the specific binding reactions between the ligand, antiligand and tracer, as described below.
Preferably, the antiligand attached to the solid support is incubated with ligand and tracer to bind both to the solid support. After a wash step to remove interfering materials, the remaining assay components may be added and the assay carried to completion as described below.
Turning now to a detailed description of the assay components, the ligand may be from any source, and may be an antigen, an antibody or a hapten. For example, the ligand may be an antigen present in a body fluid, or it may be isolated from a body fluid and subsequently introduced into a different liquid, such as buffer. In other cases, the ligand may be from a source other than a body fluid, as, for example, a culture of microorganisms or a cellular extract thereof. Preferred ligands are antigens, most preferably viral antigens present in a body fluid, such as Herpes simplex virus (HSV), Adenovirus, Influenze A virus, Parainfluenza 3 virus and Respiratory syncytial virus.
The antiligand is contacted with the ligand in the liquid to induce the immunological reaction. The antiligand may be an antigen or an antibody, either monoclonal or polyclonal, or it may be any appropriate analogue thereof which reacts specifically with the ligand. In addition, the antiligand may be an antibody complex consisting of a plurality of bound antibodies, as, for example, a second antibody bound specifically to a first antibody. Alternatively, the ligand may bind to several different antiligands, for example, an ensemble of polyclonal antibodies or a mixture of several monoclonal antibody molecules which bind simultaneously to different surface areas of the ligand. Generally, the second antibody is raised against the first antibody in a different species. The plurality of bound antibodies in the complex may contain from about two to ten or more antibodies.
In the sandwich assay of the invention, it is preferred to use excess antiligand having sufficient binding sites to bind essentially all of the ligand.
The tracer comprises two components, the first enzyme conjugated to the ligand (competitive assay as described later) or, in the preferred sandwich assay, to a second antiligand. The enzyme may be conjugated to the ligand or antiligand in any suitable way, preferably by a covalent linkage, prior to the immunological reaction. Covalent conjugation of enzymes to ligands or antiligands is conventional and well known to those skilled in the art.
Any enzyme which can remove a blocking group from a blocked modulator may be used as the first enzyme. Suitable first enzymes are generally hydrolases, such as phosphatases, peptidases, esterases, glycosidases and the like. Exemplary of, but not limited to, suitable first enzymes are trypsin, thrombin, mammalian liver esterase, acetylcholinesterase, β-galactosidase, or most preferably, alkaline phosphatase.
The liquid containing the ligand, the antiligand and the tracer may be incubated, if necessary, to induce binding. Incubation may be carried out at any temperature and for any length of time suitable to facilitate binding, preferably from about 20� to 40� for about 1 minute to 4 hours. Antiligand, ligand and tracer which are bound are hereinafter referred to as the bound fraction and antiligand, ligand and tracer which do not bind are hereinafter referred to as the free fraction. The assay may, but need not, be carried out in such a way that equilibrium is established between the bound and free fractions.
The bound fraction may be separated from the free fraction in the liquid phase of the assay mixture by any conventional method, such as filtration, decantation, centrifugation, aspiration and the like. When the immunological reaction has been carried out on a solid support, the liquid phase is conveniently decanted and the solid support washed to ensure removal of the free fraction and any other materials which would interfere with the assay and resuspended in a suitable liquid such as water, saline or buffer. The blocked modulator is then added, and the pH is adjusted to any level, preferably 6-8, which does not cause non-enzymatic removal of the blocking group, as described below.
The blocked modulator may be any material which may be converted by the first enzyme to a modulator of the second enzyme. The preferred blocked modulator has two components, the modulator and the blocking group. The most preferred blocked modulator is a blocked inhibitor which is unreactive toward the second enzyme until its blocking group is removed by the first enzyme and the inhibitor is liberated into the assay medium. Thus, the choice of the components of the blocked inhibitor depends on the first and second enzymes to be used. The blocking group should be one which can be covalently conjugated to the inhibitor by a bond which can be cleaved substantially selectively by the first enzyme, and the inhibitor component should inhibit the activity of the second enzyme while having substantially no effect on the first enzyme. Thus, the nature of the second enzyme and its substrate will be discussed prior to further description of the blocked inhibitor and the inhibitor.
In the assay of the invention, the second enzyme is generally a hydrolase which converts the substrate to the product. Suitable hydrolases are, for example, phosphatases, peptidases such as trypsin, chymotrypsin and pepsin, or preferably esterases such as acetyl cholinesterase (AChE) and butyl cholinesterase. The most preferred second enzyme is a carboxyesterase, such as rabbit liver esterase (RLE).
The substrate may be any substance containing a group which can be cleaved by the second enzyme to provide a product detectable by a signal associated with color. Thus, in one embodiment of the invention, the signal detected is the development or disappearance of a color, or a change from one color to another. In another embodiment of the invention, the signal may be a change in the rate at which the substrate is converted to the product, for example, the color of a substrate may be observed to remain unchanged for a specific length of time. Thus, measurements of the signal may be made under either kinetic or thermodynamic conditions. Kinetic measurements determine the rate of change which occurs over a period of time, and are generally carried out by making a series of measurements at various times after combining the assay reagents. Thermodynamic measurements determine the extent of change which has occurred when equilibrium has been reached between the substrate and the product of the indicator reaction. Measurements may be made either instrumentally or, preferably, with the naked eye.
It is preferred that the substrate be colorless until cleaved by the second enzyme to give a colored product. Suitable substrates are indoxyl esters and, preferably, esters of nitrophenols, such as ortho and para nitrophenyl acetates or butyrates. These substrates are colorless until cleavage of the acetyl or butyryl groups by carboxyesterase occurs to give colored nitrophenols. Thus, when the modulator is an inhibitor and the substrate is an ester of a nitrophenol, the signal which is measured is inhibition of color formation.
It is evident that the method of the invention may also be used in a fluorescence immunoassay. In this embodiment of the invention, the second enzyme may convert a nonfluorogenic substrate to a fluorogenic product wherein the signal measured is modulation of fluorescence. For this embodiment of the invention, it is preferred that the modulator be an inhibitor and the second enzyme be an esterase.
As mentioned above, the first enzyme component of the tracer cleaves the blocking group from the blocked inhibitor to provide the inhibitor of the second enzyme, and another aspect of the invention is a new class of enzyme inhibitors and blocked enzyme inhibitors of the general formulas I-IV, set forth below, wherein the nature of group B, as described later, determines whether the compound is an inhibitor or a blocked inhibitor: ##STR2##
In formulas I-IV, R1 may be H, lower alkyl of 1-6 carbon atoms, branched or unbranched, or ##STR3## wherein R2 may be lower alkyl of 1-6 carbon atoms; R3 may be H, nitro, alkoxy, halogen and the like; R4 may be an alkyl group of 1-10 carbon atoms or an alkenyl or alkynyl group of 2-10 carbon atoms optionally substituted with an aryl group or an aryl group substituted with a nitro, hydroxyl, mercapto, alkyloxy, haloalkyl, hydroxyalkyl, mercaptoalkyl group; Y and Z may independently be H or F wherein at least one of Y and Z is F; X may be O, S or NR5 wherein R5 may be H or R2 ; n may be 1-6; m may be 2-6; A may be F or CF3 ; and B may be H, a phosphoric acid or salt, a glycosyl group, an amino acid residue, such as a lysine or arginine residue covalently conjugated to X through the amino acid carboxyl group, an acyl group of 2-4 carbon atoms such as an acetyl or butyryl group, or a peptide of the formula ##STR4## R7 may be H or lower alkyl of 1 to 6 carbon atoms, branched or unbranched; R8 may be H, lower alkyl or hydroxy-lower alkyl of 1 to 4 carbon atoms, branched or unbranched, CH2 COOH or (CH2)2 COOH; R9 may be lower alkyl or lower alkoxy of 1 to 4 carbon atoms, branched or unbranched, phenyl, or benzyloxy; and q may be 0-10.
When B is H, formulas I to IV represent enzyme inhibitors. When B is any group other than H, formulas I to IV represent blocked enzyme inhibitors. When B is a phosphoric acid or salt thereof, it is intended that B have the formula ##STR5## wherein P is bonded to X and n may be as described above.
