text:
|Publication number
|Publication type
|Application number
|Publication date
|Jul 26, 1988
|Filing date
|Nov 17, 1986
|Priority date
|Mar 6, 1981
|Fee status
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|Publication number
|Edwin S. Lennox, Steven H. Sacks
|Original Assignee
|Celltech Limited
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|Patent Citations (3), Non-Patent Citations (37), Referenced by (25), Classifications (32), Legal Events (9)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of application Ser. No. 438,896, filed Nov. 1, 1982, as PCT GB82/00076 on Mar. 8, 1982, published as WO82/03089 on Sept. 16, 1982 now abandoned.
This invention relates to the field of biotechnology and concerns particularly the use of a monoclonal antibody as a blood grouping reagent.
The introduction of foreign material (antigenic material) into the body of a vertebrate animal provokes an immune reaction, the intent of which is to prevent the antigenic material causing damage to the body and to facilitate the removal of such material from the body. The immune system achieves this by producing immunoglobulin molecules (hereafter referred to as antibodies) which have the property of selectively recognising and binding to characteristic sites on the antigenic material. These sites are known as determinants and an antigen may possess one or more such determinants. Antibodies generated by the immune system each have specificity to only one determinant but a number of different antibodies may be produced if the antigenic material against which antibodies are raised possesses more than one determinant.
The primary function of antibodies is to protect the body from harmful foreign material, by agglutinating it, thereby assisting the normal body processes to remove the material.
The agglutination of antigenic material by antibodies does however have a practical use outside the body in the field of blood grouping.
Red blood cells (erythrocytes) have on their surface a number of different, and distinctive antigenic determinants, the character of which determinants allows the classification of blood into groups, or types (for example, A, B, O, A1 B, A2 B, Bcord). It is essential in the transfusion of blood from a donor to a recipient that the transfused blood be of the same group as that of the recipient's blood, for if it is not, the immune system of the recipient will generate antibodies against the unfamiliar determinants upon the surface of the transfused erythrocytes. The reaction of such antibodies with foreign erythrocytes forms the basis of the technique of blood grouping.
In broad terms the grouping of blood samples relies upon the detection of agglutination or otherwise of erythrocytes in the sample when antiserum to the determinants found on the surface of that group of erythrocytes is added. The agglutination is a macroscopic effect which can be readily discerned by eye or detected automatically by machine.
The major source of blood grouping reagents has hitherto been through the hyperimmunisation of human subjects. This involves the introduction into a human subject of a substantial, but non-lethal, dose of a blood serum of a type different from that of a human subject. This provokes the normal immunological response resulting in the production of antibodies in the blood of the subject, a sample of which blood may be subsequently removed and an antibody preparation made therefrom. Such a preparation may be used to group an unknown blood sample since it will cause a visible flocculation or agglutination of the erythrocytes if the unknown blood sample is of the same type as originally introduced into the human serum donor.
In practice there are two types of blood grouping test. (Dunsford, F.; Bowley, C.: Techniques in blood grouping: 2nd Ed (1967). In the first, a sample of blood to be grouped is placed upon a blood grouping tile (see: British Pharmacopeoia: Determination of ABO donors). This is then mixed upon the tile with a sample of antiserum. Any subsequent agglutination indicates that the unknown sample of blood being grouped belongs to the same blood group as the group against which the antiserum was raised. In practice such tile agglutination grouping tests are routinely carried out in emergencies.
In the second type of experiment the blood sample to be grouped is placed together with an antiserum in saline solution in a tube and allowed to stand for a standard period (2 hours). The presence of agglutination may then be estimated by the sedimentation that has occurred within the standard period. Under emergency conditions a centrifuge may be used to expedite the test.
The efficacy of a particular blood grouping reagent is judged by the speed with which it forms agglutinants and by the manner in which its ability as an agglutinin varies with concentration.
The former of these criteria is commonly referred to in the art as "avidity". The avidity time of a particular blood grouping reagent is defined as the time taken for the mixing of the blood sample with the reagent to the time at which a noticeable agglutination of the sample has occurred.
In order to determine the dilution characteristics of a blood grouping reagent a saline agglutination titre may be measured. This measurement comprises preparing a number of equal volume, 2 fold, series, saline dilutions of the blood grouping reagent to be tested. To each dilution sample is added a known amount of the appropriate erythocyte suspension. The same amount of suspension is added to all dilutions. Each dilution sample is left to stand for a standard period (usually 2 hours) at the end of which an agglutination count is made under a microscope. The sample of highest dilution at which substantial agglutination occurs is determined and that dilution is termed the "saline agglutination titre". A reagent having a high saline agglutination titre is therefore a potent agglutinin.
Two problems are evident with the production of antibodies to erythrocyte determinants using the technique of hyperimmunisation of a human subject. Firstly, donations of blood serum are in limited supply and nowadays with the increasing frequency of major surgery the need for blood grouping has increased markedly. This places a strain on the supply of human blood serum which is also in damand for other medical uses. In addition, the agglutination effect of naturally produced antibodies to erythrocytes tends to give somewhat variable results. One reason for this effect is that the immune response prepares a `cocktail` of antibodies each component of which cocktail has a specific action on a determinant as discussed above. It is impossible to separate the various antibodies in this cocktail and so conventional antisera contain mixtures of antibodies and the mixtures vary from animal to animal (even within the same genus and from day to day). The same set of determinants is not present on all erythrocytes of the same group, which results in the response being in many cases very variable.
In summary a reagent suitable for blood grouping must be:
(1) specific for the appropriate antigen,
(2) sufficiently potent to give good macroscopic reactions to the weaker blood groups by emergency as well as routine methods of blood grouping,
(3) stable under the conditions of use, and
(4) readily available at reasonable cost.
Recent advances in molecular biology have provided a technique for the production of highly specific antibodies by the production of a hybrid cell (or hybridoma) from an antibody-producing spleen cell and a myeloma cell. This technique, the work of Kohler and Milstein (Eur. J. Immunol. 6, 292-295 (1976); Nature 256, 495-497 (1975); Eur. J. Immunol. 6, 511-519 (1976) provides a method for producing a limitless source of antibody. The antibodies produced are termed monoclonal antibodies since the hybrid cell elaborating them produces only one type of immunoglobulin molecule i.e. only immunoglobulin specific to one determinant. Hybridoma cells combine the two desirable features of myeloma cells and lymphocytes. That is myeloma cells have an immortal character and can self-replicate in vitro, whilst lymphocytes have the desirable property of expressing antibodies. Such hybrid cells are therefore a permanent source of pure, defined immunoglobulin. The process used for producing hybrid cells commonly comprises the steps of immunising a mouse with the appropriate antigen and after allowing sufficient time for the immune reaction to take place, sacrificing the animal and removing its spleen. A cell suspension may then be prepared from the spleen, and this suspension is mixed with a suspension of mice myeloma cells. Polyethylene glycol may be used to promote fusion of the two cells. The resulting individual hybridoma cultures, having derived from one lymphocyte cell, specifically produce one type of antibody, that is an antibody specific to one particular determinant.
This technique has been used by Voak et al (Vox Sanguinis 39 134-140 (1980)) to produce a monoclonal antibody to the determinants of A-type erythrocytes and such monoclonal anti-A's have been shown to be useful blood grouping reagents.
