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|Dec 20, 1988
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|Feb 19, 1987
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|May 21, 1982
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|Robert E. Canfield, Paul H. Ehrlich, William R. Moyle
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|The Trustees Of Columbia University In The City Of New York
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|Patent Citations (14), Non-Patent Citations (53), Referenced by (9), Classifications (24), Legal Events (4)
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The invention described herein was made in the course of work under grants numbered HD-13496, HD-15454 and CA-26636 from the National Institutes of Health, United States Department of Health and Human Services.
This is a continuation of U.S. Ser. No. 728,853, filed Apr. 30, 1985, which was a continuation of U.S. Ser. No. 380,959, filed May 21, 1982, now U.S. Pat. No. 4,514,505, issued Apr. 30, 1985.
Development of the hybridoma technology [Kohler, G., and C. Milstein, (1975) Nature 256: 495 and; Goding, J. W., (1980) Immunological Methods 39: 285] has provided immunoglobulin reagents which bind to only one antigenic site. Although these reagents have found widespread use as biochemical and immunological tools, their usefulness in radioimmunoassay has frequently been limited by their lower affinity for antigen compared with that of serum antibodies [Goding, J. W., (1980) Immunological Methods 39: 285]. In principle, the affinity of monoclonal antibodies could be enhanced by more stringent hybridoma selection procedures. The production of monoclonal antibodies has also enabled investigators to dissect the humoral immune response into its pure components [Staines, N. A. and A. M. Lew (1980), Immunology 40: 287]. This will eventually result in a more comprehensive understanding of the role of the individual antibody, especially with regard to the possibility that an antiserum may have different characteristics than the sum of its individual antibodies.
During the course of a systematic assessment of the immunochemistry of human chorionic gonadotropin (hCG), including the relative orientation of different epitopes of this molecule and the effect of several monoclonal antibodies on the hormone-receptor interaction, it has been observed that the apparent affinity of mixtures of certain monoclonal antibodies is enhanced relative to the affinity of the individual antibodies.
A mixture of monoclonal antibodies which includes an effective assaying amount of each of at least two monoclonal antibodies that bind to different antigenic sites on an antigen and are capable of doing so under appropriate conditions is useful in an enhanced sensitivity assay for the antigen.
Such mixtures are particularly useful in assays for antigens having multiple epitopes, e.g. polypeptides such as human chorionic gonadotropin, follicle stimulating hormone, thyroid stimulating hormone or luteinizing hormone where the different antigenic sites on the antigen are different amino acid sequences contained therein. By employing such mixtures in assays for antigens of clinical interest one can obtain enhanced sensitivity as compared with the sensitivity obtained with the individual monoclonal antibodies.
FIG. 1: Radioimmunoassay for human chorionic gonadotropin (hCG) by double antibody liquid phase assay. The amount of labeled hCG bound in the absence of unlabeled inhibitor was approximately the same for each antibody or antibody mixture. This was achieved by appropriate dilution of each antibody solution or mixture. Rabbit anti-mouse IgG was the second antibody. The ED50 plus/minus the standard deviation was calculated for each antibody or mixture. The ED50 for the mixture of B101 and B102 is 2.44�0.74�10-10 M, antibody B101 is 2.88�0.74�10-9 M, and antibody B102 is 1.058�0.995�10-7 M.
FIG. 2: Scatchard analysis of the binding to hCG of A, a mixture of antibodies B101 and B102; B, antibody B101 and C, antibody B102. Slopes of the lines (therefore, the equilibrium binding constants) are A: 5.4�109 ; B: 5.1�108 ; and C: 1.9�107.
FIG. 3A: Radioimmunoassay for hCG by double antibody liquid phase assay. The procedure was the same as for FIG. 1. The ED50 for antibody A102 is 8.52�2.94�10-9 M, antibody B101 is 2.88�0.74�10-9 M, and mixture of antibodies B101 and A102 is 4.66�1.99�10-9 M.
FIG. 3B: Radioimmunoassay for hCG by double antibody liquid phase assay with antibodies B101, B103, and a mixture of B101 and B103. The procedure was the same as in FIG. 1.
FIG. 4: Radioimmunoassay for hCG by solid phase assay. A: Antibodies B101, B102 and a mixture of B101 and B102. B: Antibodies B102, B103 and a mixtuure of B102 and B103.
FIG. 5: Radioimmunoassay for hCG by double antibody liquid phase assay with papain digested B102. The procedure was the same as for FIG. 1 except that the second antibody used for precipitation of the antibody-antigen complex was goat anti-mouse F(ab')2. The increase in affinity of the mixture was not affected by this change of second antibody (results not shown).
FIG. 6: Radioimmunoassay for hCG by double antibody liquid phase assay with B101-F(ab')2. The procedure was the same as for FIG. 5.
FIG. 7: Logit inhibition curves of A: a mixture of B01 and B102, B: antibody B101 and C: antibody B102 with hCG and with human luteinizing hormone (a hormone with a very similar structure).
It has been observed that mixing monoclonal antibodies directed against various epitopes of human chorionic gonadotropin can increase the sensitivity of antigen binding assays. Depending on the antibody pair chosen, the affinity of the mixture can be as much as 10-fold higher than that of the monoclonal antibodies assayed separately. This increased affinity can be detected in both a solid phase assay and a liquid phase double antibody radioimmunoassay. The mechanism for the increase in affinity depends on the formation of a multi-component complex. Mixing two antibodies which cannot bind simultaneously does not result in enhanced affinity, but combining pairs which can bind at the same time under appropriate conditions results in higher sensitivity in an antigen binding assay. If one of the antibodies of a pair which results in enhanced affinity upon mixing is replaced by its F(ab) fragment, the enhancement is no longer detectable, indicating that it is unlikely that the enhancement is due to an allosteric effect. While the F(ab')2 fragment shows some enhancement when mixed with another antibody, it is not as effective as the intact antibody.
Based upon these discoveries and observations, the present invention provides mixtures of monoclonal antibodies useful in an enhanced sensitivity assay for an antigen. The antibodies useful in the practices of the inventions are also characterized by the fact that they are capable of binding to the antigen both under normal nondenaturing conditions and under conditions described hereinafter where antibodies otherwise capable of binding to the antigen are unable to do so.
The invention described herein may in principle involve more than two monoclonal antibodies. Mixtures containing three, four or more antibodies each of which binds to a different antigenic site on the antigen may be particularly useful in assays for large molecules such as polypeptides having numerous subunits. However, for illustrative purposes the invention will be described by reference to mixtures which contain two such antibodies.
Although various types of antigens could be detected in assays employing mixtures of suitable monoclonal antibodies, the invention is particularly suited for detection of polypeptide antigens where the different antigenic sites are different and distinct amino acid sequences contained within the polypeptide. Examples of antigens of this type are the sequence related hormones human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH).
To be effective in assay, each monoclonal antibody of the assay mixture should be present in an amount sufficient to permit significant binding to the antigen, that is, above about 10 percent of the antigen to be simultaneously bound to the antibodies. Preferably the amount of antigen bound should be higher, for example an amount in the range from about 20 to about 80 percent or higher.
In order to obtain such amounts of bound antigen, the precise amount of each antibody may vary widely depending upon its affinity for the antigen so that lesser amounts of antibodies having higher affinities are required than of antibodies having lower affinities.
The amounts of each of the monoclonal antibodies relative to one another will also vary over a wide range. For a two antibody mixture, the amount may vary from about 106 :1 to 1:106. However, based upon the binding constants most likely to characterize the monoclonal antibodies of interest the range might preferably be from about 102 :1 to 1:102. In general, the preferred amount of each antibody relative to the amount of other antibodies will be substantially the same as the ratio of the binding constants of the antibodies to the antigen. Thus, if the binding constants of monoclonal antibodies A and B are 10-9 and 10-7, respectively, the relative amounts of A and B will be about 10-9 /10-7 =10-2, that is, 1 molecule of A for each 100 molecules of B.
In the case of polypeptide antigens which contain subunits such as the α and β subunits of human chorionic gonadotropin, mixtures of monoclonal antibodies in which each of the antibodies binds to an antigenic site on a different subunit are more likely to provide enhanced sensitivity in assays employing them, possibly because they are more likely to be capable of simultaneously binding to the antigen under appropriate conditions.
The mixtures may be used in assays in different forms. Thus, they may be used in solid form absorbed onto a solid matrix or in liquid form dissolved in a suitable buffered solution.
The mixtures can be employed in conventional immunoassays, including radioimmunoassays, to determine qualitatively and quantitatively the presence of antigens in samples of interest. In such methods the samples are contacted with the mixture under suitable conditions permitting formation of a detectable complex between the mixture and the antigen. One specific application would involve detection of human chorionic gonadotropic as a test for pregnancy.
To assist in an understanding of the invention the results of a series of experiments follow. Of course, these experiments are intended merely to illustrate the invention and should not in any way be construed as limiting its scope as defined by the claims which follow thereafter.
Production of Monoclonal Antibodies:
Balb/c mice were immunized monthly with an intraperitoneal injection of hCG subunits in complete Freund's adjuvant for several months according to the method of Wands and Zurawski [Wands, J. R., and V. R. Zurawski, (1981) Gastroenterology 80: 225]. Three days before the spleen was excised, additional antigen was injected (50 μg, I.V., in saline).
The spleen cells were fused with P3-NS1/1-Ag4-1 myeloma cells and hybridoma cells were isolated using published procedures [Wands, J. R., and V. R. Zurawski, (1981) Gastroenterology 80: 225; Marshak-Rothstein, A., et al., (1979) J. Immunol. 122: 2491]. Antibodies used for subsequent studies were isolated from hybridoma cell lines which were cloned twice by limiting dilution on Balb/c 3T3 monolayers. The antibodies are named with a letter and three numbers. The letter B indicates the antibody is specific for the β subunit of hCG and A indicates the antibody binds the α subunit.
Cell supernatant, partially purified, or purified antibody, or both were used. To obtain partially purified antibody the hybridoma cells were grown in serum-free medium containing 2 mg/ml bovine serum albumin and the supernatant dialyzed against 0.05M ammonium bicarbonate. After lyophilization, the powder was reconstituted in the desired volume of 0.3M potassium phosphate buffer, pH 7.5. To obtain purified antibody the same protocol was followed (except the medium contained 0.1 mg/ml bovine serum albumin) and the albumin removed with a DEAE Affi-Gel Blue column (Bio-Rad Laboratories).