The inhibitor and blocked inhibitor in accordance with formulas I to IV may be synthesized by any sequence of conventional chemical reactions as may be envisioned by one skilled in the art. Suitable and convenient methods are given in the Examples, below. The following list of effective enzyme inhibitors is intended to be exemplary only.
__________________________________________________________________________ Ki (M)*Name nmr data (Esterase)__________________________________________________________________________ 1. 1,1,1-trifluoro-3-(4-hydroxy- (CDCl3)- 3.91(s,2H), 5.21(bs,1H), 2.0 � 10-6, RLE phenyl)propanone 6.90(d,2H), 7.10(d,2H) 2. 1,1,1-trifluoro-3-(3-hydroxy- (CDCl3)- 4.00(s,2H),(4.80(bs,1H), >10-4, PLE phenyl)-2-propanone 6.80(m,3H), 7.30(m,1H) 3. 1,1,1-trifluoro-4-(4-hydroxy- (CDCl3)- 2.95(m,4H), 4.90(bs,1H), 2.0 � 10-8, RLE phenyl)-2-butanone 6.92(dd,4H)J=4,60Hz 4. 1,1,1-trifluoro-4-(3-hydroxy- (CDCl3)- 2.94(t,2H), 3.05(t,2H), 1.0 � 10-7, RLE phenyl)-2-butanone 5.70(bs,1H), 6.80(m,3H), 7.15(m,1H) 5. 1,1,1-trifluoro-5-(4-hydroxy- (CDCl3)- 1.91(t,2H), 2.59(t,2H), 1.0 � 10-8, RLE phenyl)-2-pentanone 2.68(t,2H), 5.23(bs,1H), 6.95(d,2H), 7.10(d,2H) 6. 1,1,1-trifluoro-5-(3-hydroxy- (CDCl3)- 1.95(p,2H), 2.70(t,2H), 1.7 � 10-7, RLE phenyl)-2-pentanone 2.95(t,2H), 5.40(bs,1H), 6.70(m,3H), 7.30(m,1H) 7. 1,1,1-trifluoro-6-(4-hydroxy- (CDCl3)- 1.63(m,4H), 2.59(q,2H), 2.0 � 10-8, RLE phenyl)-2-hexanone 2.70(q,2H), 5.55(bs,1H), 677(d,2H) 7.02(d,2H) 8. 1-phenyl-3,3-difluoro-10- (CDCl3)- 1.35(m,4H), 1.65(m,4H), hydroxy-4-decanone 2.25(m,2H), 2.70(q,2H), 2.75(t,2H), 3.15(bs,1H), 3.55(t,2H), 7.25(m,5H) 9. 1,1,1-trifluoro-5-hydroxy-2-pentanone (CDCl3)- 2.15(m,4H), 4.03(bs,1H), 3.0 � 10-4, PLE 4.20(m,2H)10. 1,1,1-trifluoro-6-hydroxy-2-hexanone (CDCl3)- 1.85(m,4H), 2.10(bs,1H), 4.0 � 10-7, PLE 2.30(m,4H) 1,1,1-trifluoro-8-hydroxy-2-octanone (CDCl3)- 1.30-1.80(m,8H), 2.40 -- (bs,1H), 2.75(t,2H), 3.65(m,2H) 1-hydroxy-5,5-difluoro-8,8- (CDCl3)- 0.95(s,9H), 1.45(t,2H) 1.2 � 10-6, PLE dimethyl-4-nonanone 2.00(m,6H), 3.12(bs,1H), 4.15(m,2H) 1,1,1,2,2-pentafluoro-5-(4-hydroxy- (CDCl3)- 2.94(m,2H), 3.04(m,2H), 8.0 � 10-7, RLE phenyl)-3-pentanone 4.75(bs,1H), 6.90(d,2H), 7.10(m,2H) 1,1,1-trifluoro-4-(3-hydroxy- (CDCl3)- 5.90(bs,1H), 6.90(d,1H) 1.6 � 10-7, RLE phenyl)-3-trans-buten-2-one j=16Hz, 7.25(m,4H), 7.95(d,1H) j=16Hz N,N--dimethyl-N--[2-(hydroxy)ethyl] (D20)- 1.90(m,4H), 3.13(s,6H), 5.0 � 10-9, AChE 5,5,5-trifluoro-4-oxopentanaminium, 3.30(m,2H), 3.48(m,2H), 4.01(bs,2H). hydroxide salt N,N--dimethyl-N--[4-(hydroxy)butyl] (D20)- 1.95(m,4H), 2.54(m,4H) 6.5 � 10-8, ACHE 5,5,5-trifluoro-4-oxopentanaminium, 3.10(s,6H), 3.35(m,2H), 3.55(m,2H), hydroxide salt 5.25(bs,1H)__________________________________________________________________________ *PLE, Pig Liver Esterase (E.C. 126.96.36.199) RLE, Rabbit Liver Esterase (E.C. 188.8.131.52) AChE, Acetyl Choline Esterase (E.C. 188.8.131.52)
In the most preferred embodiment of the invention, the tracer is a second antiligand having alkaline phosphatase covalently conjugated thereto. After separation and washing of the solid support and resuspension thereof in a suitable assay liquid, RLE or AChE (second enzyme) and the blocked inhibitor of formula V is added. If antigen is present in the unknown fluid, it and the tracer are captured on the support, and the alkaline phosphatase causes cleavage of the phosphate ester bond of V to give inhibitor of formula VI. The assay is completed by addition of o-nitrophenylbutyrate or o-nitrophenylacetate, VII. ##STR6##
If inhibitor VI formed due to the presence of antigen in the unknown fluid, the activity of the esterase is inhibited, and colorless substrate VII is not converted to colored product VIII. If no antigen is present in the unknown fluid, no alkaline phosphatase is captured on the solid support, no inhibitor VI is formed and colored product VIII therefore forms because the esterase is not inhibited.
In this most preferred embodiment of the invention using 1�10-12 M alkaline phosphatase and 3�10-9 M RLE as first and second enzymes respectively, the presence or absence of an antigen in the unknown fluid down to a level of 1�10-12 M can be detected in about seven minutes. If antigen is present in the unknown fluid, no color detectable with the naked eye (ca 1�10-5 M of product VIII) develops in this time. If no antigen is present, color does develop. In contrast, by conventional immunoassay using first enzyme (alkaline phosphatase) alone, about 104 minutes are required for the enzyme to cleave sufficient phosphorylated nitrophenol to provide detectable color. Thus, the invention represents a 15 fold increase in assay sensitivity using these reagents at these levels.
As mentioned earlier, another embodiment of the invention is a competitive assay wherein the tracer is a predetermined quantity of the ligand conjugated to the first enzyme. In a competitive assay, the quantity of antiligand used is insufficient to bind all of the ligand and tracer present in the assay liquid so that ligand and tracer compete for the limited number of antiligand binding sites. Thus, in a competitive assay, the quantities of ligand and tracer which bind to the antiligand (bound fraction) are inversely proportioned to their concentrations in the assay liquid.
If additional signal amplification is desired, a multistage cascade amplification assay may be carried out wherein a plurality of reagents in the assay medium react sequentially leading ultimately to modulator unblocking. In describing this embodiment of the invention, it is convenient to consider the first enzyme described above as a primary enzyme which enzymatically converts a reagent in the assay medium to a secondary enzyme which unblocks the modulator for the above-described second enzyme. Further, the secondary enzyme, or any subsequent enzyme, may react with additional reagents to provide additional enzymes which may continue the cascade of enzymatic reactions until the modulator is unblocked. By proper selection of reagents to be added to the assay medium, any desired number of amplification stages may be carried out.
Amplification occurs in any embodiment of the invention heretofore described because the first enzyme, or any subsequently formed enzyme, and the second enzyme act as true catalysts wherein a single enzyme molecule may act on an essentially unlimited number of blocked modulator or substrate molecules respectively without being consumed. Thus, in theory, one molecule of each enzyme would be sufficient to perform the method of the invention. In practice, determination of the amounts of the enzymes to be added and the number of amplification stages to be used depend on the level of amplification desired and are well within the purview of one of ordinary skill in the art.
It is evident that an almost unlimited number of competitive and sandwich assay configurations which fall within the scope of the invention can be envisioned. Further, the invention provides assay configurations which are suitable for either detection of the ligand or determination of ligand concentration. Ligand concentration may be determined by comparing the magnitude of the signal generated with the unknown sample with the magnitude of the signal measured upon assay of a range of known quantities of the ligand assayed under essentially identical conditions. When the method of the invention is to be used for determination of ligand concentration, it is adantageous to read signal intensity by an appropriate instrument, such as a spectrophotometer, for example, a Beckman DU7 Spectrophotometer, Beckman Instruments, Inc., Irvine, Calif.
In another embodiment of the invention, the second enzyme may be affixed to the solid phase in close proximity to the antiligand. After the immunological reaction by which the ligand and tracer are captured on the solid support, the support is separated from the ligand phase, washed, and the blocked modulator is added. The first enzyme portion of the tracer reacts with and liberates the modulator for the second enzyme on the support. The substrate for the second enzyme may then be added and the assay completed as previously described.
Another aspect of the invention is a reagent kit or package of materials for performing an assay for a ligand in accordance with the method of the invention. The kit may include one or more antiligands, a first enzyme which may be conjugated to one of the antiligand, or to the ligand, a second enzyme, and a blocked modulator for the second enzyme wherein one of the antiligands may optionally be attached to a solid support. The kit may also include standards for the ligand, as, for example, one or more ligand samples of known concentration, or it may include other reagents, enzyme substrates, or other labeled or unlabeled specific ligands, antiligands or complexes thereof useful in carrying out the assay. It may include solutions, such as saline or buffers. The components of the kit may be supplied in separate containers, as, for example, vials, or two or more of the components may be combined in a single container.
