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Patent application title: Sensors and methods for detecting diseases caused by a single point mutation
Kyung Aih Kang (Louisville, KY, US)
IPC8 Class: AG01N3353FI
USPC Class:
435 71
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
Publication date: 2009-02-12
Patent application number: 20090042210
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A method for generating antibodies preferable to either a normal protein
and a mutated form of the normal protein, respectively, where a mutation
associated with the mutated form includes either a single point mutation
or a small number of point mutations where the method includes creating
first and second antigenic peptides of a predetermined length
corresponding respectively to common regions of the normal target protein
and the mutated form, where the common regions are identical to one
another except for the point mutation of the mutated form, obtaining
first and second antibodies by multiplying the first and second antigenic
peptides via hybridoma methods, and identifying the respective affinities
of the first and second antibodies for the normal target protein and the
mutated form. Also included are methods of using the first and second
antibodies to detect and quantify respective amounts of a normal target
protein and a mutated form of the target protein. Also included are
immunological sensors the include the first and second antibodies for
determining the presence and quantity of normal target proteins and
mutant forms of the normal target proteins.
1. A method for generating antibodies preferable to either a normal
protein and a mutated form of the normal protein, respectively, where a
mutation associated with the mutated form includes a single point
mutation, said method comprising:creating first and second antigenic
peptides of a predetermined length corresponding respectively to common
regions of the normal target protein and the mutated form, where the
common regions are identical to one another except for the point mutation
of the mutated form;obtaining first and second antibodies by multiplying
the first and second antigenic peptides via hybridoma methods;
andidentifying the respective affinities of the first and second
antibodies for the normal target protein and the mutated form.
2. The method of claim 1 wherein the first antigenic peptide is created to correspond to a normal sequence of predetermined length in the heavy chain of FVN and the second antigenic peptide is created to correspond to a mutated sequence in the heavy chain of FVL that corresponds to the mutated sequence of FVL.
3. The method of claim 2 wherein the first and second antigenic peptides are 20-mers.
4. A method for determining the presence and respective quantities of a normal target protein and a mutated form of the normal target protein, where the mutated form of the normal target protein includes a single point mutation or a small number of point mutations in a common region otherwise identical in both of the normal target protein and the mutated form, said method comprising:obtaining, using the method recited in claim 1, first and second antibodies that are respectively preferable to the normal target protein and the mutated form;exposing the sample to at least a first and a second optical sensor, wherein the first optical sensor includes the first antibodies disposed thereon and the second optical sensor includes the second antibodies disposed thereon;probing the first and second optical sensors with a fluorophore-tagged 2.degree. Mab;observing signal intensities from each of the first and second optical sensors;determining the presence and respective quantities of each of the normal target protein and the mutated form thereof by solving a system of equations with at least two unknowns.
5. The method of claim 4 wherein the presence and respective quantities of FVN and FVL are determined according to the following system of equations:SITOTAL for FVL preferable sensor=AFVL×CFVL+AFVN×CFVN SITOTAL for FVN preferable sensor=BFVL×CFVL+BFVN×CFVN
6. The method of claim 4 further comprising providing a third antibody optical sensor having known respective affinities for each of FVN and FVL.
7. The method of claim 6 further comprising providing a third optical sensor having the third antibody disposed thereon and exposing the sample to the third antibody.
8. The method of claim 7 further comprising solving the following system of equations to determine the concentrations of FVN and FVL:SITOTAL for FVL preferable sensor=AFVL×CFVL+AFVN×CFVN SITOTAL for FVN preferable sensor=BFVL×CFVL+BFVN×CFVN SITOTAL for the third sensor=DFVL×CFVL+DFVN×CFVN
9. The method of claim 4 further comprising determining a disease condition and severity by comparing values obtained for the concentrations of FVL and FVN with known values associated with the disease condition and severity.
10. A method of determining the presence and quantity of FVL and FVN in a sample comprising:creating FVN-preferable and FVL-preferable antibodies to a predetermined length of the heavy chain of each of FVL and FVN;immobilizing the FVN-preferable and FVL-preferable antibodies on first and second optical sensing fibers, respectively;exposing the sample to each of the antibodies separately;probing the first and second optical sensing fibers with a fluorophore-tagged 2.degree. Mab;obtaining signal intensity values for each of FVL and FVN; andsolving a system of equations to determine the unknown values associated with respective concentrations of FVN and FVL.
11. An immunological sensor for determining the presence and quantity of FVL and FVN in a sample comprising:a housing having a microfluidic network disposed therein;first and second optical sensing fibers disposed within said microfluidic network and configured to be in fluid communication with one another, said first optical sensing fiber having FVN-preferable antibodies immobilized thereon and a second optical sensing fiber having FVL-preferable antibodies immobilized thereon;an inlet and an outlet for discharging the sample following analysis, said inlet and said outlet configured to be in fluid communication with both said first and second optical sensing fibers and with one another; anda signal detector for detecting respective signal emissions of said first and second optical sensing fibers following exposure of said first and second optical sensing fibers first to the sample and subsequently to a fluorophore-tagged 2.degree. Mab.
12. A method of diagnosing Factor V deficiency using a sample comprising:generating FVN-preferable and FVL-preferable antibodies to a predetermined length of the heavy chain of each of FVL and FVN;immobilizing the FVN-preferable and FVL-preferable antibodies on first and second optical sensing fibers, respectively;exposing the sample to each of the antibodies separately;probing the first and second optical sensing fibers with a fluorophore-tagged 2.degree. Mab;obtaining signal intensity values for each of FVL and FVN; andbased on said obtained signal intensity values, determining if Factor V deficiency exists.
13. The method of claim 12, wherein said determining comprises:comparing said obtained signal values to a set of predetermined values.
14. The method of claim 12, wherein said determining comprises:using said obtained signal values, solving a system of equations to determine the unknown values associated with respective concentrations of FVN and FVL;based on said determined unknown values, determining if Factor V deficiency exists.
[0001]Detection and diagnosis of disease is usually a necessary prerequisite to treatment and/or curing of the disease. With many inherited diseases, definitive diagnosis must often occur at a molecular level, and typically includes DNA analysis, which can be impractical and expensive owing to the complex and time-consuming procedure inherent to DNA testing. This is especially true of diseases caused by single point mutations, where the historical difficulty in obtaining purified monoclonal antibodies leaves DNA testing as the only viable method of diagnosing disease. While this is true for most diseases caused by single point mutations, one exemplary disease is a clotting disorder associated with a single point mutation of the gene encoding Factor V, known as Factor V Leiden.
[0002]To maintain a normal physiological system, it is crucial for blood to travel in an unobstructed manner through the vascular system. When injury occurs to the body, hemostasis assists in clot formation to prevent the loss of blood, while conversely, an anti-coagulant system ensures that the clot is localized at the site of damage, i.e., on the vessel wall, rather than inside blood vessels. Naturally, disturbances in the hemostatic system result in diminished ability to dissolve clots in blood vessels, which can cause traumatic thromboembolitic results. Thromboembolism may cause a variety of dangerous conditions within the body, such as deep vein thrombosis, lung embolism, stroke, and heart attack as normal blood flow from the heart to the body organs is blocked.
[0003]The most well-recognized inherited thrombophilic conditions include a resistance to the anti-coagulant Activated Protein C, as well as deficiencies of anti-coagulants, such as Protein C, Protein S, and Antithrombin III deficiencies. For example, Factor V is a blood coagulant that is inhibited by Activated Protein C, which, in turn, prevents blood coagulation. The single point mutation to Factor V, i.e., Factor V Leiden (FVL), causes a resistance to Activated Protein C, thereby preventing the inhibition of blood clotting.
[0004]FVL is the most common hereditary blood coagulation disorder in the United States. It is present in 5-8% of the Caucasian population and 1.2% of the African American population. FVL increases the risk of venous thrombosis approximately 3-8 fold for heterozygous and 30-140 folds for homozygous individuals.
[0005]Annually, as many as 600,000 hospitalizations and approximately 50,000 deaths are caused by pulmonary embolism alone. It has been estimated that death from pulmonary embolism results within 30 minutes on onset. Despite the widespread belief that FVL is responsible for a significant number of these hospitalizations and fatalities, clinicians do not routinely screen for FVL. This failure to screen may result, in part, from the lack of widely accepted detection methods.
[0006]Presently, most accurate FVL detection methods require DNA analysis, which are impractical for routine screening, as they are very expensive and time-consuming, and they do not provide a level for FVL in plasma. Consequently, FVL is usually screened with a clotting assay that is not sufficiently specific for FVL. Further, it can be difficult for a clotting assay to distinguish between FVL and other types of blood clotting disorders, such as deficiencies in Protein C or Antithrombin III. In fact, because FVL results in a resistance to Activated Protein C, FVL and Protein C deficiency may be indistinguishable with current assay protocols.