However, it has widely been thought that the immunisation of mice wth B-type human blood cells will not produce spleen cells capable of fusion with myeloma cells to form hybridoma cells elaborating anti-B immunoglobulin.
We have surprisingly found that this is not the case and that successful fusions may be readily obtained, resulting in hybridoma cells expressing monoclonal anti-B with high efficiency thereby producing a high avidity specific blood grouping reagent with a useful saline agglutination titre profile. Furthermore, it has proven possible to define an equilibrium constant and a dissociation rate constant for the immunocomplex formed between the monoclonal anti-B and B type blood cells. Hitherto such quantitative analysis of the strength of the immunocomplex formed between a blood grouping reagent and red blood cells has not been possible since previously known blood grouping reagents have comprised mixtures of immunoglobulin of differing specificity. The best figure that could be achieved formerly was therefore an average value.
According to the present invention we provide a monoclonal antibody which has specificity for an antigenic determinant of a B-type blood cell.
In this context the term B-type blood cell is to be taken to include all red blood cells possessing one or more antigenic determinants found solely upon B-type blood cells. Examples of blood types which have such determinants are B, A1 B, A2 B and Bcord.
The monoclonal antibody is preferably a mouse, IgM, monoclonal anti-B immunoglobulin.
Preferably the monoclonal antibody is capable of causing agglutination of a sample of B-type blood having the said determinant. We have found that particularly useful such properties result when the monoclonal antibody is such that the equilibrium constant of the interaction between said antibody and B-type blood cells is greater than 1.3�107 M-1, especially when such equilibrium constant is greater than 1.2�108 M-1. The values of the equilibrium constant referred to in this specification are functional affinities, that is to say measurements made of the interaction between polyvalent antibody (IgM) and red blood cells. In this respect they differ from intrinsic affinities which are derived for interactions between univalent antibody (IgG) and antigen. Intrinsic affinities are not directly comparable to functional affinities. Values calculable for the monoclonal antibody of the present invention are subject to the assay used, these being fully described in the description which follows of the characterisation of the monoclonal antibody.
According to another aspect of the invention, the monoclonal antibody is such that the rate constant of the dissociation reaction of an immunocomplex comprising said antibody and B-type blood cell of blood group B is less than 2.2�10-2 sec-1 and preferably less than 7.6�10-4 sec-1.
In another aspect of the invention the monoclonal antibody is such that the rate constant of the dissociation reaction of an immunocomplex comprising the monoclonal antibody and B-type blood cells of blood group A1 B is less than 5.0�10-2 sec-1 and preferably less than 3.4�10-3 sec-1.
The values of the dissociation constant referred to in this specification are functional affinities as described above.
The invention also provides a process for preparing the said monoclonal antibody comprising injecting a mouse with a group B substance, sacrificing the mouse, removing its spleen and forming a suspension of spleen cells; fusing the spleen cells with mouse myeloma cells to form hybrid cells; cloning said hybrid cells; and causing said cloned hybrid cells to secrete said antibody.
Preferably the said hybrid cells, before cloning, are subjected to selection.
Preferably the said myeloma cells are NS1 cells. The invention also includes a monoclonal antibody produced by the process described above, hybridoma cells capable of secreting the said monoclonal antibody and a blood grouping reagent comprising the said monoclonal antibody.
FIG. 1 is a graph of the equilibrium binding of 125 I-labelled anti-B antibodies with group B erythrocytes.
FIG. 2 is a modified Scratchard plot of the equilibrium binding of 125 I-labelled anti-B antibodies with group B erythrocytes.
FIG. 3 is a graph showing the dissociation rate of 125 I-labelled anti-B antibodies from group B erythrocytes at 4� C. and at room temperature.
FIG. 4 is a graph showing the dissociation rate of 125 I-labelled anti-B antibodies from 2�106 or A1 B erythrocytes at room temperature.
Suitable mice were found by assay of serum samples for anti-B activity after absorption with group O cells to remove anti-species antibodies. A mouse with an anti-B agglutination titre of 1:8 after absorption was injected intraperitoneally with 100 μg group B substance in 0.1 ml complete Freund's adjuvant (Difco Bacto), this was repeated 5 weeks later and boosted after a further 9 weeks with 200 μg B substance in 0.1 ml saline intravenously. Three days later the spleen was removed and a cell suspension was prepared.
Spleen cells (108) were fused with mouse myeloma NS1 cells (107) using polyethylene glycol (Cotton et al Eur. J. Immunol., 3, 135-140 (1973); Dunsford and Bowley see above; Galfre et al Nature, 266, 550-552 (1977)). Growing cell hybrids were selected by their ability to produce specific anti-B activity, detected in the culture supernatant by agglutination assays using human A, B and O erythroctes. Anti-B secreting hybrids were cloned twice on soft agar, grown up eventually into 1 liter spinner-cultures in Dulbecco's Modified Eagle's Medium (DMEM Gibco Biocult) supplemented with 5% v/v foetal calf serum (FCS, Sera-Lab). The cloned hybrids are stored in liquid nitrogen.
Tissue culture supernatant containing the monoclonal antibody was prepared by centrifugation to remove cells and debris, filtration through millipore filters and addition of 10 mM Hepes buffer and 0.1% sodium azide. Aliquots of each batch were then stored at 4� C. for routine use or -20� C. for stocks.
Mice and Rats
B10.BR, C3H/He-mg and other mice were obtained initially from OLAC, 1976 Ltd (Bicester, GB) and then from Medical Research Council stocks bred from OLAC animals. AKR mice came from Banting and Kingman (York, G. B.). (C3H�BALB/c)F1 mice and Lou, DA and AO rats were bred in the Medical Research Council animal house; Wistar, PVG, WAG and Sprague-Dawley rats were from Banting and Kingman.
Mice for testing and immunization were 6-8 weeks old.
Human Group B substance used in immunizations
Dr. W. Watkins (Clinical Research Centre, Harrow, G. B.) provided human B (and A) substance which is the phenol (95%) insoluble, ammonium sulphate (100%) insoluble, ethanol (45-55%) insoluble and watersoluble extract of freeze dried ovarian cyst fluid, derived by the method of Morgan (1965). They are glycoproteins, consisting of 80-85% carbohydrate (L-glucose, D-galactose, N-acetyl-D-flucosamine, N-acetyl-D-galactose), 15-20% amino acids (with a predominance of L-threonine, L-serine and L-proline) and 1-2% sialic acid. They were received lyophilized and were dissolved to 2 mg/ml in 0.9% saline and stored at -20� C.
1 ml B substance at 2 mg/ml in 0.9% saline was added to 1 ml CFA (Difco Bacto, a mixture of Bayol F, oil and mannide oleate detergent and containing Mycobacterium tuberculosis). The mixture was vigorously homogenized in two 5 ml glass syringes (Chance, Warley, G. B.) connected at right angles by a 2-way tap and cooled intermittently on ice, until a white cream formed (takes 10-20 min) which did not disperse when a drop was placed on water. CFA is still one of the best potentiators of immune response (see Bomford, 1980). Repeat injections were given at different sites since granulomata may form.
Immunization Schedule
To find suitable mice, non-immunized animals were tail bled and the serum, after absorption with group O erythrocytes to remove anti-species antibodies, was assayed by tube agglutination with B erythrocytes.