Routine Analytical Techniques--Flash Silica gel chromatography was performed on ICN silica gel 32-63 mesh at 3-7 psi. Analytical TLC was performed on 0.25 mm 5�20 cm aluminum-backed silica gel plates from EM Scientific. Preparative TLC was performed on 2.0 mm 20�20 cm glass-back silica gel plates from EM Scientific. Melting points were performed on a Thomas Hoover capillary melting point apparatus and are uncorrected. NMR spectra were recorded on an IBM WP-200SY spectrophotometer and chemical shifts are reported in ppm relative to trimethylsilane. HPLC was performed on a Waters 510 two pump system with UV detection using one of two solvent systems on a Brownlee AX-300 7�250 mm column; System (A) initial hold for 5 minutes at 30 mM NH40OAc pH 6.5 followed by a linear gradient to 2.0M NH4OAc over a 30 minute period followed by a hold at 1.0M NH4OAc for 5 minutes. System (B) used as isocratic buffer system of 30 mM NH4OAc pH 6.5 for 40 minutes. Flow rates were 1.0 mL/minute. Gas Chromatography was performed on a H.P. 5840A Gas Chromatograph equipped with a FID and an automatic injector using a 30M DB-1 Megabore column purchased from J&W Scientific, Inc. GC conditions were as follows; A three minute hold at 100� C. followed by a 10� C./minute gradient to 250� C. followed by a 3.0 minute hold at 250� C. at 16.0 mL/minute flow rate.
Inhibition constants were measured in 50 mM Tris pH=8.0. Enzyme and inhibitor were incubated at ambient temperature for 20 minutes. Substrate for the enzyme was then added and the rate of hydrolysis was followed by spectrophotometrically. The substrate for PLE and RLE was o-nitro-phenylbutrate and for AChE was acetyl thiocholine and Ellman's reagent.
A. Ethyl 3-(4-methoxyphenyl)-2-(1-oxo-2,2,2-trifluoroethyl)propionate
A 1 L four neck round bottom flask, fitted with reflux condenser, dropping funnel, argon inlet, and magnetic stirrer was charged with 7.17 g (0.149M) of a 50% oil dispersion of sodium hydride and 300 mL of dry ethyl ether. Nine mL of absolute ethanol was slowly added to the stirring solution. After the evolution of hydrogen stopped, a mixture of 25 g (0.136M) of ethyl 4,4,4-trifluoromethyl acetoacetate and 21.3 g (0.136M) of 4-methoxybenzyl chloride was added over a 1 hour period. The mixture was then heated at reflux overnight. The reaction mixture was then cooled, extracted with water, 1N HCl, dried (anhydrous MgSO4), and solvent removed under reduced pressure. The crude reaction mixture, 33.5 g, was chromatographed on a 60�300 mm silica gel column with a 1:3 ethyl acetate:hexane mixture. Similar fractions were combined and gave 9.0 g (27%) of the desired product as an oil. nmr(CDCl3)--α2.12(m, 3H), 2.67(m, 2H), 3.85(m, 3H), 3.90(S, 3H), 7.24(q, 4H).
B. 1,1,1-Trifluoro-4-(4-hydroxyphenyl)-2-butanone
A 100 mL round bottom flask, fitted with reflux condenser, magnetic stirrer and argon inlet was charged with 2.05 g (6.7 mM) of ethyl 3-(4-methoxyphenyl)-2-(1-oxo-2,2,2-trifluoroethyl)propionate, 20 mL of 31% HBr in AcOH, and 10 mL of water. This mixture was heated overnight at 120� C., concentrated under reduced pressure and partitioned between dichloromethane and water. The organic layer was extracted with aqueous bisulfite, saturated sodium bicarbonate, dried (anhydrous MgSO4), and the solvent removed under reduced pressure. The crude reaction mixture was chromatographed on a 50�300 mm silica gel column with 1:1 ethyl acetate:hexane. Similar fractions were combined and solvent was removed under reduced pressure to yield 600 mg (41%) as a clear oil. nmr(CDCl3)--α2.95(m, 4H), 4.90(bs, 1H), 6.93(dd, 4H) J=4, 60 Hz.
C. Diethyl [4-(3-oxo-4,4,4-trifluorobutyl)phenyl]phosphate
A 10 mL one neck round bottom flask, fitted with argon inlet and magnetic stirrer was placed in an ice bath and charged with 400 mg (1.8 mM) of 1,1,1-trifluoro-4-(4-hydroxyphenyl)-2-butanone, 400 mg (2.4 mM) of diethyl chlorophosphate, 0.15 mL of dry pyridine and 5 mL of dichloromethane at 5� C. The mixture was stirred overnight at ambient temperature, filtered to remove pyridine HCl, extracted with 0.2N HCl, extracted with water, and dried (anhydrous MgSO4). Solvent removal under reduced pressure gave a crude yield of 600 mg of a brown oil. Preparative TLC using 1:1 ethyl acetate:hexane gave 600 mg (92%) of a clear oil nmr(CDCl3)--α1.50(m, 6H), 3.0(m, 4H), 4.20(m, 4H), 7.15(s, 4H).
D. Diammonium [4-(3-oxo-4,4,4-trifluorobutyl)phenyl]phosphate
A 25 mL one neck round bottom flask, fitted with argon inlet and magnetic stirrer was charged with 5.0 mL of dichloromethane, 140 mg (0.4 mM) of diethyl [4-(3-oxo-4,4,4-trifluorobutyl)phenyl]phosphate and 2.0 mL of bromotrimethylsilane. After stirring this mixture for 3 hours at ambient temperature, 10 mL of methanol was added and the volatile materials were removed under reduced pressure. The residue was dissolved in water and the pH adjusted to 7.3 with 1.0N NaOH. The aqueous solution was extracted with ethyl ether and lyophilized to give 190 mg of a white solid. This material was dissolved in 10 mL of 30 mM NH4OAc buffer and purified by HPLC using system A. Yield of the product after lyophilization was 50 mg (37%). mp 235-240 C. nmr(D2 O)--α1.90(m, 2H), 2.56(m, 2H), 4.65(s, DOH), 6.88(dd, 4H) J=6, 82 Hz.
A. Ethyl 2,2-difluoro-5,5-dimethylhexanoate
A 100 mL three neck round bottom flask fitted with dropping funnel, argon inlet, ice bath, and magnetic stirrer was charged with 8.0 mL of dichloromethane and 2.21 g (20 mM) of ethyl 2-oxo-5,5-dimethylhexanoate. Diethylaminosulfur trifluoride, 2.11 g (13 mM), in 5 ml of dichloromethane was added to the reaction mixture over a 15 minute period and the mixture was stirred overnight at ambient temperature. The reaction mixture was partitioned between water and dichloromethane, the organic layer dried (anhydrous MgSO4), and the solvent removed under reduced pressure. The oily residue was distilled at 83-88 C. at 20 mm to give 1.0 g (25%). nmr(CDCl3)--α0.95(s. 9H), 1.40(m, 0.4H), 2.10(m, 2H), 4.35(q, 2H).
B. 2,3,4,5-Tetrahydro-2-oxo-3-[(5,5-dimethyl-2,2-difluoro-1-oxo)hexyl]furan
A 25 mL three neck round bottom flask, fitted with drying tube, dropping funnel, heating mantle, and magnetic stirrer was charged with 0.24 g (5.0 mM) of sodium hydride in a 50% oil dispersion. The sodium hydride was washed with dry hexane (2�20 mL) and 5.0 mL of ethyl ether was added to the flask. A mixture of 5 drops of absolute ethanol and 5 mL of ether was slowly added to the sodium hydride suspension. After the evolution of hydrogen had stopped, a mixture of 1.0 g (5.0 mM) of ethyl 2,2-difluoro-5,5-dimethylhexanoate and 0.43 g (5.0 mM) of γ-butyrolactone in 5.0 mL of ethyl ether was added over a 20 minute period. The solution was refluxed for 3 hours and allowed to stir at ambient temperature overnight. The reaction mixture was partitioned with 1N HCL, the organic layer washed with water (2�50 mL), dried (anhydrous MgSO4), and the solvent removed under reduced pressure. The oily residue, 0.88 g, was crystallized from a hexane:ethyl acetate mixture, 0.66 g (53%). nmr--(CDCl3)--α0.95(s, 9H), 1.35(m, 2H), 2.00(m, 3H), 2.50(m, 2H), 3.00(m1H), 4.25(m, 1H), 4.5(m, 1H).
C. 1-Hydroxy-5,5-difluoro-8,8-dimethyl-4-nonanone
A 10 mL one neck round bottom flask, fitted with argon inlet, magnetic stirrer, and reflux condenser was charged with 1.0 mL glacial acetic acid, 4 drops of concentrated HCl and 200 mg (0.81 mM) of 2,3,4,5-tetrahydro-2-oxo-3-(5,5-dimethyl-2,2-difluoro-1-oxohexyl)furan. The reaction mixture was heated at 110 C. overnight under a blanket of argon. The reaction was extracted with ethyl ether, and the ether was cross washed with water, dried (anhydrous MgSO4), and solvent removed under reduced pressure. The residue was chromatographed on a 10�60 mm silica gel column using a 9:1 hexane:ethyl acetate mixture to give a clear oil. nmr(CDCl3)--α0.95(s, 9H), 1.45(t, 2H), 2.00(m, 6H), 3.12(bs, 1H), 4.15(m, 1H)
A. 2,3,4,5-Tetrahydro-2-oxo-3-[(2,2,2-tri-fluoro-1,1-dihydroxy)ethyl]furan
A 3 L three neck round bottom flask, fitted with reflux condenser, dropping funnel, argon inlet, magnetic stirrer, and heating mantle was charged with 36 g (0.75M) of a 50% oil dispersion of sodium hydride. The sodium hydride was washed (2�200 mL) with dry hexane and then suspended in 800 mL of ethyl ether and 2 mL of absolute ethanol. A mixture of 60.2 g (0.7M) of γ-butyrolactone and 99.4 g (0.7M) of ethyl trifluoroacetate in 750 mL of ether was added to the suspension at a rate to keep the reaction at gentle reflux. The mixture was refluxed for an additional two hours and then stirred at ambient temperature overnight. The reaction was cooled in an ice bath and 1N HCl (250 mL) was added. The organic layer was separated, washed with water (2�200 mL), dried (anhydrous MgSO4), and solvent removed under reduced pressure. The residue was crystallized from hexane:ethyl acetate to give 64.0 g (45.4%), mp--87- 90 C. nmr(DMSO-d6)--α2.33(m, 2H), 3.09(t, 1H) J=7 Hz, 4.20(m, 2h), 7.00(s, 1H), 7.52(s, 1H).
B. Preparation of 1,1,1-trifluoro-5-hydroxy-2-pentanone
A 500 mL three neck round bottom flask, fitted with reflux condenser, heating mantle, and magnetic stirrer was charged with 61.5 g (0.31M) of 2,3,4,5-tetrahydro-2-oxo-3-(2,2,2-trifluoro-1,1-dihydroxyethyl)furan, 6.4 mL of concentrated HCl, 10 mL of water, and 80 mL of acetic acid. The reaction mixture was heated at 125 C. overnight. An additional 3.0 mL of concentrated HCl and 5.0 mL of water was added and the reaction mixture was heated for an additional 10 hour. The reaction was partitioned between water and ethyl ether while the water layer was neutralized with solid sodium bicarbonate (100 g). The ether solution was washed with water (2�20 mL), dried (anhydrous MgSO4), and solvent removed under reduced pressure to give 60 g (45%) of a pale yellow oil. nmr(CDCl3)--α2.15 m, 4H), 4.03(m, 1H), 4.20(m, 1H).
C. 1,1,1-Trifluoro-5-bromo-2-pentanone