Double Antibody Radioimmunoassay:
50 μl 125 I-hCG and 50 μl of unlabeled hCG (both in 1 percent horse serum, 99 percent phosphate buffered saline) were mixed with 100 μl 0.3M potassium phosphate (pH 7.5). Subsequently, 100 μl antibody (diluted in 1 percent horse serum) were added, the tubes incubated one hour at 37� C. followed by 18 hours at 5� C., and the complex precipitated by adding 10 μl of 50 percent normal mouse serum (in phosphate buffered saline) and an appropriate amount of rabbit anti-mouse IgG or goat anti-mouse F(ab')2. Once precipitation was complete (i.e. 10 minutes at 37� C. and then 1 hour at room temperature), the precipitate was sedimented and counted.
Solid Phase Radioimmunoassay:
To coat plastic microtiter wells with antibody, 50 μl purified antibody was incubated for 18 hours at 5� C. in a Cooke microtiter plate ("U" wells) and excess non-bound antibody removed by washing the wells three times with distilled water. To saturate all the plastic sites which would bind proteins, the plates were treated with 10 percent γ-G free horse serum (90 percent phosphate buffered saline) for 2 hours and the excess removed with distilled water. HCG binding was quantified by adding 50 μl 125 I-hCG in 1 percent horse serum containing varying amounts of hCG. After an incubation of 18 hours at 5� C., excess radioactivity was removed, the plates washed with distilled water and the wells counted.
Sandwich Assay:
50 μl containing at least 30 μg/ml of monoclonal antibody were added to the 96-well microtiter plates to permit the antibody to adsorb to the surface of the plastic. After 4 hours at 37� C. the solution was removed and the plates immersed in 150 mM NaCl solution containing 1 mg/ml bovine serum albumin (BSA-saline) to fill remaining non-specific adsorption sites on the plastic surface. To saturate the antibodies with hCG, the hormone (1 μg in 50 μl BSA-saline) was added for 2 hours to each microtiter well. Under these conditions most of the hCG which became insolubilized was bound to antibody adsorbed to the plastic. Excess hCG was removed by washing the plate in BSA-saline solution. After addition of radiolabeled antibody (50,000-100,000 cpm in BSA-saline solution for 2 hours at room temperature), the non-bound label was removed by washing the plate in BSA-saline, the microtiter plates cut apart with scissors, and the radioactivity adsorbed to each well was measured.
Other Materials and Methods:
Digestion of antibody B102 was performed by methods previously described [Edelman, G. M., and J. J. Marchalonis, (1967) Methods in Immunology and Immunochemistry, Vol. I, pp. 405, Academic Press, New York]. Sodium dodecyl sulfate (NaDodSO4) polyacrylamide gel electrophoresis [Weber, K., and M. Osborn, (1969) J. Biol. Chem. 244: 4406] of the digestion products indicated that essentially no complete heavy chain remained. The F(ab')2 fragment of antibody B101 was prepared by pepsin digestion [Gorini, G., et al., (1969) J. Immunol. 103: 1132], except that the amount of pepsin was 21/2 percent by weight of the amount of antibody. Antibody which remained undigested was removed by incubation with Protein A-Sepharose (Pharmacia Fine Chemicals) at pH 8.1 and centrifugation of the Protein A-Sepharose-antibody complex. Conditions were similar to those employed by Ey, et al. [Ey, P. L., et al., (1978) Immunochemistry 15: 429] except that a batch process instead of column chromatography was employed. NaDodSO4 polyacrylamide gel electrophoresis in the absence of reducing agents revealed only one major band at a molecular weight of 105,000. The concentration of hCG was determined by the optical density at 280 nm and amino acid analysis. Chloramine-T was used to iodinate hCG according to the procedure described by Greenwood, et al. [Greenwood, F., et al., (1963) Biochem. J. 89: 114].
The ability of hCG to inhibit the binding of radioiodinated hCG to monoclonal antibodies B101, B102, and a mixture of B101 and B102 was compared (FIG. 1). HCG was 20-fold more active in inhibiting binding of radiolabel when the mixture was employed. Scatchard analysis of the data showed a similar increase in affinity of the mixture over B101 alone and a much larger increase over B102 alone (FIG. 2). The greater affinity enabled us to dilute a 1:1 mixture of the two antibodies 9-fold and achieve the same amount of tracer binding as the separate antibodies. These results were highly reproducible, as shown in Table I. While the data are not presented here, the ability of two antibodies to show this cooperative effect is not limited to pairs of antibodies which bind the same subunit since A102 and B102 also have this cooperative interaction. Other combinations of antibodies did not produce the same result. Thus, a similar cooperative effect between B101 and A102 (FIG. 3A) or between B101 and B103 (FIG. 3B) was not observed.
TABLE I______________________________________Statistical Analysis of the Affinity of Antibodies B101 andB102 Compared with a Mixture of Antibodies B101 plus B102 Antibody or EnhancementExperiment Mixture Keq*(L/nMole) Factor** P______________________________________1 B101 0.51 � 0.02 10.6 <.01 B102 0.019 � 0.002 B101 + B102 5.4 � 0.52 B101 0.41 � 0.09 7.3 <.01 B102 0.018 � 0.002 B101 + B102 3.0 � 0.23 B101 0.88 � 0.08 5.7 <.01 B101 + B102 5.0 � 0.074 B101 0.81 � 0.11 6.0 <.01 B101 + B102 5.5 � 1.2______________________________________ *Determined using Scatchard plots [Scatchard, G., (1949) Ann. N.Y. Acad. Sci. 51:660. **Keq for a mixture of B101 and B102 divided by Keq for B101. For the purposes of calculating the enhancement, the affinity of the mixture is compared to the affinity of the antibody with the higher affinity.
Mixtures of B101 and A102 (or B101 and B103) gave inhibition curves intermediate between those antibodies. Similar results are apparent with a solid phase radioimmunoassay (FIG. 4). A mixture of B101 and B102 was observed to have a higher affinity of hCG than either of the antibodies separately, although the difference between the mixture and antibody B101 was only about 4-fold. The combination of antibody B102 and B103 did not result in an increase in affinity.
Sandwich Assays:
In order to obtain data bearing on the mechanism of the affinity change, the ability of the antibodies to bind the different sites on hCG was measured using sandwich assays (Table II). If unlabeled and radiolabeled antibody bind to the same site, the unlabeled antibody will inhibit binding of the radiolabel. Conversely, if both bind to different sites, a large amount of radiolabel will be bound to the plastic. Labeled and unlabeled B101 cannot bind the antigen at the same time; therefore, little radiolabel was observed bound to the plastic. Similar results were observed using labeled and unlabeled B102. Antibodies B102 and B103 bound to hCG at a site remote from that for B101 since both allowed binding of B101 to the plastic. Further, labeled B102 bound to hCG attached to unlabeled B101. In contrast, antibody A102 prevented binding of radioactive B101 indicating that A102 and B101 cannot bind simultaneously to hCG. Similarly, it was found that antibodies B102 and B103 cannot bind simultaneously. Thus, it appeared that simultaneous binding of the antibodies on two different hCG sites was necessary but not sufficient for cooperative interactions.
Effect of Antibody Fragments:
To study the influence of the structure of the antibodies on cooperativity, fragments of the monoclonal antibodies were prepared. The capability of antibody B102 to enhance the affinity of B101 was eliminated when antibody B102 was digested with papain (FIG. 5) although papain digestion of B102 had no detectable effect on the affinity of this antibody for hCG. This indicated that either the F(c) region or the bivalency of the antibody was necessary for enhanced affinity. The bivalent F(ab')2 results in some enhancement of binding affinity but it is not as effective as the intact antibody (FIG. 6 and Table III).
TABLE II______________________________________Sandwich Assays Using Pairs of Monoclonal Antibodies Unlabeled AntibodyLabelled Antibody B101 B102 B103 A102______________________________________B101 17 9438* 11128* 415B102 5136* -202 144 5453*______________________________________ Values are cpm (means of triplicate determinations) of antibody specifically bound to hCGunlabelled antibody complexes adsorbed to the surface of the plastic microtiter plates. Radioactivity bound nonspecificaly (i.e. that which became bound to the plastic in the presence of a monoclonal antibody which does not bind hCG) was subtracted *p < .001; all the others are not significantly different from zero.
TABLE III______________________________________Statistical Analysis of the Affinity of B101 F(ab')2Compared with the Affinity of B101 F(ab')2 + B102Ex- Enhance-peri- mentment Antibody or Mixture Keq*(L/nMole) Factor** P______________________________________1 B101 F(ab')2 1.61 � 0.18*** 1.5 <.1B101 F(ab')2 + B102 2.4 � 0.42 B101 F(ab')2 0.77 � 0.11 2.2 <.01B101 F(ab')2 + B102 1.7 � 0.13 B101 F(ab')2 0.97 � 0.17 2.0 <.01B101 F(ab')2 + B102 1.9 � 0.24 B101 F(ab')2 0.72 � 0.06 6.4 <.01B101 F(ab')2 + B102 4.6 � 0.8______________________________________ *Determined using Scatchard plots [Scatchard, G., (1949) Ann. N.Y. Acad. Sci. 52:660. **Keq for a mixture of B101 F(ab')2 and B102 divided by Keq for B101 ***Keq values for B101 F(ab')2 are not significantly different than Keq for B101 shown in Table I.
Mixtures of monoclonal antibodies may have significantly different properties than the individual components. It has been shown that the affinity of a mixture of hybridoma antibodies can be enhanced. Other properties of monoclonal antibodies have previously been improved by utilizing more than one hybridoma antibody. Haber, et al. [Haber, E. et al., (1980) "Resolving antigenic sites and purifying proteins with monoclonal antibodies", Monoclonal Antibodies in Endocrine Research, Raven Press] developed a radioimmunometric assay with two monoclonal antibodies which had greater specificity than an assay employing either antibody separately. Howard, et al. [Howard, J. C., et al., (1979) Immunological Rev. 47: 139] showed that combinations of monoclonal antibodies can be synergistic in lysing red blood cells. Although some of the advantages of monoclonal antibodies may be lost upon mixing, such as the ability to react with a single determinant, many advantages may result in producing what is, in effect, a synthetic antiserum. These results may also have implications for the control of antibody synthesis and the immune response in vivo. Since mixtures of antibodies have different properties than the individual antibodies (depending on which ones are mixed), it seems likely that the immune response should result in mixtures of compatible antibodies. Thus, the maturation of the affinity of antisera [Karush, E., (1978) Comprehensive Immunology 5. Immunoglobulins pp. 85, Plenum Medical Book Co., New York] may not only be due to the fact that a high affinity clone has been stimulated but that an antibody has been produced which can enhance the affinity of another antibody already present in the serum.