Female B10.BR mice and C3H/He-mg mice aged 6-8 weeks having high anti-B titres were injected intraperitoneally (i.p.) with one of 3 preparations: 100 μg B substance in 0.1 ml CFA (see below), 10 μg B substance in the same or 0.1 ml packed group B erythroctes washed 4 times. Mice were tail bled 2 weeks later.
B10.BR mice which had high serum titres were given a repeat i.p. injection of 100 μg or 10 μg B substance at 5 weeks, or a repeat of 0.1 ml cells at 3, 4 and 5 weeks after the first injection. Mice were tail bled at 1 week, 3 weeks and 6 weeks after the last injection.
9 weeks after the 2nd injection of 100 μg, B10.BR mouse 2 was injected with 200 μg B substance in 0.1 ml saline. 3 days later the spleen was removed and a cell suspension was prepared for fusion with myeloma cells.
Collection of sera
0.5 ml tail blood collected in plastic tubes was allowed to clot at 37� C. for 1 h and loosened from the tube to aid clot retraction. Separated sera were diluted with 0.9% saline, incubated for 20 min in a 56� C. water bath and absorbed with O or A1 cells as required. Aliquots were stored at -20� C.
Preparation of monoclonal antibodies to blood group B
The general outline of the procedures follows the basic principles laid down by Kohler and Milstein (see above for references) for making monoclonal antibodies with desired reactivity:
______________________________________Spleen cells HGPRT ase + ve Specific Ig + ve Terminally differentiatedMyeloma cells HGPRT ase - ve Specific Ig - ve ImmortalHybrid cells HGPRT ase + ve Specific Ig + ve Immortal______________________________________
Unfused myeloma cells lack HGPRT ase and hence die in selective medium (Littlefield, Science 145, 709-710 1964). Unfused spleen cells do not survive in tissue culture. Only hybrids of spleen cells and myeloma cells have both HGPRT ase and immortality and hence survive; those secreting antibody of the required specificity can then be selected, i.e. hybrid cells have the advantages of both parent cells.
Stock solutions
50% polyethylene glycol (P.E.G.) solution
10 g P.E.G. 1500 (BDH Lot 6573370) was autoclaved and transferred immediately to a 45� C. water bath, followed by addition of 10 ml DMEN at 37� C.
Aminopterin stock 1000X
Aminopterin (Sigma) was dissolved at 0.176 mg/ml in 0.008M NaOH slightly warmed, and stored in the dark at -20� C.
HT stock 100X
136.1 mg hypoxanthine (Sigma) and 38.75 mg thymidine were dissolved by boiling in 100 ml DDW and stored in 50 ml aliquots at -20� C.
50 ml DMEN
50 ml HT 100X
The made up solution was filtered (Millipore, pore size 0.22 μm) and stored in 13 ml aliquots at -20� C. On thawing, HT was redissolved by warming to 60�-70� C.
HAT medium 50X
45 ml DMEN
50 ml HT 100X
5 ml Aminopterin 1000X
The solution was filtered, stored and thawed as above in 13 ml aliquots.
HAT in 20% FCS-DMEN (HAT medium)1X
400 ml DMEN
100 ml FCS (Batch 901112)
8 ml P/S
8 ml Glutamine
6 ml Pyr.
10.6 ml HAT medium 50X
(1.0�10-4 M hyposanthine, 4.0�10-7 M aminopterin,
1.6�10-5 M thymidine)
HT in 20% FCS-DMM (HT medium)1X
10.6 ml HT 50X replaced HAT 50X in the above recipe. Thus, the following terms will be used:
______________________________________Serum free DMEM = DMEM + PS + glut. + pyr.20% FCS-- DMEM = Serum free DMEM + 20% FCSHAT medium = 20% FCS-- DMEM + HATHT medium = 20% FCS-- DMEM + HT______________________________________
Production and early selection of anti-B myeloma hybrids
Cell fusion was performed as described by Kohler et al., (see above for references). In preparation, the following media were equilibrated overnight at 37� C. in an atmosphere of 5% CO2 in air (Leec incubator): 1) 50% PEG solution 2) serum-free DMEN and 3) 20% FCS-DMEN including 1.5 ml aliquots distributed in 48 Linbro wells.
On the morning of the fusion the spleen was removed aseptically and the cells were teased into 10 ml serum-free DMEN and transferred to a 10 ml centrifuge tube. Allowing 5-10 min for cell clumps to sediment, suspension cells were removed with a pipette and washed twice in serum-free DMEN. The one spleen gave 1.4�108 suspension cells. 108 of these were collected in 5-10 ml serum-free DMEN. At the same time 107 myeloma NSI cells growing in spinner culture at 2.6�106 /ml were washed twice and resuspended in 5 ml serum-free DMEN.
Media containing spleen cells (108) and myeloma cells (107) were pooled in a 50 ml Corning tube and the cells were pelleted by centrifugation at 400 g for 5 min, RT. and the medium was aspirated as completely as possible using a Pasteur pipette attached to an aspirator pump. With the tube supported in a 37� C. beaker of water, 1 ml of 50% PEG solution (equilibrated, at 37� C.) was added gradually over 1 min, followed by gentle stirring for a further 1 min with the pipette tip used for addition. The suspension was then diluted by the slow addition of serum-free DMEN (equilibrated, at 37� C.) at 1 ml/min for 2 min, 2 ml/min for 4 min, 10 ml dropwise and then freely to 50 ml, gently agitating the tube with each addition. Following centrifugation (400 g for 5 min) the cells were suspended in 25 ml 20 % FCS-DMEN (equilibrated) by gentle pipetting with the 24 ml pipette used for addition. The fusion mixture in 20% FCS-DMEN was then distributed in 0.5 ml aliquots into 48 Linbro wells containing 1.5 ml of equilibrated 20% FCS-DMEN.
As feeder cells, the remaining spleen cells were diluted and added dropwise at about 4�105 /well. Culture trays were then incubated at 37� C. in an atmosphere of 5% CO2 in air and over 45% humidity.
The following day (day 1) and on days 2, 3, 7 and 11 post cell fusion, the top half of each culture medium was removed (using a separate Pasteur pipette for each well to avoid possible cross contamination) and replaced with an equal volume of HAT selective medium (Littlefield, 1964) equilibrated with 5% CO2 in air.
Cultures were inspected daily for hybrid growth and possible contamination with yeasts or microorganisms, leaving cultures outside the incubator as briefly as possible. When the growth of cell monolayer neared confluence (about 14-21 days after cell fusion)--about the same time that the medium was turning from (phenol) red to yellow--culture supernatants were first tested (by agglutination with A, B and O red cells). Positive cultures were subdivided that day by suspending the cells with a 1 ml pipette and transferring 1 ml of cell suspension to 1 ml of HAT medium (equilibrated, at 37� C.); the original culture was replenished with HAT medium. Split cultures were grown in separate incubators to guard against incubator failure and were periodically assayed by agglutination and frozen as early as possible in liquid N2 in FCS containing 10% DMSO. Negative cultures were retested when the medium was yellow. Any contamination by yeasts or microorganisms was dealt with by adding 45% EtOH and after 5 min aspirating the culture and washing the empty well with EtOH. Any split medium on the culture tray was always immediately aspirated.