A 500 mL three neck round bottom flask, fitted with dropping funnel, magnetic stirrer, ice-salt bath, and argon inlet is charged with 125 mL of dry dimethylformamide, 29 g (35.7 mM) of tributyl phosphine, and 11.2 g (54.9 mM) of 1,1,1-trifluoro-5-hydroxy-2-pentanone. This mixture was cooled to -5 C. and 11.5 g (71.6 mM) of bromine was added dropwise over a 2 hour period. After stirring overnight at ambient temperature the reaction mixture was distilled though a 30 cm vigreaux column at 2.0 mm of pressure. Two fractions were collected; the first fraction from 27-35 C. and the second fraction at 35-70 C. The second fraction was partitioned between water and ethyl ether, the organic layer was washed with water (3�100 mL, dried (anhydrous MgSO4), and evaporated under reduced pressure at ambient temperature to give 30 g of a colorless oily mixture of dimethyl formamide, ether, and the desired product which was used in the next reaction without further purification.
D. N,N-dimethyl-5,5,5-trifluoro-4-oxopentanamine
A 500 mL three neck round bottom flask, fitted with dropping funnel, argon inlet, magnetic stirrer, and ice-salt bath was charged with 21.5 g (0.45M) of anhydrous dimethylamine at -8 C. To this mixture was added 19.4 g (88.5 mM) of 1,1,1-trifluoro-5-bromo-2-pentanone in dimethyl formamide and ethyl ether dropwise at -10 C. The suspension was stirred for 2.5 hours at -10 C. The solvent was then decanted from the precipitate. The precipitate was washed with ethyl ether (3�200 mL), the organic layers were combined, washed with water (2�30 mL), dried (anhydrous MgSO4), and evaporated under reduced pressure to give 15.0 g (92%) of a pale yellow oil. nmr of HCl salt (CDCL3)--1.89 (s, 4H), 2.90 (s, 6H), 3.21 (t, 2H).
E. N,N-dimethyl-N-[2-(methoxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium, hydroxide salt
A 25 mL one neck round bottom flask was charged with 2.95 g (21.2 mM) of 2-bromoethyl methyl ether, 0.33 g (1.77 mM) of N,N-dimethyl-(5,5,5-trifluoro-4-oxopentanamine) and 2.5 mL of dimethyl formamide and the mixture was stirred at ambient temperature for 3 days. Solvent was removed under reduced pressure and the amber oil was chromatographed on a Dowex-1 (15�200 mm) column in the hydroxide form with 200 mL of water. The effluent from the column was lyophilized to yield 0.28 g (60%) of an amber oil which was the product as the hydroxide salt. nmr (D2 O)--1.94 (m, 2H), 3.13 (s, 6H), 3.39 (s, 3H), 3.41 (m, 2H), 3.60 (m, 2H), 3.88 (bs, 2H). The material was used in next reaction without further purification.
F. N,N-dimethyl-N-[2-hydroxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium, hydroxide salt
A 10 mL round bottom flask, fitted with argon inlet and reflux condenser was charged with 0.275 g (1.1 mM) of N,N-dimethyl-N-[2-(methoxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium, hydroxide salt, 4.0 mL of water and 8.0 mL of 30% HBr in acetic acid. This mixture was heated at 120 C. for five hours, cooled, and the solvent removed under reduced pressure. The residue was co-evaporated with 3�20 mL of water and the resulting oil was chromatographed on a Dowex-1 (15�200 mm) column in the hydroxide form. Evaporation of the effluent under reduced pressure gave 0.17 g (63%) of the product as the hydroxide salt. nmr (D2 O)--1.90 (m, 4H), 3.13 (s, 6H), 3.30 (m, 2H), 3.48 (m, 2H), 4.01 (bs, 2H).
A 10 mL three neck round bottom flask fitted with dropping funnel, argon inlet, magnetic stirrer, and ice salt bath is charged with 0.093 mL) (1.0 nM) of phosphorus oxychloride and 0.5 mL of trimethyl phosphate. To this mixture was added 70.0 mg (0.2 mM) of N,N-dimethyl-N-[2-(hydroxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium, hydroxide salt dropwise at -10 C. The mixture was stirred for 30 minutes and then placed in a freezer overnight. The mixture was triturated with ethyl ether followed by petroleum ether (4�50 mL) to give a resinous precipitate. This precipitate was covered with ice, neutralized to pH 6.0 with 1N NaOH, and evaporated to dryness under reduced pressure. The resulting solid, 107 mg, was dissolved in 30 mM NH40Ac buffer at pH 7.0 and purified by HPLC with system B. The product, which eluted at 12 minutes, was isolated by lyophilization of the buffer to give 9.0 mg (11%) of a colorless resin. nmr (D.sub. 2 O)--α1.90 (m, 4H), 3.14 (s, 6H), 3.42 (m, 2H), 3.60 (m, 2H), 4.19 (bs, 2H).
Half Sandwich Assay for Hexon Antigen from Adenovirus
General Procedure:
Two-fold serial dilutions of purified Adenovirus hexon protein were coated onto a polyvinyl microtiter plate in pH 9.5 carbonate buffer (10 mM) for a period of 1-18 hours. The plate was rinsed several times with a solution consisting of 0.05% polyoxyethylenesorbitan monolaurate in phosphate buffered saline. The immunological reaction was performed using an antibody (mouse-anti-hexon IgG) conjugated to alkaline phosphatase diluted to an appropriate level in 10% culture medium (containing fetal calf serum). After incubation for 1 hour, and a standard rinse procedure, a mixture of blocked modulator (10-3-10-4M) and RLE (10-8-10-9M) was added, and the plate was preincubated for 10 to 60 minutes. A chromogenic substrate (i.e. indoxyl butyrate or o-nitrophenyl butyrate) was added to the mixture. Color development typically occured within 15 minutes, and could be stabilized for a longer period of time by the addition of excess free modulator. Wells which were coated with dilutions containing 1 ng/mL of hexon protein gave a positive response with this system.
A polyvinyl chloride microtiter plate was coated with anit-adenovirus antibody by incubating the plate for 1 hour at 37� C. in a 1:900 dilution of 4.5 mg/mL stock solution of the antibody in a 10 mM carbonate 20 mM ethylenediaminetetraacetic acid (EDTA) buffer. The plate was washed 3 times with a wash buffer solution consisting of 0.5% casein 10 mM TRIS and 154 mM NaCl at pH 7.6. Adenovirus infected HeLa cells diluted to ca 1�105 plaque forming units (PFU)/ml in pH 7.4 phosphate buffered saline containing 0.05% polyoxyethylenesorbitan monolaurate, 0.1 mg/ML gentamicin, 0.5% phenol red, 0.1% bovine serum albumin, 10 mM EDTA and 100 mM ethylene bis(oxyethylenenitrilo) tetraacetic acid were serially diluted in 2-fold steps across the plate. The plate was incubated as above to bind the cells to the antibody, washed with wash buffer, incubated with a 0.005 mg/mL tracer solution of anti-adenovirus conjugated to alkaline phosphatase in wash buffer containing 0.5% gelatin and 10% inactivated fetal calf serum. Excess tracer solution was decanted and the plate was washed 3 times with TRIS buffer (without casein). A mixture of a 1�10-4 M solution of the blocked inhibitor from Example I and 5�10-9 M RLE in 50 mM TRIS buffer, pH 8.5, was added and the plate was incubated at room temperature for 10 minutes. o-Nitrophenyl butyrate (1 mM) was then added to each well of the plate.
Control wells having no antigen typically showed color detectable with the naked eye in ca 15 minutes, and addition of free inhibitor (product B from Example I) to the control wells inhibited color formation for longer periods. Test wells having antigen dilutions remained colorless for periods of time in excess of 15 minutes.
The FIGURE shows the relationship of color formation to antigen concentration as determined with a Beckman DU7 spectrophotometer 20 minutes after chromogen addition. It is seen that the optical density (OD) of the solution in the control wells is substantially constant at about 0.24, and that the OD of the solutions in the test wells is inversely proportional to antigen concentration and approaches the control value at very low antigen concentrations.
In summary, the invention provides a method for detection or determination of a ligand present in a liquid sample at very low levels. After binding the ligand, antiligand and tracer, the bound phase is separated and contacted with a second enzyme and a blocked modulator whereby a first enzyme component of the tracer removes the blocking group to provide a modulator for the second enzyme. The resulting modulator affects conversion of a substrate to a product by the second enzyme leading to a detectable signal. Detection of the signal establishes the presence or absence of the ligand in the sample. By measuring the magnitude of the signal, the concentration of the ligand may be determined. The modulator and the second enzyme provide two amplification stages whereby the signal is amplified by 106 fold or more, enabling naked eye detection of the signal in a time up to 100 fold less than by conventional EIA.
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|18 Nov 2010
|Xuedong Song
|Absorbent articles capable of indicating the presence of urinary tract infections
|31 Aug 2010
|23 Dec 2010
|Kimberly-Clark Worldwide, Inc.
|Metering Technique for Lateral Flow Assay Devices
|15 Dec 2010
|28 Apr 2011
|Kimberly-Clark Worldwide, Inc.
|Flow Control Technique for Assay Devices
|20 Jun 1990
|23 Jan 1991
|Merrell Dow Pharmaceuticals Inc.
|Novel acetylcholinesterase inhibitors
|12 Oct 2001
|25 Apr 2002
|Saunders Alex M
|Ligand based solution assay for low concentration analytes
|1 Apr 2014
|9 Oct 2014
|Becton, Dickinson And Company
|Methods and kits for the diagnosis of influenza
|U.S. Classification
|435/7.91, 435/18, 436/808, 435/810, 987/228, 435/20, 435/19, 435/7.92, 436/518, 436/537, 987/224, 435/24, 436/536, 435/21
|International Classification
|Cooperative Classification
|European Classification
|17 Feb 1989
Owner name: BECTON, DICKINSON AND COMPANY, A CORP. OF NJ.
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:MIZE, PATRICK D.;O CONNELL, JAMES P.;REEL/FRAME:005024/0029
Effective date: 19861113
|9 Nov 1992
|Fee payment
Year of fee payment: 4
|26 Nov 1996
|Fee payment
Year of fee payment: 8
|19 Dec 2000
|Maintenance fee reminder mailed
|27 May 2001
|Lapse for failure to pay maintenance fees
|31 Jul 2001
|Expired due to failure to pay maintenance fee
Effective date: 20010530