Several types of antibody mixtures have been examined. Combining antibody B101 with antibody A102 results in the expected dose-response curve in that the logic plot shows the inhibition line for the mixture to be between the inhibition curves for the separate antibodies. B101 and A102 cannot bind hCG simultaneously. With antibodies B101 and B103, which can bind to the hormone at the same time, the logit plot for the mixture follows the plot for the high affinity antibody at low antigen concentrations and approaches the line for the low affinity antibody at high antigen concentrations. The mixture of antibodies B101 and B102 is the major result of this work in that the affinity of the mixture is higher than either antibody. As shown in Table I, this result is reproducible and statistically significant.
Once the presence of a cooperative effect had been established, the question of mechanism arose. There was a loss of enhancement when the F(ab) fragment was substituted for the antibody. This result indicates two important characteristics of the cooperativity. First, a larger fragment of the antibody than F(ab) is required. Second, the possibility that the binding of one antibody changes the conformation of the hormone thus leading to a higher affinity of the second antibody (an allosteric model) became unlikely. Another possible explanation for the enhancement is that it is caused by intermolecular crosslinking of bivalent antibodies and antigen (as in formation of a circular complex consisting of two antibody and two antigen molecules, which Schumaker, et al. [Schumaker, V. N., et al., (1973) Immunochemistry 10: 521] have predicted to be very stable complexes). The results with F(ab')2 indicate that, at the very least, this cannot be the sole mechanism. Alternatively, interaction of the F(c) regions in a complex is consistent with all data from the liquid phase radioimmunoassays. The solid phase assays seem to indicate the F(c) interactions are not the only factor since the antibodies are already anchored to the plastic and enhancement still occurs. Of course, the mechanism may be different for the two types of assays. Finally, the results with antibodies B101 and B103, which can bind simultaneously to hCG but do not have a cooperative interaction, indicate that idiotype-anti-idiotype interactions may play a role (i.e., B101 and B102 may have an idiotype-anti-idiotype interaction between the free F(ab) arms left after binding to hCG while B101 and B103 are not related in this way). It should be emphasized, however, that the fact that B101 and B103 are not enhancing argues very strongly that some characteristic of the individual antibody (such as spatial orientation of the epitope or the nature of the idiotope) is extremely important in the cooperative effect. Further experiments are necessary for complete characterization of the mechanism.
The possibility that mixtures of antibodies may have higher affinity than the individual antibodies has two major implications. First, an opportunity for increasing the affinity of monoclonal antibodies is provided. Second, the capacity to mix a series of antibodies in a defined system makes possible a more complete understanding of the subtle effects which occur in a polyclonal immune sera.
By performing radioimmunoassays with all paired combinations of five monoclonal antibodies, the prevalence of synergistic interactions was measured. The following table shows the pairs of antibodies that are synergistic (have a higher affinity for antigen when mixed) measured by double antibody radioimmunoassays. A plus sign indicates that the antibody identified at the top of the column is synergistic with the antibody named at the left. A minus sign indicates no synergistic interaction.
______________________________________Antibody A103 B101 B102 B103______________________________________A102 - - + +A103 - + +B101 + -B102 -______________________________________
Thus, out of ten possible pairs, five are synergistic. Actually, the proportion is much higher because some of these antibodies cannot bind antigen simultaneously under any conditions. If these pairs are eliminated, then 5 out of 7 pairs are synergistic.
FIG. 7 shows the logit inhibition curves of a mixture of B101 and B102, antibody B101, and antibody B102 with hCG (the antigen) and hLH (a hormone with a very similar structure). Distinguishing between these two hormones is very important in the diagnosis of pregnancy and cancer. Graph A shows that the mixture of B101 and B102 can bind hCG about 170 fold stronger than hLH. Graph B shows that B101 can bind hCG about 50 fold stronger than hLH. Graph C shows that B102 is more sensitive to hCG than hLH by about 10 fold. Therefore, the mixture of antibodies is more specific than the individual antibodies.
In order to further characterize those monoclonal antibodies which when mixed provide enhanced affinity for antigen, further studies were undertaken. These studies showed that monoclonal antibodies possessing this property could be differentiated from those which do not, based upon ability to form a stable complex between the mixture of antibodies and the antigen. This complex appeared as an extra band in gel electrophoresis.
More specifically, 1-5 μg hCG and 1-5 μg antibody mixture were placed in 11 cm long tubes of 3, 4, 5, 6 and 7 percent polyacrylamide gels. The tubes were then subjected to a constant current of 4 milliamps per tube. The method used is essentially the same as that described for the second dimension in O'Farrell, P. H. (1975) J. Biol. Chem. 250: 4007-4021, except that there is no SDS. After separation, the gels are stained with a suitable dye.
The appearance of an extra band based upon the formation of a complex between the monoclonal antibody mixture and antigen indicates that use of such monoclonal antibody mixtures in assays for the appropriate antigen will provide enhanced sensitivity.
In addition to electrophoresis, antibody combinations resulting in enhanced affinity were recognized by an extra high molecular weight peak on gel filtration chromatography. More specifically, 1-5 μg hCG containing a suitable amount of radioidinated hCG (0.1-10.0 Ci) were mixed with 1 to 10 micrograms of the antibody mixture and chromatographed in 0.015M Hepes buffer, pH 7.4-0.9% NaCl--1 μg/ml BSA over a 1 cm diameter--24 cm long Sephacryl S-400 column. Mixtures of B101+B102+hCG giving enhanced affinity gave rise to an extra peak whereas mixtures of B101+B103+hCG known not to give an increase in affinity in solution failed to produce the extra peak. Thus, this procedure is also useful in determining which pairs of antibody will be effective.
Although the invention has been described with particular reference to the use of mixtures of monoclonal antibodies in assays to provide enhanced sensitivity, it is understood that the invention also embraces use of the mixtures to provide enhanced specificity and such other purposes as may subsequently be discovered, including perhaps in vivo treatment methods for various conditions.
|Cited Patent
|Filing date
|Publication date
|Nov 11, 1976
|May 9, 1978
|Bio-Rad Laboratories, Inc.
|Radioimmunoassay for thyroid-stimulating hormone (TSH)
|Oct 26, 1976
|Jul 4, 1978
|Corning Glass Works
|Reverse sandwich immunoassay
|Mar 4, 1977
|Aug 22, 1978
|Becton, Dickinson And Company
|Automated direct serum radioimmunoassay
|Sep 5, 1978
|Jan 13, 1981
|Bio-Rad Laboratories, Inc.
|Single-incubation two-site immunoassay
|Oct 3, 1977
|Feb 3, 1981
|Mochida Seiyaku Kabushiki Kaisha
|Immunochemical process of measuring physiologically active substances
|Aug 4, 1980
|Mar 8, 1983
|Hybritech, Incorporated
|Immunometric assays using monoclonal antibodies
|Jul 26, 1982
|Sep 11, 1984
|Board Of Trustees Operating Michigan State University
|Method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies
|Feb 16, 1983
|Oct 2, 1984
|Board Of Trustees Of The Leland Stanford Junior University
|Two-site immunoassays using monoclonal antibodies of different classes or subclasses and test kits for performing same
|Oct 14, 1981
|Jan 29, 1985
|Takeda Chemical Industries, Ltd.
|Method for enzyme immunoassay and production of antibody
|May 21, 1982
|Apr 30, 1985
|The Trustees Of Columbia University In The City Of New York
|Monoclonal antibody mixtures and use thereof for enhanced sensitivity immunoassays
|Jan 7, 1983
|May 28, 1985
|The United States Of America As Represented By The Department Of Health And Human Services
|Treatment of graft versus host disease using a mixture of T-lymphocyte specific monoclonal antibody: ricin conjugates
|Apr 16, 1982
|Jun 11, 1985
|Jose Carro
|Soluble assay
|Jul 13, 1981
|Jan 20, 1982
|Unilever Plc
|Methods of immuno analysis using monoclonal antibodies
|Title not available
|Aikawa, M. et al., J. Protozool., vol. 28(3), pp. 383 388 (1981) cited in Biosis Abstract No. 73068894.
|Aikawa, M. et al., J. Protozool., vol. 28(3), pp. 383-388 (1981) cited in Biosis Abstract No. 73068894.
|Berger, P. et al., Immunobiology, vol. 160(1), p. 5 (1987).
|Bosch, A. M. G. et al., Protides Biol. Fluids, vol. 29, pp. 837 842 (1982) cited in Chemical Abstract 96: 215701x.
|Bosch, A. M. G. et al., Protides Biol. Fluids, vol. 29, pp. 837-842 (1982) cited in Chemical Abstract 96: 215701x.
|Brown, J. P. et al., Clin. Chem., vol. 27(9), pp. 1592 1596 (1981).
|Brown, J. P. et al., Clin. Chem., vol. 27(9), pp. 1592-1596 (1981).
|Buchegger, F. et al., Immunology Letters, vol. 5, pp. 85 91 (1982).
|Buchegger, F. et al., Immunology Letters, vol. 5, pp. 85-91 (1982).
|Ehrlich, P. H. et al., J. of Immunology, vol. 128(6), pp. 2709 2713 (6 1982).
|Ehrlich, P. H. et al., J. of Immunology, vol. 128(6), pp. 2709-2713 (6-1982).
|Ehrlich, P. H. et al., Science, vol. 221, pp. 279 281 (7 1983).
|Ehrlich, P. H. et al., Science, vol. 221, pp. 279-281 (7-1983).
|Gupta, S. K. et al., Canadian J. Exper. Biol., vol. 18, pp. 1361 1365 (12 1980).