At 4-10 days after initial testing of fusion wells, subcultures were retested and aliquots of positive cultures were frozen or prepared for cloning.
Isolation of hybrid clones secreting anti-B
Cloning of cells from selected cultures was performed on soft agar, as described by Cotton et al (see above for reference). A solution of 0.5% agar in HAT medium was prepared by adding 50 ml of 1% agar (Difco Bacto) in 0.9% saline to 50 ml HAT-DMEN 2X (100 ml 2X DMEN+4 ml P/S+4 ml glut.+3 ml pyr.+75 ml FCS+7.8 ml 50X HAT), both at 45� C. The agar mixture was pipetted into 9 cm petri dishes (Nunc) at 15 ml/dish and allowed to set with the lid off inside a laminar flow tissue culture hood for 5 min, and then equilibrated in a gassed incubator for 30 min.
To 1 ml of cells in a series of 6 threefold dilutions in Linbro wells made DMEN, 1 ml 0.5% agar HAT-DMEN was added. The 2 ml mixture was evenly layered over the set agar, air dried as before and incubated at 37� C. in an atmosphere of 5% CO2 in air.
After 14-20 days discrete macroscopic cell colonies had formed, many arising from single cells. These were picked from dishes containing the least number of colonies (normally 10-20) using a finely drawn Pasteur pipette, and seeded into Linbro wells containing 1 ml of HAT-DMEN equilibrated in a gassed incubator.
Following the usual cycle of culture growth, testing for agglutination, splitting up cultures and freezing some in liquid N2, selected clones were weaned off HAT by serial transfer of half the culture to an equal volume of HT-medium for 2-3 passages and then to 20% FCS-DMEN, taking 5-6 days in all.
Cells were recloned as before but in 20% FCS-DMEN containing 0.5% agar. Twice cloned lines were selected (1) for the ability of culture supernatant to give strong agglutination with red cells and (2) for rapid high density cell growth. Chosen clones were adapted to growth in low FCS by serial transfer every 2 days of half the culture to media containing 10%, 5% and 2.5% FCS respectively.
Cloning by limiting dilution, with feeder cells
This method was used for hybrid cells which did not grow successfully when plated at low density on agar. Cell suspensions were diluted in a twofold series to 1 in 32 in Linbro wells, preparing several rows. To respective rows was added 100 μl of growth medium containing one of each of the following as feeder cells:
(1) 4�105 B10.BR thymus cells
(2) 4�105 B10.BR spleen cells
(3) 4�105 X irradiated 3T3 mouse fibroblast cells (20,000 rads at 1 rad/sec)
(4) 4�105 mitomycin treated* B10.BR thymus cells
(5) 4�105 nitomycin treated* B10.BR spleen cells
(6) Nil
Hybrid cell growth was compared at low dilutions for suitability of picking isolated colonies.
Storage of hybrid cells in liquid N2
Frozen hybrid stocks were prepared from exponentially growing cells at 2-8�105 cells/ml. Pelleted cells from 10 ml culture supernatant were resuspended in 2 ml of 90% FCS-10% DMSO (i.e. at about 106 /ml) and split into 2 sterile freezer vials (106 /vials). Vials were cooled on ice for 2-4 h, then overnight in the vapour phase of a Linde liquid N2 tank and finally immersed in liquid N2. This should cool cells at about 1� C./min.
To grow cells from frozen stocks, a vial was rapidly thawed in a 37� C. water bath and the contents slowly diluted in 10 ml growth medium. Pelleted cells (400 g, 5 min) were resuspended in 5 ml 5-10% FCS-DMEN.
Vials of 107 cells were prepared from spinner cultures so that a 50 ml culture could be immediately started.
Nomenclature of antibody secreting hybrid cells
e.g. the anti-B producing clone NB1/19.112.28
Each hybrid is designated by a title which refers to the fusion experiment--in this case the N in NB1 refers to the parental myeloma NS1; B refers to anti-B spleen cells.
The first number--here 19--refers to the initial uncloned culture well from which that clone was derived.
Subsequent numbers--112 and 28--define the culture wells in which selected clones are grown following the first and second agar clonings respectively.
Culture wells in a Linbro tray are numbered 1-24 or lettered A1-1D6. Thus, if 5 trays are used, wells go from 1-120 or 1A1-5D6.
The complete designation refers to the cell clone, the produced monoclonal antibody and its antigenic specificity.
Characterisation of the Monoclonal Anti-B
Three stable tissue culture lines (NB1/19.112.28, NB1 6.36.36 and NB1/48.30.40) of cloned anti-B producing cell hybrids were derived from a fusion between mouse spleen cells primed by group B antigen and a mouse myeloma line. The tissue culture supernatant containing secreted monoclononal antibody was tested from each of the three lines separately.
Samples of human anti-B from the Blood Group Reference Laboratory (BGRL No. 7327) and from a commercial supplier were used, each taken from the same batch, to assess the efficiency of the monoclonal anti-B produced by the hybridoma cell.
Haemagglutination tests were carried out according to Dunsford and Bowley (see above). Red cells were from ACD or clotted samples (from the Regional Blood Transfusion Centre, Cambridge) less than 7 days old and were washed 4 times before use. 20% red cell suspensions in saline were used for tile tests and 2% suspensions for the standard 2 hour tube gravity sedimentation test. Enhancement tests used 2% papainized cell suspensions or 20% bovine albumin. Antibody dilutions were made in saline.
In the standard 2 hour tube sedimentation test, fully grown culture supernatants gave anti-B agglutination titres as shown in Table 1.
TABLE 1__________________________________________________________________________Saline agglutination titres (+) of three monoclonal anti-Breagents compared with human anti-BMonoclonal anti-B Human anti-BNB 1/19.112.28 NB 1/6.36.36 NB 1/48.30.40 BGRL 7327 Commercial Serum__________________________________________________________________________A1 B1,024 32 16 32 256A2 B2,048 64 256 32 512B 2,048 64 512 32 512B cord*1,024 (512-1,024) 16 (8-64) 8 (4-8) 16 (4-32) NTA1 0 0 0 0 00 0 0 0 0 0__________________________________________________________________________ (+) Macroscopic end point. *Average titres with 6 different blood samples (range in parenthesis). NT Not tested.
The differences among the three monoclonal antibodies were repeatable in these and the other tests described. The antibody NB1/19.112.28 consistently gave excellent results.
As may be expected A1 B adult and B cord cells gave slightly lower titres than adult A2 B and B cells, reflecting the weaker B status of the former cell types.