text:
Written by Jamie Ortega
Photos by Ed Clinton Go
Party philes, die-hard rakistas, acoustic mavens, pop fans, and love struck teens-they wouldn't be caught dead together. But you know what they say about music- it has the power to unite even the most diverse people. Last March 8, 2012 music lovers from all walks of life met and had a SMASHING good time at Dayly Entertainment's Smash Project. The crowd bore witness to a concert of epic proportions as Typecast, Urbandub, Chicosci, One Buck Short, Slapshock, The Cab, Chris Carraba, Cobra Starship and The Used performed their hearts out, leaving the audience, thrilled, exhilarated, and yes, more than a little infatuated.
Front acts local bands Urbandub, Chicosci, and Slapshock served as the night's diesel as they warmed up the audience with some of their most popular songs. Typecast, often referred to by fans as the Dashboard Confessional of the Philippines, kicked off the 5-hour show with their famous emo-rock hit Boston Drama. Not to be outdone was Malaysian group One Buck Short whose lively rendition of Last Friday Night was met with cheers and head-bopping.
They may have called themselves "dorks" during the press conference held the day before at the Edsa Shangri-la Hotel, but they looked nothing like it as the Las Vegas band pop-rock band The Cab composed of front man Alex Marshall, Alex De Leon, Joey Thunder, David Briggs and Frank Sidoris entertained the crowd (and made many a fan girl scream) with songs "Take My Hand," "Bad," "I'll Run," "Bounce" and "La La".
The Cab fans will be delighted to know how special the Philippines is to the band- De Leon in particular. "My godfather is Filipino and I learned so much from him; like, a lot about respect and appreciation," the front man revealed during the press conference. "We've had so many fans tweet us over the years trying to get us promoted here, and I look forward to just going out after the shows and meeting them." Expect a collaboration with Justin Timberlake very soon as De Leon dished out some JT loving also during the press conference. "I specifically look up to him because he kind of does everything. He's not a one-trick pony," gushed De Leon.
Everyone here at Manila Concert Scene would like to dole out 1,000 pogi points to the boys of the Cab for two specific things: one for looking for a member of the Manila Concert Scene staff and giving her a hug after the press conference when this writer told them that her co-worker is a big fan. Another kilig-inducing backstage moment was when a 17 year-old Cab fan by the name of Patricia fainted during the second half of the band's set. When the band heard about the incident De Leon serenaded Patricia and her friends backstage with a snippet of the song "I'll Run". Of course one of Patricia's friends caught the whole thing on tape and uploaded it on Youtube. We are sure Patricia will remember that for the rest of her life!
The mood at Araneta Coliseum shifted from pop rocking to party rocking when Cobra Starship hit the stage. The fan girls, boys (and everyone in between) gyrated, jumped, danced, and sweated to the sytnthpop band's songs Hot Mess, You Make Me Feel, Guilty Pleasure and The City Is At War. Even if he was absent during the CD signing and show's meet and greet frontman and creator Gabe Saporta made up for his absence by giving the fans a little, well, fan service. In the middle of his performance Gabe's pants tore near the crotch area exposing neat pair of blue boxer briefs. Saporta never ran out of jokes and quips during the entire set, proclaiming at one instance that if he was the President of the Philippines he would have everyone's pants tear every night. Well, if everyone in the country looked like Saporta why not? Right before leaving the stage the goofy and sexy frontman jumped into the crowd doling out kisses to the girls which we are sure, like his pants, made many girls heads explode.
You could really see the change in the crowd when Dashboard Confessional frontman Chris Carraba hit the stage. People from all sections started to stand, cheer, and whoop. Couples began to hold hands while girls had wistful smiles on their faces. Sans the rest of his band Carraba serenaded the crowd with his swoon-worthy voice as he belted out his gut-wrenching and utterly romantic anthems like Belle of the Boulevard, Vindicated, and Stolen.
During the press conference The Used's Bert McCracken kept on saying "I love you" to the members of the media. The Utah American rock band lead vocalist proved just how much he loved the Filipinos as he, along with his band, Dan Whitesides, Jeph Howard, and Quin Allman delivered a truly head-banging performance that night. Fans formed rock signs with their hands as the band electrified the atmosphere with the tunes The Taste Of Ink, A Box Full Of Sharp Projects and All That I've Got. We were also delighted when we saw the Philippine flag in front of the band's drum set. It really is more fun in the Phbilippines!
What would be a rock concert be like without a mosh pit? Bert made sure that would not be missing as he ably supervised the forming of the pit prompting the group composed mostly of boys (and very brave girls) to chest bump, fist bump, and bump whatever body parts they could- all in the name of rock insanity.
International rock festivals are scarce in this country. As the Smash Project has so ably proven you can be a rakista, a pop fiend, or a party animal but when it all comes down to it- everyone in the Philippines loves and appreciates music. We hope that there will be more festivals like the Smash Project- a lively convergence of hearts and minds from all different walks of life.May music unite our hearts and minds in more ways than one.