[0007]In view of the large population of affected individuals, early screening of FVL could make affected individuals aware of their high risk for thromboembolic complications and encourage them to take preventive actions. This may help to avoid the enormous after-care expenses incurred by victims--including physical debilitation and also emotional stress.
[0008]Embodiments of the invention provide rapid and accurate diagnostic methods and immunological sensors for diagnosing diseases caused by a single point mutation. Exemplary embodiments of the invention provide rapid and accurate diagnostic methods and sensors for diagnosing single point mutations for biomolecules needed for normal physiological functions. A more particular example is blood clotting disorders associated with a genetic mutation known as Factor V Leiden (FVL). Other exemplary embodiments include methods for generating site-specific antibodies particular to a mutation site.
[0009]FIG. 1 is a schematic diagram illustrating the role of Normal Factor V (FVN), activated Normal Factor V (FVNa), Factor V Leiden (FVL), activated Factor V Leiden (FVLa), Protein C (PC), and activated Protein C (APC);
[0010]FIG. 2 is a schematic diagram illustrating the sites of proteolysis of the heavy chain of FVN molecule;
[0011]FIG. 3 is a schematic diagram illustrating the abnormal sites (mutated from Arg to Gln) of the heavy chain of FVL molecule and its proteolysis;
[0012]FIG. 4 is a schematic diagram illustrating two 20-mer peptides of FVN and FVL used to generate antibodies according to an embodiment of the invention;
[0013]FIG. 5 is a schematic diagram illustrating a sandwich immunoassay on an optical fiber according to an embodiment of the invention;
[0014]FIG. 6 is a schematic diagram of a sandwich immunoassay on an FVL-preferred sensing fiber according to an embodiment of the invention;
[0015]FIG. 7 is a schematic diagram of a sandwich immunoassay on an FVN-preferred sensing fiber according to an embodiment of the invention;
[0016]FIG. 8 is a schematic diagram of two optical fiber immunosensors, one for FVN and one for FVL, according to a preferred embodiment of the invention;
[0017]FIG. 9 is a graph illustrating an affinity of antibodies to peptides using enzyme linked immunoabsorbent assay (ELISA);
[0018]FIG. 10 illustrates the sensitivity of sensors developed with the derived antibodies, for native FVN and FVL molecules in plasma;
[0019]FIGS. 11A and 11B are graphs illustrating exemplary standard curves for exemplary FVN and FVL preferred sensors for FV or FV molecules;
[0020]FIG. 12A is a graph illustrating exemplary sensing performance of the FVN preferred sensor for the samples of various amounts of FVN only, FVL at 8 microgram/ml, and the mixture of various amounts of FVN and FVL at 8 microgram/ml; and
[0021]FIG. 12B is a graph illustrating exemplary sensing performance of the FVL preferred sensors for the samples of various amounts of FVL only, of FVN at 8 microgram/ml, and the mixture of various amounts of FVL and FVN at 8 microgram/ml.
[0022]Embodiments of the invention provide rapid and accurate diagnostic methods and immunological sensors for diagnosing diseases caused by a single or a few point mutation. Exemplary embodiments of the invention provide rapid and accurate diagnostic methods and sensors for diagnosing blood clotting disorders associated with a genetic mutation to the gene encoding normal Factor V (FVN), where the mutation is known as Factor V Leiden (FVL).
[0023]While it is contemplated that embodiments of the invention may be used to diagnose a variety of diseases, and is especially advantageous in diagnosing those diseases caused by a point mutation, the clotting disorder associated with FVL will be discussed herein for exemplary purposes.
[0024]FVN is a single-chain glycoprotein (MW=330,000) that circulates in blood at 4-14 μg/ml. Activated Normal Factor V (FVNa) is one of the necessary components in the process of clot formation. Activated Protein C (APC) is an anticoagulant and serine protease that down-regulates thrombin formation by inactivating the activated blood coagulants, FVNa and Factor VIII (FVIIIa). FIG. 1 illustrates the blood coagulation/anticoagulation pathway, including the roles of APC, FVN, and FVNa.
[0025]FVL is a mutation to the heavy chain of the Factor V molecule, and is the most common (>90%) mutation to FVN, where FVL possesses an amino acid substitution (Arg506→Gln506). This amino acid substitution results in activated FVL (FVLa), which is consequently not inactivated by the APC (as also shown in FIG. 1), thereby resulting in a condition that is accordingly referenced as "APC resistance." The FVN and FVL molecules are equally well activated by thrombin and have the same cofactor activity in prothrombin activation. Functional differences between FVN and FVL only become apparent during the inactivation cascade, i.e., FVL does not get inactivated by APC and therefore the individuals with FVL have hypercoagulation (blood clotting) problems.
[0026]The heterozygous form of FVL has been estimated to occur in 3% to 7% of Caucasians, while the homozygous state has been estimated to occur in 1 in 5000 individuals. Also, this defect has been found in approximately 30% of patients with previously unexplained thrombotic disease and associated with a number of obstetrical pathological situations such as fetal loss, preeclampsia and HELLP syndrome.
[0027]As illustrated in FIG. 2, during a normal inactivation cascade of activated FVN (FVNa), as in healthy individuals, inactivation is achieved via proteolysis of the heavy chain of FVNa by APC at three positions of FVNa, in the following order: Arg506, Arg306, and Arg679. Inactivation of FVNa appears to be a biphasic reaction that consists of a rapid phase, during which nearly 50% of the FVNa activity is lost. This is followed by a second, slow phase that completes inactivation. Cleavage at Arg506 generates a 75 kD fragment and a 28/26 kD doublet. This proteolysis is necessary for the optimum exposure of the sites for subsequent cleavage at Arg306 and Arg679.
[0028]Proteolysis at these sites leads to the appearance of 45 kD, 30 kD, and 6 kD fragments, as well as a 22/20 kD doublet. Cleavage at Arg306 is membrane-dependent and is required for complete inactivation.
[0029]In contrast, and as illustrated in FIG. 3, proteolysis of the FVLa molecule by APC occurs in the order of Arg306 and Arg679. Cleavage at Arg306 produces a 45 kD fragment and a 62/60 kD doublet. Subsequently, the 62/60 kD doublet is cleaved at Arg679 to generate a 6 kD fragment and a 56/54 kD doublet, as illustrated in FIG. 2. Notably, the FVL molecule has mutation at Arg506 that leads to Gln506. This site is not available to APC proteolysis. This results in complete loss of cofactor activity.
[0030]Since the Arg306 and Arg679 sites of FVL are not optimally available to APC (due to no cleavage at the mutated Gln506 site), inactivation of FVLa (i.e., blocking of its coagulation function) is much slower than that of FVNa. Inactivation of FVLa occurs at a rate that is approximately similar to that of the second phase of normal FVNa inactivation. For example, while FVNa loses nearly all of its cofactor activity following 5 min of incubation with APC, the FVLa molecule only loses 50% of its activity during the same period. Moreover, following 60 minutes, FVLa still retained between 10-20% of its activity. This impaired down-regulation of FVLa allows the molecule to linger at the place of vascular injury, thereby promoting the increased risk of thrombosis.
[0031]Conventional methods of FVL diagnosis involve DNA analysis, resulting in an unduly long assay time with an associated high cost. Additionally, because FVL is the result of a point mutation, it is difficult to obtain an antibody with a high degree of specificity to FVL without cross reacting with FVN.
[0032]Embodiments of the invention provide immunological methods and sensors for detecting the presence and quantities of FVN and FVL, respectively, to accurately diagnose individuals having the FVL allele(s) as being either heterozygous or homozygous for the FVL allele. By quantifying the respective FVN and FVL molecules, the extent of normality or abnormality of the individual may be determined.
[0033]The methods and sensors of the invention are especially advantageous because embodiments of the invention provide antibodies specific to FVL molecules, promoting more precise and accurate detection and quantification of FVL for diagnosis of a disease condition. Embodiments of the invention may be used in real-time to diagnose FVL and FVN deficiency insofar as a patient's plasma may be used as the sample under analysis. Additionally, embodiments of the invention provide a short assay time, on the order of approximately 3-15 minutes. Enhanced sensitivity provided by exemplary methods allows use of small-sized (as nonlimiting examples, 1.5 cm×2.0 cm) chips to be used for a microchip sensor system. Further, due to the small sensor sizes available, a small sample volume can be used (as nonlimiting examples, 0.3 ml-0.5 ml). Cost associated with diagnosis is minimized, as compared to conventional methods (e.g., DNA analysis) of diagnosis. Embodiments promote ease of use, promoting use as a general screening sensor and method. By providing knowledge of the abnormality early to the FVL patients, the patients are able to prepare for the immediate treatment methods when thromboembolic episodes occur. In return, it will reduce the healthcare cost tremendously because, if thromboembolic episodes are not immediately treated, the cost of possible traumatic consequences is enormous.