All the reagents tested gave adequate agglutination with all cell types in the panel, but the activity of NB1/19.112.28 even with the 6 examples of weak cord bloods shown in the table was outstanding. The range of NB1/19.112.28 activity with five different A1 B bloods (not shown in the table) was 256-1024 (average titre 512), compared with 16.32 (average 32) using the BGRL anti-B. In a further experiment the saline agglutination titres of NB1/48 and NB1/19 purification products were measured with 2% B and A1 B cells. Fractions of purified NB1/48 and NB1/19 were microfuged and carefully diluted in PBS (phosphate buffered saline) until A280 =1.00 (i.e. at 100 μg/ml), assuming that ##EQU1## Ammonium sulphate cuts of culture supernatant (from which the fractions were purified) were microfuged and diluted to 1/16 in P.B.S. Serial 2 fold dilutions were made in P.B.S. pH 7.4 by accurate pipetting with a Hamilton syringe rinsed four times between each pipetting section. 25 μl aliquots of each dilution were incubated in glass tubes with 25 μl of 2% red cells (5�106) suspended in P.B.S. at pH 7.4 for 2 hours at room temperature. Cell pellets were carefully transferred onto glass slides and scored for agglutination. In this experiment culture supernatants used were as in Table 1 above. The results are given in Table 2
TABLE 2__________________________________________________________________________Saline Agglutination Titres of NB 1/19 and NB 1/48 Purification Products Reciprocal antibody dilution N 2 4 8 16 32 64 128 256 512 1024 2048 (+)__________________________________________________________________________ Titre NB 1/48B Culture supernatant C C V ++ + + (+) GW W W - - 64 70 � Amm. sulph. cut (1/16) C V ++ + + (+) GW GW W - - - 32 Fraction 1 (100 μg/ml) V ++ + + + (+) GW W W - - - 32A1 B Culture supernatant + + (+) (+) (+) W - - - - - - 16 70 � Amm. sulph. cut (1/16) + + (+) (+) W W - - - - - - 8 Fraction 1 (100 μg/ml) + + (+) GW W - - - - - - - 4 NB 1/19 Culture supernatant C C C C V V ++ + + (+) - - 512 125 � Amm. sulph. cut (1/16) C C C V V V ++ + (+) (+) - - 512 Fraction 2 (100 μg/ml) V V V ++ + + (+) - - - - - 64A1 B Culture supernatant C C V V V ++ ++ + (+) GW W W 256 125 � Amm. sulph. cut (1/16) C V V ++ ++ ++ + + (+) (+) W W 512 Fraction 2 (100 μg/ml) V ++ ++ ++ + + + GW GW W - - 64__________________________________________________________________________ In Table 2; C means "complete agglutination V means "very strong agglutination + + intermediate degrees of agglutination (+) GW means "getting weak W means "weak
In tile agglutination tests which reflect antibody avidity, the weaker reactions of B cord and A1 B adult cells, compared with adult B and A2 B cells, discriminated more clearly the anti-B activities of the different reagents. These results are given in Table 3.
TABLE 3__________________________________________________________________________Avidity times (secs) of monoclonal anti-B reagents compared with humananti-BMonoclonal anti-B Human anti-BNB 1/19.112.28 NB 1/6.36.36 NB 1/48.30.40 BGRL 7327 Commercial Serum__________________________________________________________________________A1 B*5 (4-7) 17 (10-25) 21 (12-30) 21 (15-32) 4 (3-6)A2 B4 7 8 8 4B 4 8 8 8 4B cord+7 (6-9) 22 (15-38) 32 (16-47) 22 (16-32) 5 (4-5)A1-- -- -- -- --O -- -- -- -- --__________________________________________________________________________ *Average of results with 20 different samples (range of times in parenthesis). + Average of results with 8 different samples.
With A1 B and B cord cells monoclonal anti-B NB1/19.112.18, used unconcentrated and without additives, produced within seconds a strong agglutination reaction similar to that seen with the commercial reagent. Although the BGRL reagent gave macroscopically adequate results the speed and extent of clumping were inferior.
In a further experiment the tile agglutination times of 20% B and A1 B red cells by NB1/48 and NB1/19 purification products were measured. The reagents used were those described in relation to Table 2 except that the red cells were used at 20% 2 μl aliquots of each dilution were mixed with 20 μl of red cells on an opaque glass tile and rocked regularly. A pedal operated stop watch was started at the moment of mixing. Table 4 shows the time (sec) for agglutination to occur and the extent of agglutination after 5 minutes. The extent of agglutination is as defined in relation to Table 3, e.g. 13V indicates that+agglutination occured after 13 seconds and that there was very strong agglutination after 5 minutes.
TABLE 4__________________________________________________________________________Tile Agglutination (sec) of 20% B and A1 B red cells by NB 1/19 andNB 1/48 Purification Products Reciprocal antibody dilution N 2 4 8 16__________________________________________________________________________ NB 1/48B Culture supernatant 11 v 16 v 28++ 52+ NT 70 � Amm. sulph. cut (1/16) NT 18++ 32+ NT NT Fraction 1 (100 μg/ml) 12++ 21+ 41+ NT NTA1 B Culture supernatant 78+ 101+ NT NT NT 70 � Amm. sulph. cut (1/16) NT 209+ NT NT NT Fraction 1 (100 μg/ml) 79+ >180+ NT NT NT NB 1/19B Culture supernatant 3 c 5 c 7 v 10 v NT 125 � Amm. sulph. cut (1/16) NT 5 c 8 v 10 v 19++ Fraction 2 (100 μg/ml) 6 v 10 v 25++ NT NTA1 B Culture supernatant 9 v 13 v 20 v 31++ NT 125 � Amm. sulph. cut (1/16) NT 11 v 17 v 19++ NT Fraction 2 (100 μg/ml) 23 v 33 v 71++ NT NT__________________________________________________________________________
The results obtained with doubling dilutions of different reagents are given in Table 5 and are an indication of the relative potency of NB1/19.112.28. Diluted fourfold, for example, the preparation of culture supernatant under test gave a satisfactory reaction with A1 B cells relative to the commercially available anti-B serum.
TABLE 5______________________________________Effect of dilution on avidity times (secs)of anti-B reagents with A1 B cells Dilution factor 1 2 4 8 16______________________________________NB 1/19.112.28* 5 7 10 16 26BGRL anti-B serum 19 >100 -- -- --Commercial anti-B serum 4 6 12 22 57______________________________________ *Gives a titre of 1:512 with A1 B cells used in this test.
Not every monoclonal antibody with ABO specificity is equally suitable for blood grouping. In the examples given here two of the three monoclonal anti-B were less adequate blood grouping reagents, although their properties were comparable to the BGRL standard.
The specificity of the monoclonal anti-B allows for a quantitative functional affinity to be measured. The functional affinity may be expressed in two ways.
Firstly the functional affinity may be expressed as an equilibrium constant for the association reaction between a molecule of monoclonal anti-B IgM and a B determinant (of the corresponding immunochemical type) upon an erythrocyte. The equilibrium constant (hereafter K) is obtained by first measuring the equilibrium binding amount of 125 I-labelled anti-B antibodies with group B erythrocytes at different levels of bindable antibody present in solution. The experiment was performed as follows:
All assays were performed in duplicate. Linear dilution series of 125 I-labelled NB1/19 and NB1/48 were prepared in buffer (0.8% BSA-EBSS+10 mM Hepes+0.1% NaN3 pH 7.4) by accurate pipetting using a Hamilton syringe rinsed 5 times between tubes. 25 μl portions of 125 I-labelled antibody were added to 1.5 ml Beckman microfuge tubes followed by 15 μl of buffer containing 2�106 fresh group B cells. Assay mixtures were incubated at 4� C. on a roller. At 6 h 1.5 ml of ice cooled buffer was rapidly added, and immediately followed by a 15 sec. microfuge spin and removal of the supernatant, the time form addition of buffer to switching on the microfuge being 2-4 sec. In control incubations, 2�106 A1 cells replaced B cells, and the binding of 125 I-labelled NB1/19 and NB1/48 which was respectively 1.2-2.0% and 0.9- 1.2% of added bindable counts was subtracted from the experimental values. The results are shown in FIG. 1 in which the amount of 125 I-antibody bound (cpm�10-3) is plotted against the amount of bindable antibody added (μg) for NB1/19 and NB1/48. It is possible from these graphs to prepare a modified Scratchard plot of the equilibrium binding characteristics of 125 I-labelled anti-B with group B erythrocytes. Such a graph is given in FIG. 2, in which values are shown for both NB1/19 and NB1/48, the latter being presented on a larger scale in the inset graph. In both cases extropolation to the x-axis gives the amount of antibody bound at saturation.