subset: commoncrawl


text:
Members of the City Council are:
Back Row (left to right): Council Member TJ Thomson, Council Member Ben Quintana, Council President Pro Tem Lauren McLean, Council Member Scot LudwigFront Row (left to right): Council President Elaine Clegg, Mayor David H. Bieter, Council Member Maryanne Jordan
Phone: (208) 384-4422
Email the Mayor
Email the City Council
150 N Capitol Blvd
Boise, ID 83702
William L. Bones
Chief of Police
Contact: BPD Media Relations Office
Boise, June 23, 2006 – The Boise Police Department is thankful for citizens help in locating a 10 year old boy who was missing from his home in the area of Nth 34th street in Boise.
Damion White, 10 years old was discovered missing by his parents this morning. Officers have been involved in an area search since 7:30 am when Damion was reported missing.
The Boise Police Department received several tips from citizens reporting that they had seen the boy. Officers were able to use the information gained from those calls in order to help with the search. One tip lead the officers right to where the boy was located (near Winstead Park).
The boy appeared to be unharmed and was reunited with his parents at around 7:15 p.m.
The Boise Police Department would like to thank all of the citizens who responded to our requests for help in locating the boy. We would also like to thank the media for playing a vital role in getting the information to the public.
News Release prepared by:
Sergeant Doug Schoenborn
Boise Police Watch Commander


text:
PokerListings.com is the world's largest and most trusted online poker guide, offering the best online poker bonus deals guaranteed, over $1m in exclusive freerolls every year and the most free poker content available on the Web.
Esfandiari On Brink of One Drop Repeat, Johnson Denies Ho
Already poker's all-time leading money winner, Antonio Esfandiari is on the cusp of another huge score in the $111k One Drop High Roller.
The winner of the inaugural $1m Big One for One Drop last year for $18m, Esfandiari resurrected his One Drop magic to stand on the edge of back-to-back titles with a spot in the final four after play last night.
Elsewhere at the Rio Marco Johnson denied Maria Ho her first bracelet, the Ladies event got underway and Phil Galfond got off to a big start in the $25k Six-Max as the 2013 WSOP wound ever closer to the Main Event.
Esfandiari Conjures Another Magic Run in One Drop
The event all eyes were focused on last night played down into the money and to the final four with Esfandiari the biggest name still in the hunt.
Businessman Bill Perkins is technically the chip leader with 14,700,000 but with everybody fairly short compared to the blinds it's any man's game.
Anthony Gregg, the 2009 PCA Main Event runner-up, and Chris Klodnicki, runner-up in last year's $50k Player's Championship, are at 13,900,000 and 12,000,000 respectively.
Esfandiari is the short stack on 9,200,000 - and needed a huge re-suckout late to get there - but with his magic in this event he might still be the favorite to claim the $4.8m first-place prize. Everyone left is now guaranteed at least $1.4m.
Check our feature live coverage from the Rio for a rundown of all the action and tune in today at 1 pm as they play down to a winner. The final four:
- 1. Bill Perkins 14,700,000
- 2. Anthony Gregg 13,900,000
- 3. Chris Klodnicki 12,000,000
- 4. Antonio Esfandiari 9,200,000
Johnson Grabs First Bracelet in $2,500 Limit Six-Max
Vegas high-stakes regular Marco Johnson has been on the brink of a WSOP bracelet himself three times but come up short heads-up each time.
Not last night, however, as he finally locked down his first bracelet in Event #48, $2,500 Limit Hold'em Six-Max.
This time Johnson finished off Jeff Thompson heads-up and outlasted a tough final table that included Juha Helppi and Maria Ho.
Ho, who also knows the disappointment of a runner-up finish at the WSOP, came into the final table as the chip leader but had the wheels fall off to bust in sixth.
- 1. Marco Johnson $206,796
- 2. Jeff Thompson $127,801
- 3. Juha Helppi $82,956
- 4. Danny Warchol $55,457
- 5. Mike Schiffman $38,095
- 6. Maria Ho $26,858
Ylitalo, Abrams Edge Closer to First Bracelets
Two more events played down to the edge of final tables last night with Swede Robin Ylitalo and Scott Abrams chip leaders in their respective events.
Ylitalo leads Event #49, $1,500 No-Limit Hold'em, with 21 players left, among them Barny Boatman and Kevin MacPhee.
Abrams leads Event #50, $2,500 10-Game Mix, with 20 left including Robert Williamson III and Greg Raymer.
Both events will play down to a winner today starting at 2 pm. The top 10 in each event:
Event #49, $1,500 No-Limit Hold'em
- 1. Robin Ylitalo 1,151,000
- 2. Paul Dasilva 1,129,000
- 3. Taras Kripps 945,000
- 4. Valentin Messina 905,000
- 5. Brian O'Donoghue 772,000
- 6. Walter Treccarichi 757,000
- 7. J. Austin Hijar 638,000
- 8. Noah Sandler 616,000
- 9. Jan Eric Schwippert 459,000
- 10. Sergei Stazhkov 410,000
Event #50, $2,500 10-Game Mix
- 1. Scott Abrams 351,700
- 2. Sebastian Saffari 294,300
- 3. Philip Sternheimer 229,900
- 4. Brian Tate 211,800
- 5. Robert Williamson III 203,100
- 6. Christopher George 197,400
- 7. Michael Jarmark 192,400
- 8. Loren Klein 152,300
- 9. Brandon Wong 145,000
- 10. Tom McCormick 112,500
Anderson and Boeree Advance, Galfond Crushes $25k
Two more marquee events got underway in Vegas yesterday with the kickoff of the 2013 Ladies Championship and the $25k No-Limit Hold'em Six-Max.
Importantly for the Ladies, the $10k buy-in for men steered off any interlopers and 954 ladies were the only entrants. Chris Priday is the Day 1 leader while Danielle "dmoongirl" Andersen and Liv Boeree were two of the 122 to survive.
Our feature live coverage from the Rio meanwhile focused on the $25k and the big-name superstars battling it out at each table.
Online high-stakes legend Phil Galfond ruled the day, pretty much, bagging up 335,000 for second overall behind Max Lehmanski.
His high-stakes cohort Ben "Sauce123" Sulsky also bagged up a top 5 stack. Tune in to our WSOP Live Updates today at 2 pm for more great action from the 101 remaining players. The top 10:
- 1. Max Lehmanski 406,000
- 2. Phil Galfond 338,700
- 3. Craig Casino 333,000
- 4. Ben Sulsky 330,300
- 5. Marvin Rettenmaier 289,400
- 6. Brandon Meyers 258,100
- 7. Michael Lipman 250,000
- 8. Francois Safieddine 250,000
- 9. Alex Uskov 236,000
- 10. David "Bakes" Baker 223,700
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text: There are a lot of these people I run into a few times a week at my job (Staples), but today's customer was pretty bad. The conversation goes like this.. Me: Can I help you find anything? Her: Yes, I have a uh *long pause* canon.. EOS? Me: Rebel XT, or just a rebel? Her: Yes thats it! Me: .....Okay what can I help you with? Her: Does this work for my camera? *Holds up 1gb CF card* I already have 3 1 gigs at home, and how much does it cost? Me: Yes, and it costs $25, so you get double the storage for less money than the 512 (go go gadget selling techniques) Her: Whats a 512? *note* I had pointed at the 512mb CF card before she asked Me: ..512 megabites.. half a gig Her: Okay! Now.. can I use this card over and over? My friend said It is good to put the files on a CD but I am afraid I will lose them from the card! Rebel XT Kit, and trying to buy a 4th 1gb compact flash, and she already had 2 Sandisk Ultra II 1gig CFs... Too much money to blow? $800 + $70 + $70 + $60 + $25 (after rebate) = ~$1025 spent on camera equipment, and she still thinks you'll lose your pictures if you take them off your card... DOH! Anyone else run into people like this?