|Gupta, S. K. et al., Canadian J. Exper. Biol., vol. 18, pp. 1361-1365 (12-1980).
|Holmes, N. J. et al., J. Biol. Chem., vol. 288(1), pp. 1580 1582 (2 1983).
|Holmes, N. J. et al., J. Biol. Chem., vol. 288(1), pp. 1580-1582 (2-1983).
|Howard, J. C. et al., Immunol. Rev., vol. 47, pp. 139 174 (1977). See pp. 147, 148 160 168.
|Howard, J. C. et al., Immunol. Rev., vol. 47, pp. 139-174 (1977). See pp. 147, 148 160-168.
|Hurrell, J. G. R. et al., J. Immunological Methods, vol. 45, pp. 249 254 (1981).
|Hurrell, J. G. R. et al., J. Immunological Methods, vol. 45, pp. 249-254 (1981).
|Imai, M. et al., J. of Immunology, vol. 128(1), pp. 69 72 (1 1982).
|Imai, M. et al., J. of Immunology, vol. 128(1), pp. 69-72 (1-1982).
|Jefferis, R. et al., J. Immunol. Methods, vol. 39(4), pp. 355 362 (1980).
|Jefferis, R. et al., J. Immunol. Methods, vol. 39(4), pp. 355-362 (1980).
|Khazaeli, M. B. et al., Endocrinology, vol. 109(4), pp. 1290 1292 (1981).
|Khazaeli, M. B. et al., Endocrinology, vol. 109(4), pp. 1290-1292 (1981).
|Koprowski, H. et al., Proc. Natl. Acad. Sci., U.S.A., vol. 75(7), pp. 3405 3409 (1978) cited in Biocis Abstract No. 67016885.
|Koprowski, H. et al., Proc. Natl. Acad. Sci., U.S.A., vol. 75(7), pp. 3405-3409 (1978) cited in Biocis Abstract No. 67016885.
|Lemke, H. et al., Research Monographs in Immunology, vol. 3, pp. 102 109 (1981).
|Lemke, H. et al., Research Monographs in Immunology, vol. 3, pp. 102-109 (1981).
|Moyle, W. R. et al., Molecular Immunology, vol. 20, pp. 439 452 (4 1983).
|Moyle, W. R. et al., Molecular Immunology, vol. 20, pp. 439-452 (4-1983).
|Moyle, W. R. et al., Proc. Natl. Acad. Sci., U.S.A., vol. 79, pp. 2245 2249 (4 1982).
|Moyle, W. R. et al., Proc. Natl. Acad. Sci., U.S.A., vol. 79, pp. 2245-2249 (4-1982).
|Schonherr, O. T. et al., Develop. Biol. Standards, vol. 50, pp. 235 242 (1982).
|Schonherr, O. T. et al., Develop. Biol. Standards, vol. 50, pp. 235-242 (1982).
|Shimuzu, S. Y. et al., Clinical Chemistry, vol. 28(3), pp. 546 547 (1982).
|Shimuzu, S. Y. et al., Clinical Chemistry, vol. 28(3), pp. 546-547 (1982).
|Sprent, J. et al., J. Exp. Med., vol. 154(1), pp. 188 192 (1981) cited in Biosis Abstract 73032175.
|Sprent, J. et al., J. Exp. Med., vol. 154(1), pp. 188-192 (1981) cited in Biosis Abstract 73032175.
|Steensgaard, J. et al., Immunology, vol. 32, pp. 445 456 (1977).
|Steensgaard, J. et al., Immunology, vol. 32, pp. 445-456 (1977).
|Steensgaard, J. et al., Molecular Immunology, vol. 17, pp. 1315 1318 (10 1980).
|Steensgaard, J. et al., Molecular Immunology, vol. 17, pp. 1315-1318 (10-1980).
|Tosi, R. et al., European J. Immunology, vol. 11, pp. 721 726 (9 1981).
|Tosi, R. et al., European J. Immunology, vol. 11, pp. 721-726 (9-1981).
|Uotilla, M. et al., J. Immunol. Methods, vol. 42, pp. 11 15 (1981).
|Uotilla, M. et al., J. Immunol. Methods, vol. 42, pp. 11-15 (1981).
|Wada, H. G. et al., Clin. Chem., vol. 28(9), pp. 1862 1866 (9 1982).
|Wada, H. G. et al., Clin. Chem., vol. 28(9), pp. 1862-1866 (9-1982).
|Webster, R. G. et al., Virology, vol. 104(1), pp. 139 148 (1980) cited in Biosis Abstract No. 70061846.
|Webster, R. G. et al., Virology, vol. 104(1), pp. 139-148 (1980) cited in Biosis Abstract No. 70061846.
|Citing Patent
|Filing date
|Publication date
|Jun 29, 1992
|Sep 28, 1993
|Boehringer Mannheim Gmbh
|Process and reagent for the determination of the luteinizing hormone and monoclonal antibodies suitable thereof
|Jul 22, 1993
|May 14, 1996
|Mayo Foundation For Medical Education And Research
|Antibodies specific for human prostate glandular kallkrein
|Dec 17, 1996
|Jan 11, 2000
|Hybritech Incorporated
|DNA encoding hK2 variant polypeptides
|Dec 17, 1996
|Jul 25, 2000
|Mayo Foundation For Medical Education And Research
|Recombinant hK2 polypeptide
|Mar 26, 1996
|Aug 15, 2000
|Hybritech Incorporated
|Stable variant hK2 polypeptide
|Jun 19, 1998
|May 22, 2001
|Mayo Foundation For Medical Education & Research
|Method for detection of breast cancer
|Apr 11, 1997
|Nov 12, 2002
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|Method for detection of micrometastatic prostate cancer
|Dec 23, 2003
|Dec 23, 2004
|Mayo Foundation For Medical Education And Research, Hybritech Incorporated
|Method for detection of metastatic prostate cancer
|Apr 2, 1990
|Oct 3, 1990
|Synbiotics Corporation
|Drug production using non-cross reactive antibodies
|U.S. Classification
|436/515, 436/516, 436/548, 436/536, 530/388.1, 530/388.24, 435/70.21, 436/518, 436/510
|International Classification
|Cooperative Classification
|European Classification
|Jun 16, 1992
|Fee payment
Year of fee payment: 4
|Jun 18, 1996
|Fee payment
Year of fee payment: 8
|Jun 19, 2000
|Fee payment
Year of fee payment: 12
|Dec 11, 2008
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF
Free format text: EXECUTIVE ORDER 9424, CONFIRMATORY LICENSE;ASSIGNOR:COLUMBIA UNIVERSITY NEW YORK MORNINGSIDE;REEL/FRAME:021966/0698
Effective date: 20060727


text:
TC TO ALEXANDER CARLYLE ; 30 September 1845; DOI: 10.1215/lt-18450930-TC-AC-01; CL 19: 229-232
Scotsbrig, 30 Septr, 1845—
My dear Brother,—The time for the Packet drawing nigh, I, being here where your chief interests all lie, cannot but get a line ready for you. I got to Scotsbrig almost three weeks ago, and have been loitering about, doing very little; but I can at least tell you the news of this Household, which I am very sure will be a welcome service to you.
Our dear old Mother was in waiting for me that morning Jamie brought me up from the Steamer; she has run about unweariedly ever since, and nothing can prevent her from the most restless endeavour to make her guest better and better off,—far better than he has any need or wish of. She is in, what one must call very good health for her; not much weaker than when I saw her last: her hands shake a little more, I think, and that is almost all the change one notices in her. She varies considerably, however, from day to day; has slept indifferently last night, for instance,—we cannot expect her now to be strong. She does not mourn audibly for your absence; indeed she says expressly with thankfulness, You seem for all your sadness of mind to be doing really better than there was any clear chance of here. Good Mother! She is quite cheery yet, when moderately well in health; looks back with still resignation on many a sorrow, and forward with humble pious trust. It is beautiful to see how in the gradual decay of all other strength, the strength of her heart and affections still survives, as it were, fresher than ever;—the soul of Life refuses to grow old with the body of Life; one of the most affecting sights! We were talking last night of the death of Margaret,—that unforgettable night when you and I rode down from Craigenputtoch;1—and were all again, as it were, brought together, the dead of us and the absent of us; in a sad but to me very solemn and profitable manner.— I am far from happy here; indeed, in real fact, had I consulted but my own lazy feelings I had much rather not have come: nobody knows what inexpressible sorrow and confusion it is to me to look on this old Annandale in these sick humours of mine. However, I am very thankful I did come. Amid the muddy confusions outward and inward, there are things taught me here which I can learn nowhere else. Let us be thankful, for many blessings such as fall to the lot of few. Good Parents whom you can honour, it is the foundation of all good whatsoever for a man.
Jenny is here with her two children, doing the house-work. She seems to me stronger and much cheerfuller than two years ago. She is a very handy active lassie; and conducts herself with much wisdom and discretion. Very diligent; does all the sewing of the kindred, or nearly all; has her two bits of bairns clean as two pennies, in thrifty neatness; has them good readers, the eldest is even a good sewer. She never speaks of her misfortunes, nor does anybody else: indeed I am very glad to see the figure she cuts. Perhaps all that she has suffered may turn to her for good yet.— Jean and her Husband seem very prosperous, Jamie getting more and more employment (has twelve journeymen this year, I think): they were very kind to my Mother and me two days last week;—a terrible tumult of children always round them. Mary who has been unwell for a good while, with some rather dangerous tendency in the bowels, as I understand, is now very decidedly better; she was looking really well, the other day, with her children and house all clean and snug:—I believe she still complains a little, internally; but everybody admits she is very much better. Austin was in the middle of his harvest: four ricks in, some six more expected; a really tolerable crop, better than ever he has had, they say,—and now it is expected he will be able to go on without staggering henceforth.— — At Scotsbrig here, Jamie in so far as his farming affairs go has nothing bad to report of himself: prices rather high (owing to the railway, the expectation of which has already raised the price of all kinds of labour); prices very fair; a reasonable crop, stock all thriving; rent well reduced. His harvest is backward, as all the country is;—mowing hard today, in the interval of windy showers, and not half done yet. Indeed he is getting to diminish his crop, year after year; and depends now more and more upon sheep, which I think may prove good husbandry for such a climate. Will Easton and his squad are here just now, rebuilding the Stable;—exactly where it stood, only some feet higher,—and longer, close to the corner of old Betty's house. Poor old Pate Easton2 is dead, last winter. Wull has the premises all full of mortar-heaps and lumber ever since I came; and seems to me to get on very slowly. But the saddest feature for Jamie is poor Isabella, who is really a heavy load, to herself and others. She cannot speak, except with great effort a word or two in a scarcely audible whisper; is obliged to lie on a sofa, and do what she can for directing her house by signs thro' one of her nieces from Potstown:3 she is in terror whenever Jamie leaves her; she suffers much, in mind if not in body; and he has a very sad time of it, I think, so far as this goes. One does not hear him complain; but I notice him much graver than he used to be in former times. Nothing can be done to help his poor wife or him: Patience and quiet waiting is the only remedy.