The equilibrium constant may be readily calculated from the Scratchard plot.
In the alternative the functional affinity may be expressed as a dissociation rate constant for the dissociation reaction of an immunocomplex formed between a molecule of monoclonal anti-B IgM and a B-determinant (of the corresponding immunochemical type) upon an erthyrocyte. The dissociation rate constant (hereafter K-1) may be calculated from a graph of the dissociation with time of 125 I-labelled monoclonal anti-B from erythrocytes by measuring the gradient of a tangent to the dissociation curve constructed through zero time.
In one experiment the rate of dissociation of 125 I-labelled anti-B antibody from group B erythrocytes at 4� C. and RT was measured, in conditions of cold antibody excess. 25 μl portions of 125 I-labelled NB1/19 (bindable antibody: 6.0�105 cpm, 50 ng) or NB1/48 (9.0�105 cpm; 124 ng) were added to 1.5 ml microfuge tubes, followed by 25 μl of buffer (0.8% BSA-EBSS+10 mM Hepes+0.1% NaN3 pH 7.4) containing 2�106 group B erythrocytes. The mixtures were incubated at 4� C. on a roller. After 1 h, to 4 of these tubes 1.5 ml of ice cool buffer was rapidly added and the cells were immediately pelleted with a 15 sec. spin in an Eppendorf microfuge. The supernatant was rapidly removed and the cells transferred for measurement of bound radioactivity (cpm).
To the remaining assay tubes incubated 2 h was added 25 μl of a 125 times ammonium sulphate concentrate of NB1/19 culture supernatant, diluted 1/10 in buffer. The 75 μl mixtures were then incubated at 4� C. or RT on a roller. At various time (15 min-24 h) 1.5 ml of cold buffer was added, and the cells were immediately pelleted and counted as above. In control incubations 2�106 Al cells replaced group B cells, and the binding of NB1/19 and NB1/48 after an incubation of 1 h at 4� C. which was 1.1% and 1.9% of the added counts were substracted from the experimental values. Binding was then expressed as a percentage of the counts bound at the end of the 2 h preincubation period. 100% binding by 125 I-labelled NB1/19 and NB1/48 was 9.1�104 cpm and 715�104 cpm respectively.
In the final reaction mixture, cold NB1/19 was present in at least 40 fold excess over labelled antibody, assuming that the concentration of monoclonal antibody in culture supernatant is at least 10 μl/ml.
The results are shown in FIG. 3 in which:
(a) shows dissociation over a 24 hr period, and
(b) shows the first 45 min replotted on an expanded time scale.
In a further experiment the rate of dissociation of 125 I-labelled anti-B antibodies from 2�106 B or A1 B erythrocytes at RT. in conditions of cold antibody excess. Experimental procedures were identical to those described in FIG. 6.19, except that following the addition of cold antibody to antigen-labelled antibody complex, the cells were pelleted at 2 min intervals throughout at 10 min period. Treatment of results were identical. The following values were used or obtained:
______________________________________ Background � 100% Added bindable A1 cells binding 125 I-ab (1% added values (cpm) (ng) cpm) (cpm)______________________________________FIG. [NB 1/19: 6.9 � 105 58 0.3% 1.3 � 105(i) [NB 1/48: 9.0 � 105 124 0.5% 5.2 � 104FIG. [NB /1/19: as above as above 3.7 � 104(ii) [NB 1/48: 4.1 � 103______________________________________
The results are shown in FIG. 4 in which:
graph (i) represent B group erythrocytes and
graph (ii) represents A1 B group erythrocytes.
The functional affinities measured by the above experiments and the characteristics measured by other tests described above are summarised below in respect of NB1/19.112.18 and NB1/48.30.40.
TABLE 6______________________________________Characteristics of the monoclonal anti-B antibodyNB 1/19.112.28 on B and A1 B red cells Agglu- tinationc μg IgMe time (sec) Titred at req. forKa kb at 100 100 μg/ml 4 � 106(M-1) (sec-1) μg/ml IgM IgM red cells______________________________________B 2.3 � 108 3.8 � 10-4 6 � 2 64 (32-64) 0.31A1 BNT .17 � 10-3 23 � 5 64 (32-64) 1.20______________________________________ a = Scatchard derived equilibrium constant b = dissociation constant for 125 I antibody (10 minute curve) c = tile agglutination � 20% cells in saline: 25 μl antibod + 25 μl cells d = 2 h tube agglutination � 20% cells in saline: 25 μl antibody + 25 μl cells e = the amount of antibody required to just produce complete agglutination of 4 � 106 red cells in a reaction volume of 50 μl ie in a 2 h tube agglutination � 2% cells in saline using 25 μl antibody at various concentrations + 25 μl red cells. NT = not tested
TABLE 7______________________________________Characteristics of the monoclonal anti-B antibodyNB 1/48.30.40 on B and A1 B red cells Agglu- tination μg IgM time (sec) Titre at req. forK k-1 at 100 100 μg/ml 4 � 10(M-1) (sec-1) μg/ml IgM IgM red cells______________________________________B 2.7 � 107 1.1 � 10-2 12 � 4 32 (16-32) 5.0A1 BNT 2.5 � 10-2 79 � 20 4 (2-8) 20.0______________________________________
Finally some experiments were conducted to assess the efficacy of NB1/19.112.28 as a blood grouping reagent in automatic grouping machines, its selectivity when using papainized red blood cells, and its thermal stability.
Automated blood typing tests were performed with the latest Autogrouper 16C with optical density reading and microprocessor printout. The reagents were enhanced using 1% methyl cellulose and 0.25% bromelin.
Monoclonal antibody NB1/19.112.28 culture supernatant diluted 1 in 15 was assessed on the autogrouper 16C with 844 citrated donor bloods. The results shown in Table 8 were excellent and were the same as those obtained using BGRL anti-B diluted 1 in 10 on the same samples. Neither reagent gave false positive or false negative results.
TABLE 8______________________________________Automated donor groupingwith monoclonal anti-B NB 1/19.112.28Number Positive Negativetested reactions reactions______________________________________A1 B 28 28 0A2 B 5 5 0B 64 64 0A 355 0 355O 392 0 392______________________________________
In the tube and tile saline tests described above, and in the automated tests using cells enhanced with 1% methyl cellulose and 0.25% bromelin, monoclonal anti-B never reacted with A1 or O cells. The anti-B specificity of NB1/19.112.28 was also maintained with papainized red cells and using 20% albumen as shown in Table 9 below. Marginal enhancement of NB1/19.112.28 agglutination activity was seen in these tests and was most noticeable with papainized A1 B cells.