text:
I've always related better to guys — how can I make female friends?
By Cait Robinson
Have a question? Email firstname.lastname@example.com. Letters may be edited for length, content, and clarity.
Dear Miss Information.
I am a thirty-one-year-old woman, and I've always been kind of quiet. When I was younger, it really pained me that I didn't have any "real" friendships, the way females often do — the kind that are so close, they almost seem like relationships. As a little girl, I always had way more fun playing with boys rather than girls, and that tendency has followed me right up to this day; I just find it easier to talk to guys. Whenever I talk to girls, I find that there's so much going on between the lines, that I'm simply put off or rejected (for what reason I'm never quite sure) and I've often wondered if there's something wrong with me.
I'm in a long term-relationship, and there's no problem clicking with my boyfriend's friends. The problem is I sometimes miss going out with girls, since my boyfriend often goes out with his guy friends. I've tried a few times with my sister-in-law and her friends, but I really don't know how to relate to them. Is there something wrong with me? I'm normally fine, and I see my boyfriend's friends as my friends too. That probably sounds sad, but I really don't think it is — I feel genuinely connected with these guys, and things are great otherwise. What is your input here?
— Totally the Miranda
Dear Totally the Miranda,
This question hits home, because nearly every female friend I have has given me some variation on the phrase, "I don't have many girl friends — it's so much easier to talk to guys." (This is usually said while we we're working on our cars and gutting fish, natch. I can crush a beer can on my head!) Some of us just aren't cut out for Sex and the City-style pods of friendettes, and that is absolutely okay. It doesn't mean something is wrong with you or you won't make female friends; it just means you may want to shift your goals a bit.
It doesn't sound like you're a girl-on-girl hater, given that you've tried to make female friends before. That's a great start. Now comes the trickier business of finding the girls who click with you. Think of it kind of like dating: a good number of guys you chat up aren't going to hold your attention, which is neither a flaw of yours nor of theirs; it's just a chemistry thing. Friendships aren't much different. To that end, increase your odds of finding cool girls who share your values. Pick a thing you care deeply about, and do that. Are you really into animals? Fantastic, volunteer at an animal shelter. Are you passionate about gluten-free cooking? Take a class. Do you wish you actually knew how to gut a fish? Take a number, sister.
Choose activities that will put you in contact with a self-selecting population. This is why "at a trendy yoga studio" or "at a bar" could be hit-or-miss locales for friendmaking — if a place appeals to a wide range of people, your odds of finding ones who share your values may be lower. A meditation group or a creative writing class or a wilderness club, on the other hand, will more likely attract the kind of people you want to get to know. Getting involved in an activity has the added bonus of giving you a common interest and history with anyone you meet, which is like Duraflame for friendships.
Low-key, accessible girls are out there. (I have can marks on my forehead to prove it.) Follow your interests and keep an open mind. You'll be sipping Flirtinis with your girl-pod before you know it.
Dear Miss Information,
My best friend and I are extremely close, and she recently opened up to me about something that's been bugging her. Lately she's been feeling ostracised and demeaned by her friends because she's a virgin and they're "more experienced." These friends in particular are boy-crazy and really uninhibited, which is not my BFF's style. They've also been belittling her, saying things like, "Don't worry, I'm sure you'll find the chance to lose it soon." She confided in me because we have the same opinions on love and sex. She knows that I understand her, because I'm not boy-crazy, and because I'm also a virgin who's in no rush to sleep around.
Only problem: I'm not a virgin, and she doesn't know that.
I'm a private person and generally don't talk about things like that with other people. Furthermore, I didn't have any intention of telling my BFF about my lack of virginity because 1) she hates the guy I lost it to and have been sleeping with for almost a year, 2) I don't want her to think that I'm some kind of idiot or floozy for sleeping with "an asshole," and, 3) I understand how alienated she must feel and I don't want her to feel even more alone if I confess to her that I'm not a virgin.
I'm worried about what might happen if/when she finds out in the future and she realizes that I had lied to her or kept something from her. Is it better to just keep it to myself?
— Shitty BFF
Dear Shitty BFF,
Of all of the decrepit mores I wish would limp to a gutter to die, the concept of "virginity" is probably the worst. In its old-school sense, women who maintain their virtue fetch a higher bride-price at market (fifteen pence and a fine, fatted calf! A girl can dream!) But the faux-sexual-empowerment backlash against old-school virginity — the idea that having sex is a mark of how popular and/or well-adjusted you are — is no less damaging.
Having sex isn't about popularity; it isn't in itself a badge of pride if someone wants to fuck you and you say yes. Taking control of your actions and doing what is right for you, however, is a point of pride. This means holding out for a situation where you feel safe and comfortable, which may happen next week or may happen when you're thirty-seven. "Not having sex" does not equal "not being lovable," and rushing into anything that doesn't feel organic will likely do more harm than good.
As far as you're concerned, let your friend know that you are no longer a virgin, simply because lies compounded on lies will only lead her to distrust you. But break the news to her with a big "Who cares?" shrug. Your having had sex isn't a betrayal of your friend, just like her virginity isn't somehow an affront to you. It's just a thing, like you're a brunette and she's a blonde, or you're five-foot-four while she's five-foot-six. Who has had sex and when is thoroughly a non-issue.
It's unfortunate that she hates the guy you've been sleeping with. She may feel threatened by this information initially, but stay on-message: you care about her, you happen to have had sex, it is not a big deal, and you can still love and support her. This information alone will set you apart from her other friends, who don't sound like the "loving, supportive" type. As long as you're happy in your relationship with the guy, her opinion doesn't matter. You have nothing to apologize for.
If you really want to flip your moniker around (and, while we're at it, I highly doubt you are a "Shitty BFF"), hit home the message of "to each their own." Because it's true. There's a lot of strength in choosing when and under what circumstances you say yes. She should recognize and appreciate that power, rather than feel like she needs to get it over with to appease her oddly passive-aggressive friends.


text:
P – Australia's Will Power has placed third behind New Zealand ace Scott Dixon in the Indycar race in Phoenix to secure mhis first points of the season.
Power, the 2014 series champion, missed the season opener in St Petersburg with an ear infection.
However he could not match Dixon who dominated after taking the lead on a midrace caution and crossed under another yellow flag to win from Simon Pagenaud in IndyCar's first race at Phoenix International Raceway in 11 years on Saturday night.
Defending and four-times series champion Dixon started ninth and took advantage of tyre trouble for Power's Team Penske teammates Helio Castroneves and Juan Pablo Montoya to move within range of the lead on the fast mile oval.
The Chip Ganassi Racing driver moved past teammate Tony Kanaan out of the pits and led the final 155 laps of IndyCar's first oval race of the season.
The win was Dixon's 39th of his career, tying him with Al Unser for fourth on IndyCar's all-time list.
Kanaan finished fourth to give Chevy the top four spots and Honda driver Graham Rahal was fifth.
IndyCar returned to PIR for the first time since 2005 and the drivers faced a reconfigured-and-repaved track that was much faster than before.
Maybe too fast.
Drivers began breaking track records, at least unofficially, during a test in February and during the first practice session on Friday, when 16 of 22 drivers eclipsed Arie Luyendyk's 20-year-old record.
Six more drivers blew past the record during qualifying, led by pole sitter Helio Castroneves' lap of more than 192 mph.
With the race coming under the lights, the concern was that lower temperatures could lead to even more speed and dangerous conditions with little room to pass.
Instead of carnage, the race was mostly clean – except for Team Penske.
Castroneves led the first 39 laps, but dropped out after his right front tyre blew out.
Juan Pablo Montoya took the lead when Castroneves was forced into the pits, then he suffered the same problem to the same tyre and was forced to the pits after leading 56 laps.


text:
All sports should be popularised via TV, says Shah Rukh Khan
Superstar Shah Rukh Khan, who co-owns Indian Premier League (IPL) franchise Kolkata Knight Riders (KKR), is happy that several actors are encouraging different sports in the country, but feels the only way to make sports popular is through TV.
Actor Shah Rukh Khan
Appreciating the efforts of actors like Abhishek Bachchan, who has invested in a kabaddi team as well as in a football franchise, SRK said at a press conference yesterday: "We should be supporting every kind of sport and I'm glad some actors have come forward. But it can only happen through TV. Till the time a sport is not popular on TV, it won't get the required encouragement," he added.


text:
Whether you'd like to join in, volunteer, or organise an event of your own, we'd love to hear from you.
Events Calendar
We welcome your questions or suggestions. Please contact Emma Nelson on 01252 375581 or email@example.com.
You can also join in a local run, walk or challenge event for Parity. View the list by clicking here.