This is nearly all the news I have to give of Scotsbrig. As to the rest, I have seen almost nobody, nobody at all that I could help seeing; and can only report in general that the people seem all busy harvesting; that the weather is very plashy and wet. Indeed our summer over all the Island was unusually dim and cold; people say the harvest is much better than was once expected. A very sad feature of it, however, is the Disease of the Potatoes: almost everywhere the Potatoes when nearly ripe are seized by a kind of murrain; wither in the stems; the Potatoe itself gets black-spotted, soft, and slowly or fast inevitably goes to mush. Jamie thinks he has lost about half of his: he has put up his swine to eat them as fast as possible out of the way. It is thought by some the Potatoe is about refusing to grow any more in these climates; which will be a very frightful business indeed! All over Holland, Germany &c as well as in Britain and Ireland this Potatoe-Epedemic4 is prevalent; to a great extent, I fear, tho' nobody as yet can tell how great.— Near Frank Graham the Tailor's house (a little on the Scalewood5 side) is seen a heap of dug earth among the corn: it marks the course of the railway in that quarter; which is just to be begun; and has raised wages, as I was saying: for two years there will likely be a greater briskness of demand for labour, and then the demand will end again; indeed it will mainly be the Irish that do the digging, I suppose; they are already crowding in great quantities into this quarter.— One Corry (Andrew Corry, I think they called him), a drunken Drover once well known hereabouts overturned his gig on the road from Dumfries lately, and killed himself on the spot: it was broad daylight, but the poor wretch was utterly drunk.
Dear Brother, you must write a Letter to us soon; you will have more leisure now that the winter is coming. A Letter of Tom's giving a minute account of all the domestic stock is still spoken of with much praise here! Tell him to try it again; and Jane too: you cannot be too minute. I must be off homewards again, in some week or ten days. The Book is coming out, I believe; you shall hear of me again. Farewell
All here salute you; I design to make my Mother write her own salutations, tho' her hand shake. My blessings on you one and all. Be diligent, patient: "Work and hope!"
[Margaret A. Carlyle's Postscript]
My dear Alick I would like to write to you if I could as this will testify but send you my best wishes thank Tom for his good letter and teell Jane and him to write soon and tell me all their news.
May the God keep and guid you all in His ways and fear which is the prayer of your old Mother
give my kindest love to Jenny and all the dear little ones. May you all be in Gods keeping

subset: commoncrawl


text:
Jan. 12, 2013
Box Score | Box Score (PDF) | Postgame Quotes
UCLA's Season Stats | 2012-13 UCLA Media Guide
BOULDER, Colo. - Travis Wear scored 14 of his career-high 23 points in the second half and Jordan Adams' two free throws with seven seconds left sealed UCLA's ninth straight victory, 78-75, at Colorado on Saturday.
UCLA (14-3, 4-0 Pac-12) also received 18 points from Jordan Adams and 14 from Shabazz Muhammad. Kyle Anderson registered his fifth double-double of the season, totaling 12 points and a team-best 10 rebounds.
The Bruins' nine-game winning streak is their longest since winning 10 straight during the 2008-09 season.
Spencer Dinwiddie led Colorado (11-5, 1-3) with 23 points. The host Buffaloes also received 18 points each from Josh Scott and Askia Booker.
UCLA improved to 6-1 this season in games decided by five points or fewer. The Bruins improved to 2-0 on the road this season, after having defeated Utah in Salt Lake City, 57-53, on Thursday night.
The Bruins led at halftime, 35-34, and pushed their margin to 40-34 early in the second quarter before Colorado tie the game, 42-42, with 16:09 remaining.
Leading 44-43, UCLA used a 17-5 scoring run over the next 6:49 to secure a 61-48 margin. That scoring run was capped by a three-pointer from Adams, who was fouled on the shot and converted the ensuing free throw.
Colorado chipped away, closing its deficit to 66-61 with 2:15 left in regulation. The Buffaloes got as close as one point, trailing UCLA by a 76-75 margin with seven seconds left, before Jordan Adams made two free throws.
Booker's three-point attempt to tie the game in the final seconds was unsuccessful.
Game Notes: This marks the fourth time in head coach Ben Howland's 10 seasons at UCLA in which the Bruins have opened conference play with a 4-0 mark (also in 2003-04, 2007-08, 2008-09) ... Shabazz Muhammad finished with 14 points, scoring in double figures for the 13th time in 14 games ... Jordan Adams has scored in double figures in 13 of 17 games played ... UCLA improved its all-time record against Colorado to 6-1 ... the Bruins had not played at Colorado since Dec. 7, 1962 ... Travis Wear scored nine consecutive points for UCLA in the final 10 minutes of the game ... UCLA did not trail Colorado in the second half (game was tied, 42-42, at the 16:09 mark).


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text:
May 29, 2001
By DENNIS WASZAK Jr.
AP Sports Writer
College baseball fans are expected to flock to the super regional series between longtime rivals Louisiana State and Tulane, and the NCAA did its best to accommodate them.
Defending national champion Louisiana State will renew a 108-year-old rivalry against Tulane at Zephyr Field in Metairie, La., in one of the eight super regionals, which begin Friday.
Tulane (53-10), which has the most wins among Division I teams this season, put in bids for Zephyr Field, home of the Houston Astros' Triple-A minor league affiliate, and its home field, Turchin Stadium. The Division I baseball committee chose Zephyr Field because it seats up to 11,000, which is 6,000 more than Turchin Stadium.
"The matchup between these teams may be one of the premier in the super regionals," said Dennis Poppe, the NCAA's senior director for baseball and football. "Tulane obviously earned the right to host, and to accommodate the expected fan interest the committee opted to go for the larger field."
The Tigers (43-20-1) lead the series between the schools, located about 70 miles apart in southern Louisiana, 150-112-3. But the Green Wave won both of this year's meetings.
"The Tulane-LSU rivalry showcases the best in college baseball, and this series will put the eyes of the college baseball world on Louisiana," Tulane athletic director Rick Dickson said.
All eight of the national seeds will play host to the best-of-three super regionals, and the winner of each advances to the College World Series, which starts June 8 in Omaha, Neb. The sites were announced Monday by the committee, which set up brackets at the beginning of the tournament to determine the super regional pairings.
Wally Groff, chairman of the committee, said the national seeds were given highest priority to play host.
The other sites and pairings are: Mississippi State (39-22) at Cal State-Fullerton (44-16), South Carolina (48-18) at Stanford (46-15), Clemson (41-20) at Miami (47-12), Florida State (46-17) at Georgia (45-19), Tennessee (44-18) at East Carolina (47-11), Rice (47-18) at Nebraska (48-14), and Florida International (43-19) at Southern California (51-12).
Each of the remaining teams placed bids to play host to a super regional, and the range of bids ranged from the required minimum of $35,000 to a high of about $180,000.
All of the super regionals will be played on campus fields except for LSU-Tulane and Tennessee-East Carolina, which will be at Grainger Stadium, home of Cleveland's Single-A team in Kinston, N.C.
The Triple-A New Orleans Zephyrs adjusted their home schedule to allow for the super regional to be played at Zephyr Field.
"The Zephyrs have been very accommodating to make sure the super regional happens in an expeditious fashion," Poppe said.
"I'm excited about hosting," Tulane coach Rick Jones said. "The great thing about Zephyr Field is it means so many more people will see the games."
The Tigers are trying for their sixth national championship in the last 11 years under coach Skip Bertman, who is retiring at the end of the season after 18 years. Meanwhile, the Green Wave are looking for their first trip to the College World Series.
"This is unmarked territory," Jones said.


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MEDINA — A 15-year-old Medina girl and the 22-year-old man she is believed to have run away with are in custody in Florida.
"They are safe and unharmed. Their families have been notified," Medina police Chief Patrick Berarducci said.
Jessica Kollar and Maxwell Leibsia were taken into custody Saturday by Palm Beach County Sheriff's Office officers, the chief said.
Both face charges in Florida and Medina County.
An arrest warrant had been issued for Leibsla on the charge of interference with custody, a first-degree misdemeanor that becomes a fifth-degree felony if the child is removed from the state, according to Medina Municipal Court records.
Kollar was last seen by her family on Jan. 24. The girl had contacted friends saying she is OK, but she refused to say where she is.
Berarducci said the couple were tracked by Medina police detectives Amy Kerr and Mary Gross to Akron; Miami, Fla.; and Lake Worth, in Palm Beach County, Fla.


text:
Based in Washington, D.C., the Councils mission is to help redefine and restructure U.S. tax, trade, and environmental policies so that this country can increase its pace of economic growth. (http://www.accf.org/Mission.htm; accessed 10/11/01)
The Councils Center for Policy Research received $80,000 from ExxonMobil. (http://www.exxonmobil.com/contributions/public_info.html; accessed 6/27/01)
Board of Trustees
Maxine C. Champion, President, Champion Strategies; Paul R. Huard, Executive Vice President of Finance and Management, National Association of Manufacturers; Larry W. Pollock, Vice President and Director of Taxes, Weyerhaeuser. (http://www.accf.org/Mission.htm; accessed 6/29/01)


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Jan 6, 2011 8:25 AM
It doesn't matter if you have migraines, sinus headaches, tension headaches, spend too much time at the computer, or just need a little "down time" each day, The Temple and Eye Massager ($150) is like a trip to the spa. Strap it on and you'll feel a combination of vibration massage and air-bag massage (during which the device expands and contracts against your eye sockets). There's also heat compression to stimulate blood flow and soothe sore muscles around the eyes. The timer can be set for 5, 10, or 15 minutes, during which you'll hear the soothing sounds of nature as they play through and integrated speaker system. Ahhh...