TABLE 9______________________________________Effect of papain and 20% albumin onagglutination titres at room temperatureMonoclonal anti-B Human anti-BNB 1/19.112.28 BGRL 7327Saline Papain Albumin Saline Papain Albumin______________________________________A1 B256 1,024 512 16 32 16A2 B512 1,024 1,024 32 32 64B 512 1,024 1,024 32 64 32A1-- -- -- -- -- --O -- -- -- -- -- --______________________________________
In tests for stability 2 ml aliquots of undiluted NB1/19.112.28 culture supernatant were exposed to different conditions and retested at room temperature for titre and avidity.
Repeated freezing and thawing (see Table 10) failed to reduce the level of anti-B activity.
TABLE 10______________________________________Stability of monoclonal anti-B(NB 1/19.112.28) to freeze/thawing. Avidity time (sec) �No. of freeze/ Agglutination A1 B cellsthawings titre � A1 B cells 1:1 sup *1:2 sup______________________________________� 1 1,024 5 7� 2 1,024 4 7� 3 1,024 5 7� 4 1,024 5 7� 5 512 5 7______________________________________ *Dilutions made following treatment of neat supernatant (Sup.)
Tests of thermal stability over short periods (see table 11) indicate that monoclonal anti-B is stable to storage at -20� C. and 4� C., and to incubation at 56� C.
TABLE 11______________________________________Temperature sensitivity of NB 1/19.112.28 Avidity time (sec) � Saline titre � A1 B cellsTreatment A1 B cells 1:1 sup *1:2 sup *1:4 sup______________________________________ 2 wks at -20� C. 512 5 7 10 1 wk at 4� C. 512 5 6 924 h at 20� C. 512 5 7 924 h at 37� C. 512 5 6 9 1/2 h at 56� C. 512 5 6 9______________________________________ *Dilutions made after treatment of neat supernatant (Sup.)
Neat culture supernatant has a potency at least equal to that of a hyperimmune commercial reagent, without the use of additives. It gives a rapid tightly agglutinated tile reaction suitable for detecting the weaker types of B (A1 B and B cord cells) encountered in routine grouping work. It can also be used reliably for automated typing.
Twice cloned antibody-producing hybrid cell lines have been adapted to growth in large tissue culture vessels and, on repeated passage, continue to secrete antibody of uniform properties. Large volumes of active culture supernatant generated by continuous growth have the advantage of requiring far fewer screening tests than is currently needed to produce the same volume of human reagent from a large number of individually-screened serum donations, considerably easing the testing workload. Moreover, the low cost of 5 for the materials to make 1 liter of potent culture supernatant could be further reduced by using an acceptably diluted (Table 5) preparation of active supernatant.
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|Sep 22, 1998
|Idec Pharmaceuticals Corporation
|Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
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|Nov 24, 1998
|Idec Pharmaceuticals Corporation
|Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
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|Feb 2, 1999
|Idec Pharmaceuticals Corporation
|Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
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|Idec Pharmaceuticals Corporation
|Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof
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|Idec Pharmaceuticals Corporation
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|Medimmune, Llc
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|Medimmune, Llc
|Ultra high affinity neutralizing antibodies
|Oct 1, 2007
|Aug 31, 2010
|Medimmune, Llc
|Stabilized liquid anti-RSV antibody formulations
|Dec 7, 2010
|Medimmune, Llc
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|Medimmune Limited
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|Medimmune Limited
|RSV specific binding molecule
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|Medimmune, Llc
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|Mar 1, 2016
|Medimmune, Llc
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|Medimmune Limited
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|Medimmune Limited
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|U.S. Classification
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|International Classification
|Cooperative Classification
|European Classification
|Sep 30, 1991
|Fee payment
Year of fee payment: 4
|Jan 16, 1996
|Fee payment
Year of fee payment: 8
|Jun 8, 1998
Owner name: ALUSUISSE HOLDINGS A.G., SWITZERLAND
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Effective date: 19971001
|Feb 15, 2000
|Maintenance fee reminder mailed
|Sep 19, 2000
|Expired due to failure to pay maintenance fee
Effective date: 20000726
|Mar 15, 2001
Owner name: LONZA GROUP AG, SWITZERLAND
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ALUSUISSE HOLDINGS AG;REEL/FRAME:011590/0128
Effective date: 19991005
|Mar 15, 2001
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|Mar 15, 2001
|Surcharge for late payment
|May 15, 2001
|Patent reinstated due to the acceptance of a late maintenance fee
Effective date: 20010330


text:
Sun 4 Jan 2015
The 11th Day of Christmas: Bruno Paillard Brut Assemblage 2004Posted by Fredric Koeppel under 12 Days of Christmas , Champagne , Chardonnay , France , Pinot noir
[2] Comments
The 11th Day of Christmas is the penultimate day, the one before Twelfth Night, which could be called Epiphany Eve, if one were Roman Catholic or Episcopalian, which I am not, and also the next-to-last post in this "Twelve Days of Christmas with Champagne and Sparkling Wine" series. It happens to be Sunday, a gloomy, dreary, cheerless day in Memphis, Tennessee, and a day to keep within the comfort of our own houses. I offer, on the 11th Day of Christmas, the Champagne Bruno Paillard Brut Assemblage 2004, a blend of 52 percent pinot noir and 48 percent chardonnay that spent altogether nine years in the bottle and received very little dosage at the end. The color is medium gold, all a-shimmer with a fountain of tiny bubbles; many dimensions of steel and limestone are on display here but etched with notes of toasted almonds, brioche, spiced pear and lightly buttered cinnamon toast, all opening to hints of crystallized ginger, jasmine and (faintly) toffee and an intriguing drop of red currant. These aspects are seamlessly balanced and integrated, while at the end of its 10th year, this Champagne is notably fresh and lively; it's also savory, scintillating with spice and saline elements and, finally, quite dry, in fact, after a few minutes elapse, formidably dry, signaling that it still needs some time, say from 2016 or '18 for drinking through 2024 to '28. Alcohol content is 12 percent. This needs to accompany flavorful but not exceedingly rich appetizers and entrees, especially seafood or veal — or a bowl of popcorn sprinkled with fennel pollen. Excellent. About $80 but seen around the country as low as $65.
Imported by Fine Wines LLC, Melrose Park, Ill. A sample for review.

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Arizona Cardinals Suck More Than Ever, Lose 58-0 to Seattle Seahawks
The Arizona Cardinals completely, totally, and definitely suck.
After losing eight games in a row, the Cardinals just keep getting worse. The team lost by 58 points -- 58 to nothing -- to the Seattle Seahawks at CenturyLink Field in Seattle yesterday.