text: UGG Australia brings you the Dakota moccasin style slipper this season. Classy details like leather laces, precision stitching and an UGG Australia embossed logo grace this charming slipper for travels around your home or down to the coffeehouse. Suede upper. Leather lace and bow detailing. Embossed UGG® logo. Genuine sheepskin sock liner that naturally wicks away moisture and helps keep your feet dry. Molded rubber outsoles. Color: brown


text:
Created in Melbourne, inspired by Asia.
Located at the Paris end of Little Collins Street, Burma Lane brings guests a modern Australian dining experience with distinct Asian flair.
The style of dining is designed around light tapas bites moving to the more substantial, all delicious and perfect for sharing a quick bite or a lingering meal. The menu itself is concise, with regularly changing, seasonal fare celebrating local produce. Fresh, vibrant and deliciously different with a hand sourced wine list and sensational cocktails to match!
The venue has a beautifully designed interior, stunning Mezzanine level and high ceilings. The awe-inspiring floor to ceiling artwork by renowned Melbourne street artist, Mike Eleven, incorporates moving projected images, bringing the modern Asian-Australian feel of the restaurant to life.
Take a 360° look around our lovely venue by clicking on the image above or find out more at burmalane.com
"Took a couple of work colleagues here for dinner last night, one from out of town, the other a local who doesn't venture into the city much. We all loved it, the food was magnificent and the service outstanding. We shared a number of dishes and all disappeared really quickly. It would be unfair to isolate any one dish because they were all really, really good. We had a couple of dishes from each of the "sizes" meaning we eventually shared about 6 – 7 dishes, a great way to get a real taste of the menu. My recommendation….Just Go!" | Trip Advisor
Burma Lane
Modern Australian fare with a distinct Asian flair
118 Little Collins Street
Melbourne VIC 3000
Reservations: (03) 9615 8500
Opening Hours: Mon – Sat 12-3pm / 5pm-Late
"The welcome is warm and the flavours are fresh and interesting – a combination which would seem to be a recipe for success." – Where Magazine


text:
7 things to do for a happy and healthy relationship
Those who are looking for tips to a healthy relationship, here are seven such things you can do to make your bond stronger
Those in a relationship always look for the right spark and those who are willing to be in a relationship, look for the right goal. It may take years for a relationship to turn into a 'lived happily ever after' story, but it takes only few minutes to break the walls and part ways off. So for those who are looking for tips to a healthy relationship, here are seven such things you can do to make your bond stronger.
1. Put mobiles away when you are together: Now day's social networking happens mostly through mobile phones. Keep your mobiles and social networking tools like Facebook, Instagram, WhatsApp etc aside when you two are lazing around and spending quality time together. Mobile phones can bring a lot of distraction in communication. Forget the gadget world and indulge in some special talks. You will surely have a lot to share and lot to express to each other.
2. Spend less time in front of the television: It is good for couples to watch TV together, but spending too much time in front of the idiot box can shift your mind from other important things or the person next to you. When both of you are together at home, try to spend time together by sitting over a cup of tea/coffee, listening to music, engaging in romantic acts like cuddling and caressing each other, and making vacation plans.
3. Explore your culinary skills together: Love can happen in kitchen! Try to cook a dish together as it will keep both of you engaged in the same act and in the same place for a long time. In the middle of cooking, share a warm hug and a gentle kiss to make the environment more romantic and not boring. Know each other's food choices before heading for a cooking and dining date.
4. Sit together over a cuppa in the morning: Sipping morning tea/coffee together can do wonders to a relationship. Before you head for a busy day and hectic work schedule, spend some time together in the morning and keep yourself engaged in small talks. If time permits, have breakfast together and proceed for work like usual. Keep stress and worries away, and start your day on a good and romantic note.
5. Go for a vacation: Take short vacations to bring spark to your relation. Every time you may not get a long leave from work, so head for weekend getaways or short trips. Being together on a holiday away from daily chores rejuvenates you and takes togetherness a step higher. Enjoy adventure activates, plan romantic dinners, explore local life and have a pleasant vacation together.
6. Learn to forget and forgive: Do not nag or sit over a small fight for long as it can make things bitter. If you have done something wrong, seek apology, bring some small gifts for your better half and resolve the matter at the earliest. Even if you cannot forgive at times, just let it go. Start a new day on a new and good note, because that's the way you can heal an ailing relation.
7. Share a warm hug at least once a day: You may not head for an intercourse every night, because you may be either tired or not in mood. However, share a gentle hug at least once in a day or a small kiss to make each other feel better. Try to get close to each other whenever possible. The warmth of your gentle hug can be quite powerful to mend your relationship. Even if everything is lovey-dovey, try to retain the physical intimacy between each other.
Relationship is not always a bed of roses and only a couple can make things work for them. Avoid distraction of phones, talking about third parties, and dedicate more time to your you-and-me world.


text:
Separate names with a comma.
Discussion in 'Suspension Parts' started by trance, Oct 31, 2009.
NOT including shipping
2inch drop springs $50
what brand are they?
dropzone? idk its whats printed them
how much shipped to 10509?
still have these?
still have?
Do you still have the springs if so how much to ship to 07828 or where are you located if close to nj i can pick up this [email protected]
do you have any photos of how the car looked with them installed? i ordered a set yesterday on ebay.
shipping to 40336? pm me if u still have them
springs still available? Ill buy them ASAP! ready now
fit 96 gt? how much shipped to 80014


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Break through the battle formations on the basketball court with the responsive energy of these ???Jordan Extra Fly' men's basketball shoes from Nike. Built to induce and endure game-changing tactics, these black lace-ups have features like:
- Moulded synthetic upper with perforated forefoot shroud for breathable comfort and proper support.
- Zoom Air unit for explosive lift-off.
- Flywire cables for dynamic lockdown through the midfoot.
- Injected Phylon midsole for responsive cushioning.
- Rubber outsole for multidirectional traction control and explosive acceleration.
- Sole MaterialRubber
- ClosingLace-up
- Product warranty6 Months Warranty by Brand/Manufacturer Against Manufacturing Defects And Not Valid On More Than 20% Discounted Products
- Shoe CareSurface Dirt can be cleaned with a good quality brush or a damp cloth.
- FitRegular
- Ankle HeightLow Ankle
- ColorBlack
- Upper Material DetailsSynthetic
Sold by -Do you want to contact Jabong to know more about the seller?


text: Bet you are all thinking here's another idiot that doesn't know how to turn off notifications in the default android sms app? After installing Handcent I immediately disabled notifications in the default app so that only Handcent was generating them. This worked fine up until about 2 weeks ago when the default app started generating them. The option is definitely 100% disabled so I have no idea why it is still generating them. To make it worse even if I go into all messages with both Handcent and the default app my LED remains blue. Has anyone any idea why this might be happening? Thanks in advance tooner


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nonce (number used once or number once)
A nonce, in information technology, is a number generated for a specific use, such as session authentication. In this context, "nonce" stands for "number used once" or "number once."
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Typically, a nonce is some value that varies with time, although a very large random number is sometimes used. A nonce can be a time stamp, a visit counter on a Web page, or a special marker intended to limit or prevent the unauthorized replay or reproduction of a file.
An initialization vector (IV) is a nonce used for data encryption. The IV, used only once in any session, prevents repetition of sequences in encrypted text. Identifying such repetitions can help an attacker break a cipher.
In general usage, nonce means "for the immediate occasion" or "for now." Here's an explanation of the word's development from Merriam-Webster:
"Nonce" first appeared in Middle English as a noun spelled "nanes." The spelling likely came about from a misdivision of the phrase "then anes" ("Then" was the Middle English equivalent of "the" and "anes" meant "one purpose.") The word was especially used in the phrase "for the nonce," meaning "for the one purpose," as in Geoffrey Chaucer's Prologue of "Canterbury Tales:" A cook they hadde with hem for the nones To boille the chiknes with the marybones.
A nonce word is a neologism (newly coined word) that is created for a single use, such as inclusion in a work of fiction. However, once created, nonce words often make their way into common language. For example, Lewis Carroll coined the word "chortle" for the poem "Jabberwocky" and James Joyce created the word "quark" for his novel "Finnegan's Wake."
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Go Kart Style Ride On Car - 6 Volt Twin Motor - White
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Item: 1456503689217
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Go Kart Style Ride On Car - 6 Volt Twin Motor - White
The Go Kart Style Ride On Car - 6 Volt Twin Motor - White: This stylish ride on car is suitable for children aged between 3-7 years. This car has safety seat belt, gear shift control for choice of forward and reverse driving and working front lights. Also, it has a motor size of twin 6V 30W and it makes the perfect gift!
Go Kart Style Ride On Car - 6 Volt Twin Motor - White Extra Info
- Ride-on Car Size: 1070 X 480 X 645mm
- Ride-on Car Motor Size: Twin 6V 30W
- Suitable Age: 3-7 years (max 30kg)
- Ride-on car Speed: 2-3 Km/h
- Working front lights
Age Group: 3-7 years Approximate Weight (KG): 12 Gender: Unisex Maximum Rider Weight: 30 kg Maximum Speed: N/A Recharge Time: N/A Run Time: 1 Hour Voltage: Twin 6V 30W
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