[0034]A first preferred embodiment of the invention provides for a method of generating antibodies specific to the site with the abnormality (a single amino acid mutation site) of the molecule. In this manner, antibodies may be created with a high specificity and high accuracy to particular mutation sites of particular target molecules, such as FVN and FVL, thus enabling one to distinguish between the normal protein and the mutated variant with greater specificity. While it is contemplated that the method may be used to generate antibodies specific to a variety of genetic sequences that include single point mutations, for exemplary purposes, antibodies specific to FVN and FVL will be discussed in conjunction with the first preferred method.
[0035]According to the first preferred embodiment, non-mutant-preferable and mutant-preferable antibodies are obtained by creating antigenic synthetic precursor peptides having a predetermined length that correspond individually to normal and mutated molecules, respectively. Preferably, the respective antibodies approach total mutual exclusivity to one or the other of the normal or mutant molecules, such that there is little to no cross reactivity. In this way, each antibody ideally approaches mutually exclusive reactivity with only one of a normal or mutated molecule.
[0036]For exemplary purposes, the instant embodiment will be shown and described in connection with the FVN and FVL molecules. For example, FVN-preferable and FVL-preferable antibodies are obtained by creating antigenic synthetic precursor peptides having a predetermined length and corresponding individually to FVN and FVL, herein referenced as the "FVN peptide" and the "FVL peptide." While the number of amino acid residues in each of the FVN and FVL peptides may vary to suit individual applications, the peptides include the target cleavage site, either in its non-mutated form for the FVN peptide (Arg506) or its mutated form for the FVL peptide (Gln506). In the preferred embodiment, each of the FVN and FVL peptides are 20 amino acid residues ("20-mers") in length, as illustrated in FIG. 4. The FVN peptides and FVL peptides are then conjugated to carrier molecules via a terminal cysteine residue to serve as immunogens, with which multiple hybridoma cell lines may be derived, such as from immunized mice.
[0037]A hybridoma is a hybrid cell produced by injecting a specific foreign antigen into a host animal, such as a mouse, collecting an antibody-producing cell from the host's spleen, and fusing it with a long-lived cancerous immune cell called a myeloma cell. Individual hybridoma cells are cloned and tested to find those that produce the desired antibody. Their many identical daughter clones will secrete, over a long period of time, millions of identical copies of made-to-order "monoclonal" antibodies.
[0038]The antibodies resulting from the hybridoma technique yield the FVN-preferable and FVL-preferable antibodies, as confirmed by initially screening the antibodies with the 20-mers. After the initial screening, the selected antibodies are again screened using the FVN molecule (native FVN molecule) and the FVL molecule (native FVL molecule). As a result of this additional screening, antibodies with high affinity to FVL molecules that have limited to no cross-reactivity with FVN are selected to obtain the FVL-preferable antibodies. Similarly, antibodies with high affinity to FVN molecules that have limited to no cross-reactivity with FVL are selected to obtain the FVN-preferable antibodies. The FVN-preferable and FVL-preferable antibodies are therefore more specific against the mutation site rather than to the entire molecule, which gives a better probability to obtain antibodies specific to that site.
[0039]Another preferred embodiment of the invention includes a method of using non-mutant-preferable antibodies and mutant-preferable antibodies to detect and quantify non-mutant and mutant variants of a protein to diagnose individuals having a particular allele(s). For example, the instant preferred embodiment includes a method of using FVN-preferable antibodies and FVL-preferable antibodies to quantify FVN and FVL, respectively, to diagnose individuals having the FVL allele(s). Further, by quantifying FVN and FVL, methods of the invention may be used to determine the genotype of the individual (i.e., heterozygous or homozygous) and the extent of affliction, if any.
[0040]More particularly, by using known sensitivities of a combination of antibodies that include one or more of 1) commercial antibodies against the light chain (light chain antibodies) of FVL or FVN (these antibodies are reactive to epitopes of both FVL and FVN because FVL is a mutation to only the heavy chain of the molecule); 2) FVL-preferable antibodies; and 3) FVN-preferable antibodies, presence and quantity of FVL and FVN in a sample, as well as Total Factor V (FVT), may be determined. Total Factor V (FVT) is the amount of normal Factor V (FVN) plus the amount of Factor V Leiden (FVL). Once detected and quantified, the method provides for determine the genotype of the individual (i.e., heterozygous or homozygous) and degree of affliction.
[0041]The instant preferred method includes obtaining FVL-preferable antibodies and FVN-preferable antibodies and subsequently quantifying the amount of FVL and FVN in a sample, such as plasma. Using known or experimentally ascertainable affinities of each of the FVL-preferable and FVN-preferable antibodies for the respective FVL and FVN molecules, a system of equations may be employed and solved to determine values of the unknown variables that represent the respective quantities of FVL and FVN. The variables may be used to determine the degree to which a patient is afflicted with FVL, as well as make a determination as to the patient's homozygosity or heterozygosity.
[0042]Preferably, a commercial light chain antibody is also screened against the sample. Because a commercial light chain antibody has generally equal affinity for FVL and FVN, a commercial light chain antibody will be used as a second antibody for the sandwich assay. Once the amount of FVL and FVN in a sample is determined then the total amount of Factor V (FVT) will be determined. Since this sensing system provides the FVT in plasma it can also diagnose FVN deficiency.
[0043]Having obtained FVN-preferable and FVL-preferable antibodies, a sample may then be selectively exposed to each type of antibody with separate FVN and FVL optical sensors. Signal intensities are obtained from exposure of the sample to the antibodies on the respective optical sensors, which may then be used to solve for quantities of each of FVL and FVN present in the sample.
[0044]More particularly, as illustrated in FIG. 5, one preferred method includes exposing a sample serially or simultaneously to a pair of optical fibers, where each of the optical fibers bears either immobilized FVN-preferable or FVL-preferable antibodies. After exposure to the sample, the system is preferably probed with a fluorophore-tagged 2° monoclonal antibody (Mab) against FVT (antibody against the light chain), which binds both FVN and FVL ubiquitously.
[0045]While the invention contemplates use of a variety of fluorophores, one exemplary fluorophore is Alexa fluorophore 647® (AF647), such that the tagged 2° Mab is AF647-2°Mab. Light of a suitable wavelength, such as approximately 635 nm for example, is applied through the optical fibers, and the specifically bound AF647-2° Mab will generate fluorescence at a level proportional to the concentration of FVN or FVL in the sample. Fluorescence may be measured with a fluorometer, whereby the fluorescence intensity is correlated to the amount of antigen in a sample.
[0046]For example, a sample with only FVL and without FVN will exhibit a signal intensity to the sensor with FVL preferable sensor according to the following equation:
FVL only: SIFVL=AFVL×CFVL (EQ1)
where "SI" represents "signal intensity," "A" represents the slope of the standard curve showing the relationship between the FVL concentration and the signal intensity, and "CFVL" represents concentration of the FVL. The slope A may be determined, for example, by obtaining standard curves using samples with only FVN or FVL in plasma.
[0047]Similarly, a sample with only FVN and without FVL will exhibit a signal intensity according to the following equation:
FVN only: SIFVN=AFVN×CFVN (EQ2)
where "SI" represents "signal intensity," "A" represents slope of the standard curve showing the relationship between the FVN concentration and the signal intensity, and "CFVN" represents concentration of the FVN.
[0048]Accordingly, the total signal intensity obtained from both reactions (pure FVL and pure FVN) may be expressed by the following equation:
SITOTAL for FVL preferable sensor=AFVL×CFVL+AFVN×CFVN (EQ3)
[0049]Using the same concept, the total sensing signal from an FVN preferable sensor can be expressed as follows:
SITOTAL for FVN preferable sensor=BFVL×CFVL+BFVN×CFVN (EQ4)
Where BFVL and BFVN are the slopes of the standard curve for the FVN preferable sensor.
[0050]Because there are two unknowns, where the two unknowns are the respective concentrations of FVL (CFVL) and FVN (CFVN), a system of equations (EQ3 and EQ4) is employed to solve for each of the two values.
[0051]In this way, values calculated for CFVL and CFVN may be compared to values associated with healthy or disease-causing levels of FVL, with enough specificity to determine whether an individual is normal (FVN), heterozygous (FVN/FVL) or homozygous (FVL/FVL) for the disease. For example, a normal range of FVN in a healthy (not having the allele) individual is between 4 and 14 micrograms/mL with no FVL.
[0052]In another embodiment, three equations may be employed as a check of the results associated with FVN and FVL concentrations. More particularly, in addition to the FVN-preferable and FVL-preferable antibodies against respective sequences of the heavy chain, antibodies against the light chain of FVN, such as those commercially available and known in the art, may also be used. The light chain antibodies have slightly different yet ascertainable affinities for each of FVL and FVN. As such, exposure of the sample to the third, and relatively ubiquitous antibody, acts as a further check in the system of equations.