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2003-04: :MacDonald had a strong junior season, appearing in 29 games, tallying eight points (1 g, 7 a)... Also committed four penalties for eight minutes... His lone goal was a big one, a power-play game-winning tally in the second period of UMass' first-ever win over Boston University at Walter Brown Arena (Jan. 7). Also tallied an assist in the same game against the Terriers... Dished out two assists in the 5-5 tie at Vermont (Dec. 28)... Distributed assists in back-to-back games against Providence (Nov. 15) and Maine (Nov. 21)... Had helpers against New Hampshire twice (Dec. 11 and Feb. 6)... Had 25 shots on goal for the Minutemen.
2002-03: Appeared in 15 games for the Minutemen, accumulating three points (1 g, 2 a)... Scored lone goal of the season in UMass' 8-6 victory over UConn on Feb. 14... Dished out assists against Providence (Nov. 1) and Maine (Dec. 29)... Recorded 14 shots on goal and had three penalties for six minutes.
2001-02: Played in 27 of the team's 34 games... Finished the season tied for fifth on the team in scoring with six goals and six assists for 12 points... Was third on the team in goals (six) and tied for third on the team in power play goals (two)... Finished second on the team with a .182 shooting percentage... Had five penalties for 10 minutes... Netted two power play goals, including the game-winner, in the 4-0 victory over Dartmouth (Nov. 24)... Had a pair of assists in the 3-3 tie with Merrimack (Jan. 11)... Scored a goal and an assist in the 4-2 victory over Northeastern (Nov. 3)... Also had a goal and an assist against Boston College (Jan. 7)... Recorded his first collegiate point with a goal in the 4-3 victory over Providence (Oct. 28)... Netted a goal against Merrimack (Jan. 12)... Notched a single assist in the victories over Niagara (Dec. 28) and Northeastern (Jan. 26).
BEFORE UMASS: Played two seasons for Tom Resor and Tom Daccord at Noble & Greenough... Team captain senior year... Earned All-ISL honors in 2001... Played in the All-New England All-Star Game... Had 26 goals and 26 assists for 52 points in 2001... Finished Noble & Greenough and Catholic Memorial career with a combined 119 points on 52 goals and 67 assists... Played three seasons for Bill Hanson at Catholic Memorial... Member of the national champion and Massachusetts state champion Catholic Memorial team in 1997-1998 with a 24-0 record... Also state champions in 1998-1999... Earned Unsung Hero Award at Catholic Memorial in 1999. THE CRAIG MacDONALD FILE: Craig William MacDonald... Son of Robert and Barbara MacDonald... Born May 7, 1982, in Norwood, Mass.... Majoring in economics.... Was ranked 130th in the final NHL Central Scouting Service rankings for North American skaters prior to the 2001 NHL Entry Draft.
MacDONALd'S CAREER STATS... Year GP G A Pts. Pen-PIM PPG GWG SHG HT +/- 2001-02 27 6 6 12 5-10 2 1 0 0 -13 2002-03 14 1 2 3 3-6 0 0 0 0 -5 2003-04 29 1 7 8 4-8 1 1 0 0 -6 CAREER 70 8 15 23 12-24 3 2 0 0 -24
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Sheriff's son in gun theft case jailedStaff Report | February 05,2013The son of a member of the Rutland County Sheriff's Department who was charged last month with stealing his father's gun is behind bars after allegedly returning home Friday night with a handful of heroin.
Jaron Wilk, 24, of Clarendon, pleaded innocent Monday in Rutland criminal court to a felony charge of possession and misdemeanor charge of violating the conditions of his release.
Wilk was on release after pleading innocent Jan. 22 in Rutland criminal court to charges of burglary and grand larceny after allegedly robbing his parents' Clarendon home, stealing cash, checks and a .40-caliber Glock semiautomatic firearm belonging to his father, Frank Wilk Jr., a lieutenant with the Rutland County Sheriff's Department.
Jaron Wilk's conditions of release required him to maintain an evening curfew at his parents' home and to not be arrested again. But, according to an affidavit filed with the court, Friday night, police received an anonymous tip that Wilk was not at home, and when police responded to the residence on East Clarendon Road they followed Wilk as he turned into his driveway.
While police questioned Wilk, they watched him throw a small object, which police recovered from a nearby tree and found to be five bags of heroin wrapped in an elastic band, records state.
Judge Nancy Corsones rejected a request from Wilk's attorney for a 24-hour curfew, and instead imposed $15,000 bail.MORE IN This Just InMichael Chadwick adores karate, but the 27-year-old with cerebral palsy needs a lift to get into... Full StoryBENNINGTON The Vermont Department of Health has slightly altered its health advisory for PFOA... Full StoryMOUNT TABOR Vermont State Police said a two-vehicle crash on Route 7 sent a juvenile female to... Full Story
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2007 Inductees: Dan DeKaye, Al Faechner, Jodi Kruta, Anthony Taylor
Since first awarded in 1994, the Northern Alumni Association All-Sports Hall of Fame has inducted 53 individuals who have contributed to the tradition of excellence both in the classroom and on the playing field at Montana State University—Northern; a tradition that goes back to the very beginning of the school and has continued throughout its 78-year history. MSU-Northern and the Northern Alumni Association will again recognize four outstanding individuals during the Alumni Athletic Hall of Fame inductions Homecoming Week October 5-6, 2007. Dan DeKaye of Argyle, Texas, Al Faechner of Great Falls, Montana, Jodi Kruta of Douglasville, Georgia, and Anthony L. Taylor of Laurel, Maryland will be presented commemorative awards in ceremonies on the Northern campus Friday, October 5th during the Homecoming Alumni Games.
Dan DeKaye
Dan DeKaye, a Cut Bank, Montana native, competed from 1992-1996, wrestling at the 177 pound weight class for Northern. After redshirting his first year, he competed on the varsity team, placing 5th or higher three times at the NAIA National Tournament. DeKaye was ranked second nationally in 1995 and held the #1 ranking for ten weeks in 1996, finishing 2nd at the national tournament. A 3-time Northern Regional Champion, he was the team captain his junior and senior year. DeKaye won 6 of 8 tournaments his junior season and 6 of 7 his senior year, where he was voted tournament Outstanding Wrestler twice. His over-all college record was 97 wins and 23 losses. His junior and senior years he had an impressive 63-8 record with a total of 19 falls 88% win ratio. DeKaye graduated in 1996 with a degree in civil engineering technology with an associate's degree in drafting. He is currently employed with Kiewit Corporation in management in their Dallas/Fort Worth, Texas Branch and has participated in 17 different projects including construction of a dam, bridges, airport runways and highway construction throughout Montana, Texas, Colorado, New Mexico, Oklahoma and Louisiana.
Al Faechner
Receiving the Veteran Award this year is Al Faechner, of Great Falls, Montana. He attended MSU-Northern (then Northern Montana College) on a basketball scholarship for two years from 1951-53 graduating in the Pre-Business program. During his freshman year, Faechner scored 350 points in 24 games ranking second on the team to teammate Gene Espeland and led the team his second year. He set a record for scoring at the Junior College Regional Tournament at Dixie Junior College in Provo, Utah netting 37 points and a spot on the All-Tournament team in 1952. At that time, there were no 3 point shots and the quarters were only 8 minutes long. NMC was also the only Junior College in the league, so they competed against more seasoned juniors and seniors. Faechner also played baseball both years for Northern. After leaving Northern, he served two years in the Korean War. Returning to Montana, he married his college fiancée, Joyce Ferrin, and continued his education at the College of Great Falls. After graduation, he worked in banking in Glasgow, Montana and, as President of the Glasgow Sports Club, he was instrumental in arranging a football game between Minot State and the NMC Lights in Glasgow which drew over 2,000 fans in 1969. He was President of Multisports which built the softball complex in Great Falls, in 1965
Jodi Kruta
Jodi Kruta was a recruit from Thief River Falls, Minnesota and played for the MSU-Northern Skylights basketball team from 1992 to 1996. As a three year starter, she played a key role in the four Frontier Conference Championships, four National Tournament appearances, and the 1993 District 12 Tournament and the 1993 National Championship for the Skylights. This is still the only National Championship to date of any Frontier Conference Women's Basketball team. During this time, the teams won 100 games with only 18 losses. Kruta is the Skylights second all-time leading scorer with 1562 points and is one of only 9 Skylights to be an NAIA All-American. Her senior year she was an All American Honorable Mention, First Team All-Conference, and First Team Pacific Northwest Region. She was named the Frontier Conference Player of the Week four times during her career. The Skylights teams she played on also won the Bahamas Tournament in 1993, The Colorado College Holiday Tournament in 1994, and the Pacific Northwest Regional Championship in 1996. Kruta graduated in 1997 with a degree in Health and Physical Education. She lived in Norway to play team handball and became a member of the 1999 USA Team Handball National Team. She participated in the 1999 Pan American Games placing 4th in an attempt to qualify for the 2000 Olympic Games. She is currently teaching 6th-8th grade Physical Education and Health in Lovinggood Middle School in Georgia.
Anthony Taylor
Anthony L. Taylor wrestled in high school in Ohio and finished with 80 wins and 15 losses. He attended Lassen Community College in California in 1990 and after injuring his right ankle, he received a medical hardship his first year in which he had already amassed a 16-2 record. In 1992, he claimed the California Junior College State Championship with a record of 30-3 and transferred to Northern in 1993 where he placed second in the NAIA National Tournament as a sophomore and as a junior, losing first place by only one point. Both years, he was 24-4 at the 158 pound weight classification. In 1994, his senior year, he received a season ending knee injury and was ranked #1 in the NAIA National Ratings at the time. He was a two-time All-American and a National runner-up at 158 pounds. His record at Northern was 60 wins and 9 losses and his over-all collegiate career record was 106-14. Taylor was instrumental in the 1992 National Team Championship and the 1993 National Team Runner-up on teams that won two NAIA District 12 and Frontier Conference Championships along the way. Anthony Taylor was also a NWCA Academic All-American. He was an assistant coach at Northern for three seasons, during the remainder of his senior year and for the following two years, 1995 and 1996, while working on his Masters degree. Taylor is now working in Washington D.C. as a Federal Probation/Parole Officer with new responsibilities for training his colleagues.