[0053]One exemplary system of equations that includes signal intensity data from a third optical sensor is as follows:
SITOTAL for FVL preferable sensor=AFVL×CFVL+AFVN×CFVN (EQ5)
SITOTAL for FVN preferable sensor=BFVL×CFVL+BFVN×CFVN (EQ6)
SITOTAL for the third sensor =DFVL×CFVL+DFVN×CFVN (EQ7)
where DFVL and DFVN are the slopes of the standard curve for the FVL and FVN respectively, for the third sensor developed using the commercially available two different monoclonal antibodies generated against the light chain of the molecule.
[0054]While embodiments of the method have been described as using two and three optical sensors, respectively, it is contemplated by the invention that an artisan may employ additional optical sensors to enhance the precision and accuracy of the sample analysis by employing a system of equations where the number of equations is commensurate with the number of optical sensors employed in the method.
[0055]Other embodiments of the invention provide immunological sensors that include mutant-preferable and non-mutant-preferable antibodies for detecting the presence and quantities of FVN and FVL, respectively, to accurately diagnose individuals heterozygous and homozygous for the FVL allele.
[0056]As illustrated in FIG. 5, preferred embodiments of the immunological sensor include two or more optical fibers configured to have a sandwich fluoro-immunoassay performed thereon. More particularly, an optical FVL-sensing fiber 10 is illustrated generally in FIG. 6, an optical FVN-sensing fiber 12 is illustrated generally in FIG. 7, and a sensor, generally at 14, that includes both the FVL-sensing fiber and FVN-sensing fiber is illustrated in FIG. 8.
[0057]A surface of the FVL-sensing fiber 10 preferably includes an FVL 1°Mab 16 against the FVL mutant cleavage site 18 ("anti-FVL"), which is designated as 6E6 or 8D7 in FIG. 9. When FVL 20 binds to the FVL 1° Mab 16, a Cy5-linked anti-FVT 2° Mab 22 binds FVL and will fluoresce to indicate the presence of FVL. Similarly, a surface of the FVN-sensing fiber 12 includes an FVN 1° Mab 24 against the FVN site 26 (anti-FVN) corresponding to the mutant cleavage site of FVN 28. A Cy5-linked anti-FVT 2° Mab 30 will bind FVN and fluoresce to indicate the presence of FVN. The order in which the 1° Mab and 2° Mab are used in the sensor may be reversed.
[0058]While the optical sensing fibers 10, 12 may be remote from one another, the preferred immunological sensor 14 (FIG. 8) is configured such that the two optical sensing fibers are in fluid communication with one another such that a sample may be passed simultaneously or serially over each optical sensing fiber.
[0059]More particularly, the two optical-sensing fibers 10, 12 are preferably placed in a platform, such as a cartridge 32, to form a dual sensing system. Since the two fiber system is preferably interconnected, the optical sensing fibers 10, 12 are preferably in close proximity, which optimizes the overall size of the system. One preferred platform is the cartridge 32 having two grooves 34, 36 disposed therein, wherein a first of the grooves 34 is configured to receive an FVN sensing unit 38 that houses the FVN-sensing fiber 12 and the other of the grooves 36 is configured to receive an FVL sensing unit 40 that houses the FVL-sensing fiber 10.
[0060]Example FVN and FVL sensing units 38, 40 may advantageously be prepared separately and subsequently coupled to the cartridge 32, which controls the timing and flow velocity, by the use of micro-electro-mechanical system (MEMS) technique. Within a particular example sensing unit 38, 40, an FVN optical-sensing fiber 12 is placed within a sample chamber such as, but not limited to, a glass tube where a sample, such as plasma, is injected. Suitable fluid inlet and outlet ports (such as, but not limited to, a valve such as a T-valve) lead into and out of the sample chamber, respectively. The sensing units 38, 40 include an immuno-optical sensor within a predetermined length of the sensing chamber, for example a 3 cm length of the glass tube. While a volume of the sensing units 38, 40 may vary, one exemplary volume is approximately 100 μl.
[0061]In FIG. 8, sensing units 38 and 40 are connected, and valves 52, 66, and 68 are provided to maintain the sample in a circulation/incubation circuit, indicated by the arrows at 58, or to alternatively discharge the sample, indicated by arrows at 60. The cartridge 32 is preferably manufactured using any one of suitable materials, such as soda lime glass, silicon or polymeric substrates as Plexiglas in the future, which promotes easy sterilization. Preferably, the cartridge 32 is a microchip.
[0062]As illustrated in FIG. 8, an inlet 62 is configured to receive a sample via injection or other means. A micro pumping system (for example, valve 52) circulates the sample through the FVN sensing unit 38 and the FVL sensing unit 40, which are fluidly coupled via channels 64, 66, for a predetermined period of time. After the sample is incubated, an outlet 68 is provided for sample discharge. The detector 50 is also preferably provided to detect and record signal intensities emitted by the optical sensing fibers.
[0063]Still other embodiments of the invention include a dual sensor embodied in a MEMS device. A MEMS device includes a minimized sized probe; automatically controlled convective sample/reagent application; minimized sensing time; sensor reusability, waste disposal to avoid the possible blood born pathogens, etc. The assay protocol will be automated to make the sensor user-friendly and to eliminate the human error. These features may be realized with MEMS scaled microchannels to deliver sample to the sensor and to permit evacuation of sample from the sensor after an analysis has been conducted.
[0064]Sensing methods associated with the immunological biosensor are also provided by the invention. Each of the FVL-sensing and FVN-sensing fibers 10, 12 in FIGS. 6 and 7 are exposed to a sample, such as a plasma sample, and incubated for a period of time sufficient for target molecules (FVL 20 and FVN 28) to bind the respective antibodies 16, 24. While incubation times may vary to suit individual applications, one preferred incubation time is approximately 3 minutes.
[0065]Following incubation, as illustrated in FIG. 6, the FVL-sensing 10 fiber is preferably washed with washing buffer. Next, the system is probed with AF647 (fluorophore) tagged 2° Mabs 22 against an epitope that is shared by both FVN and FVL ("anti-FVT" against light chain). Commercially available monoclonal antibodies developed for the light chain of FV are sufficient for use as a reagent. Excitation light will be applied through the FVL-sensing fiber 10, and AF647-2° Mabs will generate fluorescence at a level proportional to FVL 20 only.
[0066]In the FVN-sensing fiber 12, illustrated in FIG. 7, the FVN 1° Mabs 24 attached to the fiber surface will be directed to the region of FVN 28 corresponding to the FVN mutant cleavage site 26 (anti-FVN). This will allow FVN 28 to preferably bind to the fiber, while minimizing (preferably approaching excluding) the binding of FVL 20. AF647-2° Mab (anti-FVT) used in this fiber system will be the same as that used in the first system. Once excitation light is applied through the FVN-sensing fiber 12, the specifically attached AF647-2° Mabs 30 will generate fluorescence at a level proportional to the concentration of only (or nearly only) FVN 28.
[0067]It is contemplated by the invention that the two fibers 10, 12 may be integrated in a dual sensing system, providing a simultaneous quantification of FVL and FVN, thereby providing a rapid and complete quantitative information on this particular abnormality.
[0068]The ratio of FVL to FVN can provide the level of thrombophilia, because the average total FV concentration in plasma deviates significantly (4-14 μg/ml). This is also useful for the development of an appropriate treatment plan for the patient.
Results and Discussion
[0069]While various methods may be used to prepare the optical sensing fibers, sensing sample, and antibodies, exemplary methods are provided herein. To increase the probability for generating antibodies against the mutated site, 20 amino acid sequences of FVN or FVL around the mutation region were generated (FIG. 4). The 20-mers conjugated with carrier protein were injected to mice and cell lines were generated using the hybridoma technique. The resulting antibodies were initially screened with the 20-mers. Antibodies with high affinity to FVL molecules without cross-reacting with FVN, and vice verse, were selected.
[0070]For testing the affinity of the antibodies generated, ELISA was performed as follows: 96 well microtiter plates were coated with 2 μg/ml of FVN in FVN free plasma and FVL homozygous plasma. After blocking with 1% Bovine Serum Albumin (BSA), the antibodies (1 μg/ml) was applied and a 1/2 serial dilution were performed. Then 1:1000 rabbit anti-mouse IgG Fc (gamma) specific, HRP conjugated was applied. After adding OPD solution, optical density was measured at 450 nm in ELISA reader.
[0071]FIG. 10 illustrates the ratios of the signal intensity of native FVN molecules to FVL in sensing results increased from 5G3 to 1D4 antibodies. The ratio represented the affinity ratio of an antibody to FVN over that antibody to FVL. Antibody 5G3 is FVL preferred and 1D4 is FVN preferred. Therefore, 5G3 and 1D4 was selected to be 1°Mab in FVL preferred sensor and FVN preferred sensor, respectively.
[0072]Since both FVN and FVL molecules have the same amino acid sequence in light chain, each antibody against light chain is supposed to have the same binding site and the affinity to both FVN and FVL molecules. The 2°Mab for FVN preferable antibody and FVL-preferable antibody are preferably the same in a single sensing system. Therefore, a commercial light chain antibody, such as from Haematologic Tech., was determined as the 2°Mab for these sensors.
[0073]The core of the FVL and FVN sensors used in the exemplary method are tapered quartz fibers. The antibody against FVN or FVL (1° Mab) is immobilized on the fiber surface and then the fiber is enclosed in a sample chamber. When the sample is injected into the chamber, the FVN/FVL molecules are captured by the 1° Mab. After washing the fiber surface to remove unbound bio-molecules, another type of antibody (2° Mab) linked with fluorophores is applied to the sensing chamber. After the sandwich complex is formed, the emitted fluorescence is measured by a fluorometer. The fluorescence intensity is correlated with the amount of antigens in the sample.
[0074]When these generated monoclonal antibodies were tested with native FVN molecules or FVL plasma, they were found to be not specific to only one antigen but some had higher affinity to FVN and some with FVL and vice verse. The affinities of these antibodies were also expressed with the signal intensities of native FVN molecules and FVL plasma by using the fiber optical sensing system, where the generated antibodies were used as the 1°Mab in the sensor. The sensing experiments were performed with 10 cm sensor, 10/10 min incubation time and 1.2 cm/s circulation flow velocity.
[0075]These two sensors--FVL preferred and FVN preferred--could be used for quantifying FVN and FVL molecules in the sample. The abilities of these sensors to sense the FVN or FVL separately are illustrated in FIGS. 11A and 11B. The sensing experiments were performed with 3 cm sensor, 3/2 min incubation time and 1.2 cm/s circulation flow velocity.
[0076]The standard curves were achieved under the pre-described sensing conditions within the interested sensing range (0-14 μg/ml for FVN; 0-12 μg/ml for FVL). The apparent reaction in fiber optical sensing system is slight diffusion limited for Protein C sensing. Since the molecules weight of FVN is 5 times larger than Protein C and the kinetic characters in all immuno-reactions, binding reaction between antibody and antigen, are similar, the apparent reactions in FVN preferred and FVL preferred sensor should also be diffusion limited. Under the certain sensing condition, the signal intensity is approximately linear with the concentration of analytes. FIGS. 11A and 11B illustrate the relationship between signal intensity and the concentration of FV molecules was linear in FVN and FVL preferred sensors. For FVL molecules, the relationship between signal intensity and concentration was also linear under the same experimental conditions.
[0077]The FVN and FVL mixture samples with a fixed FVL concentration (8 μg/ml) were measured by using FVN-preferred sensor. FIG. 12A illustrates the signal intensity of the mixture was linear with the concentration of FV in sample with the slope 8.9, which is similar with the slope of the standard curve of FVN for FVN-preferred sensor, and the preset intercept 54.4, which is exactly same with the signal intensity of the only FVN plasma sample (8 μg/ml) for this sensor. The signal intensity of the mixture is only the physical addition of the signal intensity of FVN and FVL. Therefore, FVL molecules do not affect the affinity to FVN in FVN-preferred sensor. FIG. 12B also illustrates that FVN molecules do not affect the affinity to FVL in FVL-preferred sensor.
[0078]In summary, the similarity of the amino acid sequence of FVN and FVL molecule posed challenges to the quantification of both molecules. The fiber optical sensors and methods provide rapid and accurate quantifications of the protein (FVL and/or FVN) in blood plasma, but it requires specific antibodies. In order to increase probability for specific antibodies, 20-mers were used to generate antibodies against FVN or FVL only. Resulting antibodies with high respective affinities to FVN or FVL were chosen as 1°MAb of the FVN or FVL preferred sensor respectively. Each type sensor could sense FVN or FVL individually and standard curves for each analyte were generated. The signal intensity was linear with the concentration of each analyte in both FVN preferred and FVL preferred sensor. From sensing results with the mixture sample, the affinity of 1°Mab to FVN on the FVN-preferred sensor could not affect by the adding FVL molecules, and vice versa. Therefore, a mathematical model was provided to obtain the FV or FVL concentration simultaneously.
[0079]While various embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.
[0080]Various features of the invention are set forth in the following claims.
Patent applications by Kyung Aih Kang, Louisville, KY US
Patent applications in class Involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
Patent applications in all subclasses Involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
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text:
Detroit sports fans faced a dilemma Monday night: Watch the Tigers battle the Texas Rangers in extra innings of the American League Championship Series on Fox or watch Barry Sanders and the Selected of God choir introduce Monday Night Football on ESPN?
If you chose the latter, you're in luck. Responding to repeated demands, the four-letter network finally uploaded the MNF introduction yesterday afternoon, and it is embedded here for your viewing pleasure.
"Detroit's soul isn't in the streets of the city, it's in the hearts of the people," Sanders says in the clip as the choir and soul singer Jimmy Scott add some gravitas. "I know; I played here for ten years."
Sanders stepped up after ESPN fired longtime MNF performer Hank Williams Jr. for controversial comments he made about President Barack Obama. While Kid Rock may have missed his friend, we were happy Detroit got the opportunity to break the mold.


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Real estate agents don't want to waste time with people that are not qualify for a loan.
Look at it this way: Would you want to work for someone for a month or even more then that without eventually making money.
I don't think its fair for the real estate agent that is working so hard to find the right home and then drive you around until you will really like something out there, then suddenly understand that you can't even purchase a home at this time because of qualification issues.
You should just get pre qualify with any lender out there or let the real estate agent refer you to someone he worked with in the past and let the real estate agent feel comfortable to work hard for you and make you Happy.
Good Luck.
First, don't listed to Dunes because he has no idea what he;s talking about. Sorry.
There is no RULE that you must be pre-qualified. But be very wary of any agent that would not make sure you are. All the real professionals that would be a good advocate for you and represent your best interests - they all know that they must first take care of themselves as well and make sure you are qualified. It sounds like this agent you refer to did a horrible job explaining him/herself however. And yes, agents are making sure they don't waste their time. And yes, that's a really good thing!! If you work with an agent that is running all over town with half of their clients unqualified, that's not good for anyone. And for your own good....the loan pre-approval process these days in not as simple as it once was. Before you go shopping, you want to be 100% sure you have the buying power to purchase and are ready to go. It's the absolute best way to start your home search. Good Luck!!
Why are so many agents soliciting the consumers here...call me, email me, I'll show you that property?
They don't know if that consumer is approved for anything or has any money. It's because they NEED business and that's a way they try to find it. Like showing a home.
Showing a property is a tool used sometimes to find buyers now or for the future. Any claim that there is some standard or test that has to be passed by the consumer to view a property seems slightly off.
I guess if you are doing so well that you can't handle all the deals you have going then having a screening process might be in order. Congrads to those agents.!!
The questions that pops into my mind....When Kenny does have a loan approved, why would he call that agent back? Are any agents doing so well that they can take that kind of gamble on a business opportunity?
Kenny, I stick with my move on, find someone else and go see the property....Dunes
...and the direct answer to that question is "No, it's not true".
A real estate agent may have many reasons not to show you, and the fact that you haven't been pre-qualified or pre-approved yet, may be among them. But there is no hard & fast rule or law that states you must be pre-qualified in order to be shown a property, and the fact that your agent lied to you, is enough reason, in my mind, to kick him to the curb and find someone more interested in YOUR needs than his own.
To answer your question, the answer is no. That could also be viewed in a discriminatory manner by a court.
He dosen't want to show you a house unless you want to make an offer on it? That's crazy.
Get rid of that real estate clown now.
Also, you should do your own reseach and due dilligence and start the mortgage qualification process now. Don't depend on trusting a realtor to walk you through the process without any of your own research. It would be preferable to keep them out of the entire mortgage application process. But if you do allow the realtor (who is not a finance professional) to assist you with obtaining the best mortgage quote, at least do your own independent research.
If an agent tells you what the above agent did, then move on and find another one it's as simple as that. There are many agents available and looking for clients.
Your responsibility is to be honest and not BS the agent. They have not been placed on this earth to show every curious person properties for free.
Armchair General, Dunes
The quick answer is We ( Realtors) want be sure our buyers are qualified. It is a huge waste of time to go home shopping with buyers that cannot buy because they are not qualified or not qualified for the amount they WANT to spend.
So Realtors Prefer buyers to have financial justification.
Despite all the I'm selling 30 homes a day talk many agents are desperate.. Go to activerain or any other site (This one included) and read the blogs, comments, ect... Lot of agents talking about nothing going on and formulating strategies sure to find someone to represent. No shortage of agents desperate for business. Find someone else, simple as that. Why should you on first contact prove something to someone you are hiring.
Ask them to prove something to you, like they actually know something........Agents use the word standard a lot, but it really has little meaning in this market.
I don't remember anyone ever stopping me in the grocery store to see if I was pre-approved or refuse to show me where the beer was or tell me the price without a loan approval. Why do stores have display windows?