text:
|MMORPG Games: The Realms Public discussion forum for all MMORPG games.
|Thread Tools
|22nd December 2012, 11:36 AM
|#1 (permalink)
Age of Wushu - Sandbox martial arts mmo
( Wǔshù literally means "martial art". It is formed from the two words 武術: 武 (wǔ), meaning "martial" or "military" and 術 (shù), which translates into "discipline", "skill" or "method." )
When I saw news about Age of Wushu a while ago (like a year ago?) I shrugged it off as yet another asian grinder with flashy combat but little substance. The other day, however, I read this article about it on Massively:
First impressions from the Age of Wushu beta | Massively
That piqued my interest. Thus I went to youtube for enlightenment (aka looking for gameplay videos). I must say this game looks to be much much much more than I originally thought.
First, here's the link to the official site where you can check out details or even try the game (limited trial) or actually buy the game even though it's technically still in beta with a release date of March 3, 2013.
Age of Wushu Closed Beta : Free To Play MMORPG
Here's some videos:
This is a pretty long "first impressions" video that tells quite a bit about the game.
Here are some videos showcasing combat moves learned in the various schools. They start out showing some of the normal attacks then blocks to build up rage to do the big attack.
I want the school that has a "Get Off My Lawn" attack .
Righteous Schools
The Emei school is the female only sisterhood akin to the Shaolin
Wudang school
Shaolin school ( male only school )
Beggars school (you actually have to have the beggar profession to join this school)
Neutral Schools
Scholar school (you have to have a scholarly profession to join)
Tangmen school
Evil Schools
Royal Guards
Wanderer's Valley
That's all so far!
Last edited by Ordegar; 23rd December 2012 at 12:45 PM. Reason: I had the wrong first impressions video embedded!
|23rd December 2012, 08:20 AM
|#2 (permalink)
Things you should know about Age of Wushu
Age of Wushu is the US version of Age of Wulin (being released in Asia by Snail Game CN, and by gPotato in EU). Age of Wushu is an international version meaning it has no region limits. It is published by Snail Game US.
Age of Wushu is in BETA. Currently it is Closed Beta 2; which is the first beta that will not have character wipes, so if you play now you can keep that character after the game officially releases.
It will be a "free to play" game. You can download the game for free now, and there will not be a cost to play. There will be a cash shop with accelerator type items and cosmetic items. Nothing in the cash shop will make you superior to what you can be without it; it just might help you get there faster.
There is a VIP status, which requires a subscription of about $10 per month. Buying the Deluxe package will give you 60 days of VIP after which you will need to subscribe to continue the VIP status. VIP status gives you benefits such as faster xp to skill rate; offline skill progression; offline merchant capability.
You can only have one character per account. Your character can only join one School, but it is possible to learn skills from other Schools. You can only cultivate (level) one skill at a time; there is no queue so you need to go online to change which skill you wish to cultivate or to continue a skill that has reached a new level. VIP status lets you cultivate skills while offline.
Even if you are not VIP your character will continue in the world as an NPC doing jobs. The job he or she does depends on where your character is when you log off. He may work in a shop sweeping floors, or work for a farmer, etc.
Guilds can claim territory which crafters can then build on and make a guild village. There are limited spots, so there will be wars over territory.
Know what you want your character to be BEFORE YOU BUY.
If you try the game and want to buy the Deluxe package to be able to play the beta unlimited, there's some things that you should know first.
This is from the official forums warning about deleting your character:
Age of Wushu • View topic - Deleting Characters - Elite & Deluxe Edition Rewards
You can delete and remake your character as many times as you want as long as you do it before you upgrade the account by buying the Deluxe pack. You cannot claim the in-game rewards again.
So what I plan on doing is researching the game enough to decide how I want my character. I tend to be one of those players who wants to do everything so I end up with a bunch of alts; but since this game limits us to one character per account, I will only have one character.
The main thing to decide on is the School, as they are all very different -- it's almost like choosing a class. Each class has 3 different skill sets, which you can see by the three videos of each School that I posted above. A good place to look about the Schools is on the wiki: Schools - Age of Wushu Wiki -- click on the picture representing the school to go to the details page for that school; some of them have explanations about each of the three skill sets to give you an idea of what the strengths and weaknesses are for that school.
Last edited by Ordegar; 23rd December 2012 at 12:27 PM.
|23rd December 2012, 09:01 AM
|#3 (permalink)
Some vocabulary to help with understanding story and the game:
Last edited by Ordegar; 23rd December 2012 at 11:05 AM.
|23rd December 2012, 09:31 AM
|#4 (permalink)
Last edited by Ordegar; 23rd December 2012 at 11:06 AM.
|24th December 2012, 06:46 AM
|#6 (permalink)
is this in it:
Welcome to the Elder Scrolls Online Division
|24th December 2012, 09:25 AM
|#8 (permalink)
Keep in mind this is not a wii game, so YOU don't actually have to do the moves, your character does them with a simple button push.
On a serious note, this IS an open pvp game, but it's not a fps shooter by any means. Also, it's a sandbox world, not a pvp arena game, so there's a lot more to it than just fighting other players. You could play as a crafter and only make stuff for guild-mates if that's what you wanted to do.
I'm not really a pvp oriented player myself, though I did play Warhammer Online exclusively on the pvp side; and I do enjoy some Planetside 2 with TOG occasionally, but I'm happy with my 1/3 k/d ratio PlanetSide 2 Character Stats Ordegar - PlanetSide Universe
In a sandbox mmo, with rules and consequences to pvp, it's more about living in the world and developing your character in many aspects. In Age of Wushu, they even took character development beyond combat and crafting and went into your characters social development. You can get achievements for making friends, for example, or enemies. Guilds are more than just a chat channel; there is territory to take and hold; things to build.
It is a sandbox though, not a themepark. You do get an introductory set of quests at the beginning that chain together, but once that's done you're on your own. There are quests and events, but they don't chain together like in a themepark mmo. Some people don't like having to decide on their own where and what to do, so a game like this is not for them.
Sorry, I'm raving I know; I'm just excited about an mmo that is not just the same old thing.
|10th January 2013, 08:36 PM
|#9 (permalink)
I bought the Deluxe package to get extra time to beta test the game. I created Elondra and ran through the game about 25% to get a feeling of what to concentrate on for bug reports.
I deleted Elondra and created Olondra so I can repeat the same class line. Then I found out that the Deluxe & Elite packages are tied to the CHARACTER and not the account. So by deleting Elondra, I lost the privilage to test long hours. I am currently stuck at only 1 hour a day to test the game but I could try again if I spend more $$$ to pay-to-test.
And Snail Games USA said they are looking for great employees in the Los Angeles site in an email reply to my concern.
So I may pass on testing the game now. I've recently received an Alpha invite to an upcoming MMO and testing there has taken up much of my time.
|8th April 2013, 10:08 PM
|#10 (permalink)
Bought the deluxe edition for this game, and I am really liking it a lot.
Do we have any plans for a guild or something?
By the way, it is worth mentioning that after the beta is done, the 1 hour per day time restriction on free players will be lifted. (According to a GM post: Age of Wushu • View topic - Free Player Access Time During Closed Beta))
Last edited by Elyzion; 9th April 2013 at 11:49 AM.
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text:
Scrabble word: THEOLOGY
In which Scrabble dictionary does THEOLOGY exist?
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(66 pts)
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Hanging with friends
(50 pts)
(8 pts)
Lexulous (US)
(68 pts)
Definitions of THEOLOGY in dictionaries:
- noun - the rational and systematic study of religion and its influences and of the nature of religious truth
- noun - a particular system or school of religious beliefs and teachings
- noun - the learned profession acquired by specialized courses in religion (usually taught at a college or seminary)
- The study of the nature of God and religious truth; rational inquiry into religious questions.
- A system or school of opinions concerning God and religious questions.
- A course of specialized religious study usually at a college or seminary.
- noun - the study of religion
There are 8 letters in THEOLOGY: E G H L O O T Y
Scrabble words that can be created with an extra letter added to THEOLOGY
All anagrams that could be made from letters of word THEOLOGY plus a wildcard: THEOLOGY?
Scrabble words that can be created with letters from word THEOLOGY
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text:
Bet's Fish Fry
Boothbay, ME
Phone: 207-208-7477
Bet Finocchiaro prepares 1,500 pounds of haddock and 800 pounds of potatoes weekly at her roadside takeout. She no longer catches the fish herself, but she still uses her grandmother's breading recipe and whips up her own special tartar sauce. Be forewarned: Portions are humongous.
Please Note: This information was accurate at the time of publication. When planning a trip, please confirm details by directly contacting any company or establishment you intend to visit.
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text:
a parent in a foreign country decided that his son had spent enough time playing on Warcraft. So instead
of being an actual parent and limiting his sons activities. The parent hired a cyber hit man to grief the sons characters all over Azeroth til they became frustrated and quit.
Admitting to myself that this was both creative and wrong. It spiked the gold goblin in me to the idea that this idea wasn't the best to earn gold with. Blizzard has been haphazard with their enforcement of no griefing policies when it comes to stalking players. Many stories have been passed around about bans for 'griefing' other players by following them and killing them on PVP servers.
When my server was much stronger on world PVP there would be epic battles as groups would raid cities and people would run to the rescue. Darnassus tended to be the most popular of places to plant players in and cause a ruckus along the server world. Many cyber enemies were created with this PVP battles and many players became hated enemies along the times.
But I can say that it never crossed my mind to hire people to hunt someone til they became frustrated with the game. This would be crossing an unwritten etiquette rule of sorts that were created between players. Now I'm sure that people will read this news entry (found here) but I would hope that players would decide it isn't worth the possible loss of account access and general scorn from the server population.
Everyone wants to make real money from their gaming addiction. Diablo presented this possibility with the Real Money Auction House. And there is a black market for players to sell their characters once they are finished with them. But to me getting paid to hunt and stalk a player til they quit a game just does not feel like the gold goblin way.