I'll weigh in on this as well as a Realtor.
It's really easy for a interested buyer to throw out an amount they "think" they're qualified to purchase without getting pre-approved, which I've always found to be interesting. Buyer's need to put themselves in the seller's position and ask themselves- Is It fair to the seller for the agent to show the home to a non-qualified buyer? NO! The seller is looking for feedback on their home and definitely not looking for sightseer's. I agree with the agent below, to the buyer who doesn't want to receive pre qualifications.. they should visit open houses to get familiar with what's on the market.
It's like going to a car dealer asking to test drive a car they can't afford to purchase. I'm not really sure why buyer's think the real estate profession is any different. It's a business also.
To get pre-approved you need to fill out an application and provide some documents to your mortgage broker which could include: home address for the past two years, social security number, landlords for the past two years, name and address of employers for the past two years, w-2 forms, tax returns past two years w/ schedules, two most recent paystubs, bank statement past 3 months, checking , savings and credit union account numbers an locations, IRA/401K information and your mother-in-laws maiden name. You might also be required to provide debt info such as car payments, student loans, divorce decree or separation agreement, divorced personal debts, payments automatically drafted from your accounts along with an explanation for any late payments, judgments, liens to name a few. While these might seem lengthy all of them might not apply to you based on your situation and the type of loan your mortgage broker is trying to get you approved for.
To answer your question... no, it is not true that a real estate agent can not show you a house until you are pre-qualified with a lender, however it is true that some will not. It is also true that to take you shopping (which is what you are doing when you view homes) before knowing what your spending comfort level is, while it may be enjoyable for some folks is in truth potentially a waste of your time.
As for not wanting to show homes to clients until they are ready to make an offer on that home. That may be how some folks choose to do business but is not how I do it, nor how any of the folks I work with do either. The way that you have phrased the comment it reads to me that you are being told to go do your own legwork and when you have found what you want to contact the agent you mention to write the deal for you.
The truth is that a good buyer's agent will do a whole lot more for you than just help write up your contract. A good buyer's agent will do as much work before you cross the threshold of the first home you view as they will do after the offer is made and accepted. They will be able to tell you if the home is in distress (facing foreclosure or a similar situation), what the market is like and what the comparable homes have sold for. They will have also found out if the home has any other offers on it, will know how long it has been on the market and whether it has been on the market without selling before.
That being said, I have found that if I as your agent do my job effectively by learning what your wants and needs are, the amount of time spent walking through various homes can be minimized to the point where only two, three or four homes at the most need to be viewed in person before we write the offer for the home that is perfect for you.
Since you tell us that you are new to the process of home buying the best advice I can offer you is to interview agents you are considering working with and to select someone who you "click" with and who also knows not just the "what:'s" but also the "why's" of the transaction.
Thank you for asking the question. Good luck with your home search. Take care and have a great day!
Tisza Major-Posner, Realtor, IVPG Realty (909) 837-8922
As professionals, we get fingerprinted by the Department of Public Safety before we can even begin our careers.
The only way we can at least trace a person who wants to look at homes is through the prequalification process in which we kill 2 birds with one stone: the buyer begins the loan process, and the agent knows that he is working with a true buyer.
Although your agent is required to work in your best interest and for you; our code of ethics requires us to be fair to both parties, the buyer and the seller, too. A professional, high producer, highly experienced agent is worth hanging onto when you can see he/she will not bring just any person into a home where valuables can be assessed for a later break in. Does this make sense?
Getting prequalified first is a win-win situation for sellers, agents, and most importantly, you, the buyer. Buying a home is a process, and the very first step is to make sure you are eligible to get a loan. Looking at homes is exciting! But there are always open houses to go look at, and you don't need to pre-qualified, and you don't need a Realtor for that.
The agent has valid reasons why he can't show you homes until you get pre-qualified. . He is willing to give up his time, using up his gas and his car in order to guide you and help you find and buy your dream home. He has just one requirement as his standard of practice: please get pre-qualified. The bottom line is, the only reason why a person would not start the home buying process by going to a lender first, is because they aren't really buyers.
I believe most people are smart enough to use the mortgage calculators on line to come close to monthly payments. They can go to the Tax Assessor page to calculate the taxes for the year.
In the past it was the GREEDY banks that approved people for more than they could afford!
You need to find an agent you are comfortable working with. You should be able to ask your agent questions and expect reasonable answers. Every agent wants to show properties to a buyer who is ready to buy. Some people just take longer to make a decision! In this market things are selling in multiple offers so you really do need to be ready!
That isn't typical practice of all agents, but you should get pre-approval so you know what amount you can get a loan for and which homes to preview within your range. It's hard to guess without the pre-approval, and your agent will need it to submit an offer for you. He should also be able to recommend a good lender for you....has he? Call me if you need assistance!
In terms of professionalism and best practices...It would be in your best interest to know, in advance, exactly what you can afford to purchase, and to work with a true Professional Realtor for your home purchase.
P.S. If this agent does not have "time" to work with you...there are many of us who will!
It is not a rule or set standard of this industry for the agnet to ask you to get pre-qualify or pre-approved prior on taking you to the show the property.
now on the other hand the seller's agent could ask you to send in your offer with a Lender Pre-Approval letter. so you might want to ask your agent if that is the case.
We use a local lender that is very easy to get approved with, please see his contact info below.
Houtan Hormozian
American Guardian Home Loans
This agent could have handle this better. He could have pre-qualified the buyer in regular conversation.
Example: What do you do for a living?
How long have you been working at ()*^?
Those two questions qualify the job and the income depending on the type of job.
While an agent is not a mortgage broker and can't qualify them for a mortgage they can qualify them to view a house.Whether or not the buyer should be Pre-approved is one thing but there is also a better way to go about it.
Some good information below. I would like to re-visit your question from your perspective.
1. How would you feel if you fell in love with a home only to find out it is out of your reach financially?
2. Do you have the time to be led on a "wild goose chase" viewing homes that are either well below or well above your purchasing power?
3. If you fall in love with a home in the $1.0 million dollar price range and find out that you can only afford $500,000, will any home at the lower price ever make you as happy as the home at the higher price?
4. When you have made any major purchase in your life time (car, engagement ring, audio system, etc), did you set your expectations of what you wanted, then look at your finances, then shop around to get the best deal for the best price?
Buying a home is a major purchase, a major decision, and for many, a very emotional experience. Knowing what you are jumping into, before jumping, can make the experience much more joyful and fulfilling.
Now, regarding the agent you spoke with, obviously this person has a different communication style than you do. Do you want to spend the next few months of your life, sharing your major decisions, hopes and wishes with someone who, it appears, speaks a language that is foreign to you? Better to find out early that they may not be the best fit for you. What they might have been saying, although not in a language that was clear to you or to many of those who answered below is: (1) Find out how much you can afford. (2) Think about what you want in a house. (3) Communicate this clearly with the professionals who make up your Team (who will be helping you find and negotiate a great deal on your new home). (4) Then with everyone on the same page, lets get out there and find a home that fits your needs, your lifestyle and your pocket book. (5) And lastly, lets negotiate a great deal on this great home and make sure that you have a great loan to finance it.
If that sounds like what you want to do, feel free to contact me. Working together, we can make your goal of home ownership a reality. Dare to Dream.
Shel-lee Davis
Real Estate Consultant
RE/MAX Palos Verdes Realty.
As with all professions you want a person is serious about wanting to purchase a home, qualified is first step we dont want to show $1 mil homes person who cant qualify can only purchase $500K home. All sales offers are submitted with lender approval letters, if not your offer is not consider till mortgage approval is obtained.
Right now interest is low and asking prices are too. It's a great time to buy a house. Spend a little time learning about what financing options are available to you while you're familiarizing yourself with your market. And find yourself a Realtor who will look after your needs.
Good luck.


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These 27 happened to be a package that had only flatties. You have to eat around it, then press the meat out from between the bones with a fingertip. Or sometimes it just pulls out, depends on how well they are cooked.
This was the best lunch I've had in a long time. When I stood up to go to the oven after the timer went off, my mouth watered so much I thought I blew a saliva valve or something. Pavlov's dog, big time.
Edit: No way, same plates? That must make the jealousy of seeing my lunch even harder to take. ha ha


text:
Rob Berini Vice President, Global Service Line Lead – Retail Banking, Multichannel Customer Acquisition & Servicing, Genpact Ltd
Rob has over 20 years of consulting and solutions experience with expertise in Emerging Technology Strategy, Innovation, and Multi-Channel Transformation. He has worked with nearly all of the leading global financial services institutions, with a particular focus on consumer and small business banking, payments and wealth management. Mr. Berini is a published author, and has contributed articles to publications such as Bank Systems and Technology.
Nick Miller President, Clarity Advantage Corporation
Nick Miller and Clarity help banks generate more profitable relationships faster with small and medium-sized businesses, their owners, and employees. The firm consults on sales targeting and segmentation, value propositions, sales process, customer experience, and staff development. During the last thirty years, Nick's clients have included many of the fifty largest banks in the U.S. and Canada and their subsidiaries in the Caribbean and Latin America.
Mary Beth Sullivan Managing Partner, Capital Performance GroupMary Beth Sullivan has 25 years of experience in financial services, both as a practitioner and an advisor to senior managers and directors. Mary Beth is a founding partner of Capital Performance Group LLC, a management consulting firm that provides advice, research, analysis, and project management services to the financial services industry.
Bill Hippensteel Managing Director for Knowledge Network and Content, BAI
Bill is responsible for tracking global industry trends and opportunities for BAI and for directing related development of BAI retail banking education, publishing, training and related research to provide critical strategic resources for the retail banking industry. He has over 30 years of financial services industry experience including BBVA Compass, BMO Harris and Fifth Third Bank. Bill holds an MBA in marketing from Western Michigan University and is a graduate of the Stonier Graduate School of Banking and the Wharton Business School/BBVA Marketing Graduate Program.


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The fifth and final 2006 Oscar nominee for Best Foreign Language Film finally arrives in American theaters, and it's a serious case of too little, too late. Susanne Bier's After the Wedding, from Denmark, is fairly middlebrow and melodramatic, not as bloody awful as Rachid Bouchareb's Days of Glory or Deepa Mehta's Water, but equally unmemorable. It's a testament to how badly the Academy needs to revamp this category: instead of taking a single submission from each of a list of countries, why not simply nominate the best foreign language films that played in American theaters during a calendar year? That way we could have enjoyed such nominees as Hou Hsiao-hsien's Three Times, Claude Chabrol's The Bridesmaid, Park Chan-wook's Lady Vengeance, Cristi Puiu's The Death of Mr. Lazarescu, Patrice Chéreau's Gabrielle, or perhaps even Jean-Pierre Melville's resurrected 1969 film Army of Shadows.
Fortunately, there's another reason for After the Wedding to exist, and that's the unique and charismatic star Mads Mikkelsen, with his impossibly pointy cheekbones, beady eyes and reptilian lips that look as if they're about to slide right off his face. In this country, he's best known as James Bond's nemesis in Casino Royale, or as Clive Owen's scrungy sidekick in King Arthur (2004), basically a sadistic badass. But in his native Denmark, he's capable of all kinds of things, from black comedies (Wilbur Wants to Kill Himself, The Green Butchers) to weepy melodrama (Open Hearts). After the Wedding definitely falls into the latter category (otherwise, it wouldn't have been an Oscar nominee).
p class="MsoNormal">Mikkelsen plays Jacob, a man with a mysterious past who now runs an orphanage in India. When he is asked to return to Denmark for a meeting, his most tearful goodbye is reserved for eight year-old, Pramod (Neeral Mulchandani), to whom Jacob has more or less become a surrogate father. In Denmark, he is merely supposed to shake hands with a rich bigwig, Jørgen (Rolf Lassgård), cross his fingers and hope for more money to run his organization. But Jørgen takes a liking to Jacob and invites him to his daughter's wedding the next day. There, Jacob runs into an old flame, Helene (Sidse Babett Knudsen), now Jørgen's wife. But he soon puts two and two together and realizes that he's the birth father of the young blushing bride, Anna (Stine Fischer Christensen), the daughter he never knew existed.
Basically the rest of the film shows all these characters learning how to relate to one another given this new wrinkle in their lives. Surprisingly, Jacob and Anna actually take to one another like milk to cereal; the movie's real drama arises from elsewhere. It turns out that Anna's new betrothed, a suckup who works for Jørgen, isn't exactly a model husband. And Jørgen has a secret of his own that causes Jacob to make a tough decision. None of this stuff is particularly surprising to anyone who's ever watched a soap opera or a melodrama. And director Susanne Bier (the Dogma 95 entry Open Hearts and Brothers) and her co-writer Anders Thomas Jensen (responsible for just about every recent Danish film you've ever heard of) can't quite juggle all the various details.
For example, when Jacob first arrives in Denmark, he checks into a massive, deluxe hotel suite, complete with all the luxuries. We know that Jacob mistrusts wealthy people and that this is a metaphor, but who is paying for this suite? If his job is to ask Jørgen for money, why would Jørgen be the one to pay for his room? Moreover, why does Jacob need to stay in Denmark for over a week when his meeting lasts only a few hours? (The answer is that it gives him time to deal with all his various soap operas.) Moreover, the movie draws obvious thematic parallels -- such as a father who cheats and a husband who cheats, or a husband and wife whose separately kept secrets lead to disaster -- but fails to really take these parallels anywhere.
For example, when Anna's no-good spouse cheats on her (mere days after their nuptials), she turns to her newfound dad for solace, even though she knows that Jacob once cheated on her mom. The movie finds no irony or friction in this potentially wounding duality. Finally, the movie sets up a hard choice for Jacob, whether he should stay in Denmark or return to India, but actually refuses to make the decision. It ends on an ambiguous note. Normally, I'm all for ambiguity in movies, but in a formula genre film like this, it's frustrating. It's a bit like a Rocky movie ending just before the big fight begins. However, this tactic actually ended the movie all that much sooner, which is a good thing.As with her previous features, Bier shoots on hand-held, high-contrast digital video, which results in an ultra-realistic, immediate feel, but which also highlights and underlines the film's confusion and dramatic sidestepping. Nevertheless, in the middle of all this tentative drama, we have Mikkelsen, lost in each of his individual moods and coming out on top. Perhaps this movie's Oscar nomination will result in Mikkelsen landing a few roles in Hollywood movies and sweeping aside our own army of bland pretty boys. He could romance Julia Roberts in the summer and then turn around and do something more substantial (sleep with a suburban mom, or hunt for blood diamonds?) in the fall.


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TUC Leading Change Programme 2016/17
The TUC launched 'Leading Change', a training and development programme aimed at senior trade union officers in leadership positions in 2004. Since its launch, over 150 senior union officers have completed the programme. The programme, which will be delivered in partnership with the Birkbeck University and the Labor & Worklife programme at Harvard University, is a comprehensive package of training, discussion and support spread across four modules delivered over a 9 month period...
21 July 2016 to 17 February 2017
Austerity has failed! Demonstrate with us at the Conservative Party Conference. October 2
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02 October 2016
TUC Young Workers Forum campaign workshops
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150 years of Trades Councils - Public Rally
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TUC unionlearn Regional Learning and Skills Event
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Gender and Occupational Safety and Health
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18 October 2016
South West TUC Pensioners' Annual Conference
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Support Wear Red Day
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Young Workers & Austerity
17 November 2016 to 18 November 2016


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"Serious biz" begins today, according to Randall catcher Josh Payne.
"During the preseason, it's not about your record, or anything, but getting to work and finding out who is going to do what during district," said Payne, the Raiders' senior catcher and emotional leader. "It's to the point now where this is a tradition at Randall. Of course, every team wants to win district. But for us it's to the point where you don't want to be the class that messes up the streak. We take this serious."
Serious, for Payne and his teammates, is the District 3-4A baseball race which opens at 4:>30 p.m. today when the Raiders play host to Dumas. Randall has won five consecutive district championships, including a 14-0 district slate last season. And with a pro prospect as its pitching ace, Randall is favored to retain its crown.
Metro Capsules
Class 5A
Amarillo High (10-7, 0-0) at Lubbock High (12-5, 0-1), 4:30 p.m.: Sandies open District 3-5A season on the road. This marks the start of a busy schedule for AHS, with three games during the next five days.
Class 4A
Canyon (10-5, 0-0) at Caprock (4-7, 0-0), 4:30 p.m.: District 3-4A opener for both teams. Eagles expected to compete for district title. Caprock coming off a solid showing in Crown Tournament, which included a 12-11 loss to district favorite Randall.
Dumas (7-6, 0-0) at Randall (10-5, 0-0), 4:30 p.m.: Raiders have won five district baseball titles in a row. Dumas looking to end Raiders' 14-game district winning streak.
Pampa (6-10, 0-0) at Palo Duro (3-8, 0-0), 4:30 p.m.: Both teams looking for early momentum in the District 3-4A race.
Class 3A
River Road (6-3) at San Jacinto Christian (7-2), 4:30 p.m.: Non-district clash between two teams expected to contend for district titles in respective races.
Other district games today have Canyon visiting Caprock, Borger traveling to Hereford, and Pampa visiting Palo Duro. All three games are set for 4:30 p.m.
During the previous five years under sixth-year coach Steve Ribera, Randall has compiled a 52-6 record in district games (89.6 winning percentage). Randall enters district play with a 10-5 record. However, the Raiders have won seven in a row, including four in the Crown of Texas Tournament last weekend.
"These guys can just smell district when it gets near," explained Ribera. "Winning district is definitely something we talk about and take pride in."