Good Luck and Good Hunting
Want to get in contact with me? . Email me at Dragonbearjoe @ gmail. dOT com or find me on twitter at "@JMTCMommar" You can find me hanging out on the Shandris Server, chat me up and I will buy you a glass of juice.
Previous Entries
Time Management With Faction Grinding
Receipt Isolation Sales
Breaking Through The Wall
Transmog Me First
Pay What
Outside the New Years Box
Heat Of The Moment
Five Star Sales
The Gauntlet Thrown Down
Post Holiday Toys
Rock Star Pricing
Follow Your Strength
The Go To Gold Goblin???
Keep Your Head Up Gold Goblin
Plant Pvp Enigma Seed vs Songbell Seed
The Short List
Decking Out Your Farmer
Winter Veil Farming: Head Start
A Perfect Circle of Goblin Life
Addon Abandonment Issues
The Guild: Striking Tone For Sales
Controlling Your Reputation
Posted by Joseph Douglas at 12:10 PM
Labels: A Perfect Circle, Good Luck, Guild, Head Start Program, New Year, Shandris Server, The Go, warcraft
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text: Apr 11, 2013 17:14 Rooftop party aids Slidell hospital Rooftop party aids Slidell hospital Advocate staff report Comments SLIDELL — St. Tammany Homestead and Slidell Memorial Hospital Foundation will host Rooftop Rendezvous 2013 from 8 p.m. to midnight Saturday on the rooftop of the SMH parking garage, 1001 Gause Blvd. Dancing under the stars to the beats of MoJEAUX, food and fun are expected to attract approximately 500 people to the annual party. St. Tammany Fire Chief Larry Hess is celebrity auctioneer, auctioning off collectible painted plates, a charter fishing trip, Saints tickets and a theater ticket and dinner package. For the sixth year, Slidell Mayor Freddy Drennan and his chefs will serve 300 pounds of brisket. Many local restaurants are contributing food as well. Reservations can be made by calling (985) 280-8820. Tickets are $100 per person and available by calling or online at http://www.smhfdn.org. Funds raised will continue to support the new SMH Regional Cancer Center, the new SMH Women's Imaging Center, scholarships for children at Fit as a Firefighter summer camp, SMH Parenting Center, and health screening and education programs. Get The Advocate in your inbox Subscribe


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The ice queen in us can't resist the Ultima Thule Wine Glasses ($43 for 2). Painstakingly modeled after glaciers of Finland, designer Tapio Wirkkala even invented a new glassblowing technique to pull these off. These wine goblets are part of the clasic 1960s line from iitala, so we're thinkin' about what crystal encrusted shift mini dress we should pair 'em with.
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Santa's Apprentice
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Sacha Molitorisz AMID the avalanche of imported animation, it's heartening to see a film where the opening sequences feature the Sydney Opera House and most of the accents are unashamedly Aussie.
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Petite Women's Komarov Mixed Media A-Line Dress, Size
A floral print in a moody palette captivates day or night in a scoop-neck dress cut from lustrous charmeuse offset by matte chiffon and lace. The flattering A-line style is hand pleated for a more forgiving, stretch-like fit. 48" regular length (size Medium). Slips on over head. V-neck. Sheer three-quarter ... Learn More


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ST LOUIS (LALATE) – Poland vs Romania 2013 (live score below) battle in the only International Friendly soccer match Saturday. Both teams have impressed news with at least three wins in their last separate five games. But what will happen with two face off Saturday?
Saturday's Poland vs Romania 2013 match (start time 11:45 am PST) follows an impressive run Romania since last year. But Romania has not played since November, news analysts note. Romania's last game was an international friendly match that delivered a win against Belgium. Previously, the team battled in four World Cup qualifiers, and only lost one of them.
In September, Romania kicked off a three game winning streak in which the team gave up no goals. Romania first defeated Estonia, then Andorra and finally Turkey. The team's only recent loss has been to the Netherlands last October, officials tell news.
Poland, in contrast, last battled in December in a friendly against Macedonia, which it won. Previously the team was defeated by Uruguay in another friendly back in November. Earlier in the year, the team delivered two wins, against Moldova and South Africa, and one draw against England. For live score results Saturday, click HERE.
After Saturday's match, Poland will battle Ireland in a friendly next Wednesday and then take a break until March. Romania, in turn, will battle also next Wednesday, but against Australia.


text:
The Pest: A Healing Based Glance Tank Build for PVP
by Read
Blades / Chaos
Active Abilities:
- Escalation
- Karma
- Mockingbird
- Martial Discipline
- Sleight Of Hand
- Clearing the Path
- Art of War
Passive Abilities
- Stave Off
- Tipped Scales
- Riposte (Can swap to Circulation or another healing passive if you want more healing and less damage)
- Enervate
- Regeneration
- Resilient Defender
- 6th Sense (Can swap to Forged in Fire for 1v1 situations where 6th Sense won't proc often enough
As this is a build for PVP, there is no rotation to follow so to speak, the key is anticipating and reacting to your enemies. The most important thing to keep in mind when playing this build is that you cannot block, evade, or glance an attack while impaired, so using Sleight of Hand wisely will go a long way into keeping yourself alive.
Gearing Needs:
For this build, you'll want an even split between attack rating and heal rating. I use one major and one minor talisman of each, and the rest of my talismans are HP. You'll want to glyph to around 900 defense, then split the rest of your stat budget between evade and block. Signets of Salvation and Ablation also help the effectiveness of this build.
What makes this build Tick?
This is a glance tank build, meaning that most of its effectiveness comes from when enemy attacks glance you. I designed the build to provide both damage and survivability, so I'll talk about those aspects separately.
Riposte will damage enemies when their attacks glance you
Enervate will afflict enemies when their attacks glance you
Clearing the Path automatically penetrates when you hit an afflicted target
Those 3 skills together will provide you with good, consistent damage to multiple enemies. While you won't kill enemy healers this way, you will overwhelm them and they won't be able to keep everyone alive.
Mockingbird causes any attack that hits you to also heal you.
Resilient defender causes any attack you evade, block, or any attack that glances you to also heal you.
6th Sense will heal you for 10% of your HP after every 6 attacks that glance you.
Tipped Scales applies a Heal Over Time(HOT) whenever you hit an impaired enemy. Combined with Stave Off, this HOT will have close to 100% uptime when multiple enemies are attacking you.
When you hit an enemy with Karma, it will cause that enemy's next attack to heal you for 2% of your HP(at 5 resources). If you hit 5 enemies with it, that's 10% of your HP
Regeneration gives you a massive HoT when you use Martial Discipline.
All of the above combined will provide you with anywhere from 300 to 1500+ healing per second, depending on how many enemies are attacking you.
I posted a video of this build in action so you can kind of see how everything works together.


text:
>Brookings Oregon News, Sports, & Weather | The Curry Coastal Pilot
Tickets are now on sale for the Brookings-Harbor Community Theater youth production of "Pinocchio."
The play opens at 7:30 p.m. Friday, Jan. 13, at the Harbor Performing Arts Center. The show will run Friday, Jan. 20 as well, with Saturday matinees at 2 p.m. Jan. 14 and 21.
Author Kathryn Schultz Miller opens the story with Geppetto, a lonely wood-carver surrounded by toys and wooden marionettes, who makes a wish to his Lucky Star, for a real son of his own. Soon, a wooden toy comes to life, his own son, Pinocchio! With Hickory Cricket by his side and magical help from the Blue Fairy, Pinocchio begins a grand adventure filled with tricksters and trials along the way. Pinocchio meets Fibber the Fox, Lefty the Cat, Lampwick, and no-good buddies in the Land of Toys. Will Pinocchio save Geppetto from the belly of the whale? Will he ever prove that he is good enough to become a Real Boy?
The show is full of fun, audience participation and an old moral dilemma for the audience to help resolve, said co-directors Dori Blodgett. Of course the tale wouldn't be complete without the not-so-true tales from Pinocchio; just how big will his nose grow?
Directors Blodgett and Katherine McPherson bring this fairy tale to life on stage for a new generation of children to enjoy.
The cast includes Maya Primich, Megan Pearson, Karman Mann, Felix Fernandez, Nate Campbell, John Graves, Ally Graves and Teryn Van Maren as the toys and the fish are played by Eden Van Maren, Lauren Pieper and Amber Van Maren.
The puppets and puppeteers include Everett Van Maren, Emily Mann, Ally Moffit, Miki Dingle, Destiny LaVelle, Sarah Scott, Julia Scott, Layla Gagnon, Daisha Nidiffer, and Nikki Campbell.
Pinocchio is played by Daniel Pearson. Pinocchio's leading characters include: Daisy Moffit as Lorenza the Magnifica, Tyler Freeman as Geppetto, Silas Pieper as Hickory Cricket, Lilly Stockton as Ivana, Claire Penree as Lucky Star, Shawntisha Bailey as Blue Fairy, Brittney Lawson as Fibber the Fox, Megan Hulen as Lefty the Cat, Micah Teague as Lampwick, Ashton Chapman as Coachman, Camden Geotze as Jack and Ella Campbell as Jill.
Crew members are Alia Graves on lights, Jacque Graves on sound and Tiffany Van Maren as stage manager, assisted by Suzanne Graves.
Tickets are $8 for adults and $5 for youth younger than 18 and may be purchased in advance at Wright's Custom Frame Shop in Brookings and New Wave Video in Harbor. Harbor Performing Arts Center is located in the Brookings Harbor Shopping Center, 97900 Shopping Center Avenue in Harbor. Reservations may be made at 541-469-4700 or 541-661-2473.


text:
"The only way that we are able to make a living is by convincing people, sometimes one at a time, that this thing that we have won't exist without us, and that it is necessary."
The short film Why We Create Music examines the absolute importance of music to the individuals who create it. Fifteen musicians, ranging from Bill Withers to Aloe Blacc to Lady Antebellum, share their thoughts on songwriting and composing. Continue reading


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Food, wine and Napa Valley photos
Call us a bit biased, but we think the photos that regularly appear on this here blog are pretty darn good.
So good, in fact, that we feel the need to point you to our resident photographer's personal showcase. Go check out her website to see a few samples of stunning food, wine and various Napa Valley landscapes.
If you like what you see, hit the contact page to get in touch.
[techtags: Napa, Napa Valley, napa valley photographer, napa photographer, napa photography, napa valley photography]