Randall returns a solid nucleus of six starters - pitcher Chance Douglass, outfielder Ryan Jackson, first baseman Craig Howard, outfielder Dustin Brooks, infielder Sam Sessler and Payne.
Without question, Randall features an ace in the hole no other district team can - The Douglass Factor. Douglass, a senior who will be taking his 90-mph-plus to Rice University later this year, is considered one of the top pro prospects in Amarillo-metro history. He is 3-1 on the year and is coming off a no-hitter against Tascosa. Plus, he has hit eight home runs. However, unlike last year, Randall doesn't own a deep and experienced pitching staff. That doesn't matter, says Payne.
"We do have so much confidence with Chance on the mound," said Payne, a Seward County College (Kan.) signee. "But we also have it without him on the mound because we have a lineup that can score a lot of runs."
Although Randall is considered the favorite, certainly nobody is handing the district title over to the Raiders - especially the Canyon Eagles.
Canyon enters district play 10-5 under veteran coach Gary Hix. Ben Weston is among the district's top pitchers, and the Eagles also feature pitcher Candy Medellin, first baseman Michael Crabtree, cleanup hitter Chris McMenamy, leadoff man Cody Weatherly, shortstop Wade Conner and outfielder Evan Parker.
"I hope we are ready," Hix said. "I believe in these guys, and they work hard."
Caprock coach Gerald Nipp, who opens his 23rd district season today and 18th with the Longhorns against Canyon, says the Eagles are a team to reckoned with.
"I think Canyon is the team to beat," said Nipp, who is seven wins shy of 200 victories. "Of course, you have to make Randall a contender, too, but Canyon plays the best as a team. After those two, it's a fight."
Nipp, whose team is 4-7, added, "We are going to have to get some consistent pitching and hit the ball a little better than we have so far."
Hereford (5-6-1) returns seven starters from last year's playoff team, including four-year varsity player Andrew Villareal at shortstop and senior catcher Juan Ayala.
Dumas, under third-year coach Phil Guerra, joins Randall and Canyon as the only other 3-4A team with a winning non-district record. Dumas is 7-6 and returns four starters - outfielders Zach Wilson and Justin Kehoe, infielder/pitcher Jose Garcia and first baseman Adam Rogers.
Pampa was 6-10 during non-district and features ace hurler Adam Jones, who led the Amarillo-area in strikeouts last year.
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OMG! That's Halarious!!!
Click The Link For And Link TO IGN.
at one point there was halo for ds. put that in yo adress bar to see for yourself.
OMG! That's Halarious!!!
Click The Link For And Link TO IGN.
yeah so i don't hink that ever happend.. wy would they make games on a systems for the rivals?????
Google is your friend
Ceraintly Own:
Wii,RedSteel,Legend of Zelda,The godfather ,R4DS & DS,prince of persia
yah i played it
rare still makes ds games, and they work for microsoft. microsoft doesnt consider the ds competition because they dont have a handheld system...yet.
Thanks Star Striker for the sig.
Support the Third Parties!
Buy Zack and Wiki
No More Heroes
well the ds is probably the most successful console of 2006
yah, and your full of it tooOriginally Posted by wiiboy 27
got beef
ya, i heard of this prject too bad it never got to see the day of light...
I have the wii!!!! i know i suck at spelling!
Rep me if I did good.
Spoiler Alert!Nintendo Fanboy: 49%
Sony Fanboy: 14%
Microsoft Fanboy: 20%
This means you're: Not a fanboy
You like all the systems pretty equally, or at least don't love any of thes systesms. Good for you, you like vareiety.
yea i wish it never got cancelled


text:
Four-star Willey on song again
Watch free daily video highlights from every LV= County Championship match here on ecb.co.uk this summer
David Willey took his LV= County Championship wicket-tally to 15 in five innings with 4-71 on the opening day against Gloucestershire at Bristol.
Thanks largely to the left-arm seamer, the home side were restricted to 192 all out after winning the toss, 18-year-old wicketkeeper Cameron Herring top-scoring with 43 in his first championship innings.
Steven Crook, with 3-47, James Middlebrook - the beneficiary of two late wickets - and Trent Copeland, who conceded only 26 runs from 18 overs, provided able support for Willey, before the visitors replied with 107 for three.
The hosts delivered a tentative batting display and were nine for two in the seventh over when Willey followed up having Chris Dent taken in the slips by bowling Dan Housego.
Michael Klinger was caught behind playing forward to Crook for 23 and, with the ball moving in the air and off the seam, may have regretted his decision to bat.
Crook accounted for Alex Gidman, on 20, as he was caught at first slip by Andrew Hall and the score read 70 for five when Hamish Marshall was caught behind off Willey for eight in the final over before lunch.
Will Gidman offered a second catch to Sales to give the in-form Willey his fourth wicket.
Benny Howell looked in better form than most, but he had his stumps rearranged by Crook when pushing forward to make it 111 for seven.
Herring and Jack Taylor then put together an attractive stand of 48, which ended when off-spinner Middlebrook was introduced into the Northants attack.
The veteran accounted for Taylor, who skied to mid-off with 31 to his name, and David Payne was stumped by David Murphy off the medium-pace of Hall for a duck.
Herring, who had come in at 90 for six, was last man out, also stumped advancing to Middlebrook having hit eight fours in an 81-ball innings of immense promise.
The teenager's good day continued when he dived to catch Kyle Coetzer for 24 off Will Gidman to break a Northants opening stand of 41.
David Sales pulled a simple catch to midwicket off Liam Norwell and departed kicking himself for five.
Stephen Peters, on 34, repeated the shot against the same bowler and was brilliantly caught by Will Gidman at mid-on, but Northants could still be pleased with their day`s work.
Click here for all the latest news from the domestic cricket scene, including exclusive interviews, team news, match reports and photo galleries


text:
|Sunday, 19 January 2003
Rajiv's assassins follow computer classes
Three people who were given the death penalty for their role in the assassination of former Indian Prime Minister Rajiv Gandhi are taking computer lessons from a university named after his mother, a report said Saturday.
Murugan, Perivalan and Nalini were sentenced to death in 1998 for aiding and abetting Gandhi's murder in the Southern State of Tamil Nadu in May 1991. A female suicide bomber from Sri Lanka's Tamil Tigers killed Gandhi.
Nalini's death sentence was commuted to life imprisonment after the intervention of Gandhi's Italian-born widow, Sonia. The other two have mercy petitions with the president pending since April 2000.
A report in the Hindustan Times newspaper said Murugan and Perivalan had enrolled for undergraduate courses in computer applications, while Nalini had joined the master's degree classes. The three, who are in a Tamil Nadu jail, have correspondence classes organised by an open university named after former Prime Minister Indira Gandhi, Rajiv's mother. The report said Nalini keeps Sonia Gandhi informed about her academic progress and had received congratulatory notes from the Congress party leader.
News | Business | Features
| Editorial | Security
Produced by Lake House


text:
The head of the BBC ignored a complaint about bullying for several months, after promising to improve the way it treats staff in the wake of a critical report.
"We cocked up," Lord Hall of Birkenhead, the BBC's Director-General, told the Culture, Media and Sport Select Committee yesterday, after admitting that a complaint made by a whistleblower in July had been overlooked.
"I apologise to that person completely," Lord Hall told the MPs. "We didn't handle it properly. I take the blame. I run the shop, but we're now dealing with it."
Ben Bradshaw, the Labour MP for Exeter, said


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A&E (Sunday, March 17, 8:30 p.m. ET)
British actress Felicity Kendal as Gemma is adorable, 30 and happy. Then she discovers that her part-time roomie and lover, Danny, has succumbed to the busty charms of her best friend. So Gemma dumps him, the best friend and her job and begins a new life, minus men. "It's your age, you know. Women start to panic once they're 30," the dumped Danny tells her. "The only reason a woman would panic when she's 30," Gemma replies, "is because she's stuck with a man who's 30 and, boy, is that a dismal scene." Solo is a modern, feminist sitcom with a sense of humor. Gemma tells her mother: "If God had been a woman, we'd be out having all the fun, and they'd be sitting at home sobbing on the telephone." She speaks to the empty half of her bed—to an imaginary Danny—and says, "For you, I could have been just like my mother, all warm and soft, wearing the proud and brave hallmarks of devotion—rough hands, sacrificial smile and stretch marks." The writing, by Carla (Butterflies) Lane, is sophisticated without being snotty, relevant but not preachy. She gives you a frank and funny picture of the trouble with romance these days. And the acting...Well, the critic may as well admit it: He's had a crush on Felicity Kendal since she appeared on American TV in the imported sitcom The Good Neighbors. She is the British Mary Tyler Moore, instantly endearing with her charm and rough-edged grace and the greatest grin ever broadcast. Watch her in Solo and see if you don't fall in love too.
Picks and Pans Review: Solo
updated 03/18/1985 AT 01:00 AM EST
•originally published 03/18/1985 AT 01:00 AM EST