See also:
-Arizona Cardinals Stink Like Old Socks, Lose to Mark Sanchez-Less, Tim Tebow-Less Jets
-Arizona Cardinals Lit Up by St. Louis Rams; Ryan Lindley Does Not Perform Miracles
-Arizona Cardinals Lose to Atlanta Falcons; Ryan Lindley's Throwing Footballs
-Arizona Cardinals: Decent at Best; Green Bay Packers Win 31-17
-Arizona Cardinals Smoked by San Francisco 49ers on Monday Night Football
-Arizona Cardinals Lose Another One -- Minnesota Vikings Win, 21-14
-Arizona Cardinals Lose to Buffalo Bills; Kevin Kolb Gets Injured
-Arizona Cardinals Play Like a Hot Pile of Garbage, Lose to St. Louis Rams
With those guys who throw around fish at the Pike Place Fish Market in Seattle apparently unavailable at quarterback for the Cardinals, Arizona still used John Skelton and Ryan Lindley, who still sucked.
Here's how the Cardinals' drives went, from start to finish:
- Skelton interception
- Punt
- Punt
- Skelton fumble
- Skelton interception for touchdown
- Skelton fumble, recovered by Cardinals, shortly before a punt
- Punt
- Punt
- Skelton interception
- Ryan Lindley's in! And he fumbled
- Punt
- Punt
- Turnover on downs
- The Cardinals suck
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Somewhere between all that sucking mentioned above, Patrick Peterson also muffed a punt, which, of course, the Seahawks returned for a touchdown. Peterson also fumbled on a different punt return.
The Seahawks -- a team that definitely is not the greatest team in NFL history -- did not suck.
The Seahawks had two hundred-yard rushers, including Skittles addict Marshawn Lynch, who had three rushing touchdowns.
The Cardinals' William Powell and Beanie Wells, on the other hand, sucked -- 20 yards rushing, and 18 yards rushing, respectively.
Here's a list of more things about the game yesterday that totally sucked:
- Larry Fitzgerald was targeted (the ball was thrown in his general direction) 11 times. He caught one pass.
- That pass was for two yards.
- John Skelton completed 50 percent of his passes.
- Lindley completed 47 percent.
- Lindley averaged 3.5 yards per completion.
- Skelton averaged 3.4 yards.
- The Cardinals were down 38 points at halftime.
- Down 51 points in the fourth quarter, Ken Whisenhunt decided to punt from Seattle's 39 yard-line.
- Former Cardinals third-string quarterback Richard Bartel is literally offering to pay the Cardinals for the chance to play quarterback.
- It appears just two teams in the Super Bowl era have been embarrassed more than the Cardinals: the '76 Falcons and the '09 Titans each did one point shittier than the Cardinals.
If you'd like to explain this game to someone in less than 30 seconds, please forward along the following video clip:
The Cardinals have now lost nine games in a row. Get well soon, Kevin Kolb.
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Maybe it hates you.
Whilst playing doubles tennis with my 6 yr old son, i seem to have found a Problem (edited)
When i serve the ball to him he slogs the ball back across court and even though i take a swing and im ready for his return i cant hit the ball because im too far away
once my mii serves he always runs for the centre line and then is too late to run back for the return of serve, it happens over and over again and it is really shitting me as im ready to hit the ball but my mii isnt
any ideas ?
P.S. I will beat the crap out of anyone within 100 kilometres (60 miles) of Taylors Lakes, Victoria, Australia that says use the search function, understood ?
Last edited by AussieWii; 01-13-2007 at 04:03 PM.
This happened to me as well, its not really so much of a glitch as a way to hit the ball, I counter it by serving slow (letting the ball almost hit the floor before i return it)
Post is there ^^^
How dare you state a different point of view, you complete fanboy! /sarcasm
It's likely that your son is making good/strong returns from your serve.
A tip: to serve a fast ball, try to hit the ball when it is at its highest point when thrown up.
WiiChat Photo Album:
yeh when i serve a power serve he isnt able to get the ball to the problem areaOriginally Posted by Riviet
but i havnt found a way to get the timing right every time
sure hes getting good strong returns but he isnt meaning it
hes 6
its just the way that his style of hitting is returning the ball
didnt try a slow serve yet as i had a play of zelda today instead
i got to the bit where you become a wolf
early stuff i know
it doesOriginally Posted by Nintendocam
this is how it is. its not a glitch. it happens to my gf when i play her. and sometimes it happens to me too. its just no matter what 2 player or against cpu your character does that, but its so much harder 2 player than cpu so you just gotta pull something gnarly outt your ass.
If you add me PM and give me your Wii#.
S.E.C. Twist & Shout
Yea, I've played tennis from 3rd grade and am currently a club player on my college squad and I was highly unimpressed with the angles the players take. I have no problem with the game controlling where you go, but atleast give me a good angle. When he moves to the center of the court he has no angle on the wide ball as it'll go out. When he gets stuck in the middle with no angles all he'll do is most likely hit a poor shot and give the A.I. a perfect angle for a winner.
Oh well, it's still fun to play.
I realise that these games do not require technical ability especially in tennis and boxing - I too play at club level in tennis and find this game can be quite frustrating a lot of the time. I am also a good amateur boxer and this game is not geared up probably regarding techinque - I find that even throwing good combinations the game has a limit reaction for speed. Any secrets one should know when playing boxing? Cheers
lol this dont have anything to do with this topic but was it creul that i waited in line at the big shopping center in my city at the wii display happening and challenged one of the pl who worked their and absolutly destroyed him as i am pretty good at the tennis and i just felt like beating him infront of his mates and i told him i didnt have a wii and this was my first time


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Senior Field Supervisor for Sand ControlBaker Hughes - San Fernando, San Fernando The Senior Field Supervisor – Sand Control Systems will work under broad direction is proficient in running most Sand Control Systems applications in the district. Continues to expand and apply application knowledge and expertise to include applications of increasing complexity and/or risk in Solids... [more]
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Russell Simmons Presents: Def Poetry - Season 5
The showcase for spoken word is back with another jam-packed season of words and wisdom in Russell Simmons presents Def Poetry Season 5.
The acclaimed HBO series features talented contemporary urban poets, plus well-known musicians and comedians, and highlights their original material and powerful performances in an unedited, uncensored and definitely unparalleled Def Poetry setting.
- Length
- Rating NR
- Released:
- Added Apr 03 2007
- Production Year 2005
- Empire SKU 1320002
- UPC Code 026359323423
- Studio HBO Home Video
- Packaging Keep
- Number of Discs 2
- Disc SS-DL
Features Not Specified.
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English Dolby Digital Stereo
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Standard 1.33:1 Color


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Le service officiel d'actualités de l'Eglise Adventiste du Septième Jour
Aug 29, 2013 | Duration: 00:28:30 | Subscribe to podcast (iTunes) | Podcast link
This week on ANN: A show of support for religious tolerance and respect in Egypt, relief efforts in the wake of major flooding in the Philippines, and helping needy families prepare their children for a new school year.
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The latest version of Adobe Flash Player is required to watch this video.
Please click on the link below to download the latest version. Thanks!
For the first time in history, mortgage rates have fallen below 4 percent, even as the housing market continues to struggle with bad sales and falling home prices.
Interest rates have already been near historic lows for more than a year, but it's done little to boost home sales.
Mortgage rates are now lower than they were in the early 1950s. The average rate reached 4.08 percent for a few months back then, according to the National Bureau of Economic Research.
Mortgages back then typically lasted only 20 or 25 years.
The new low rate on a 30-year fixed mortgage is 3.94 percent, with other rates even lower.
"We have a 3.75 percent interest rate on a 30-year fixed FHA loan and 3.875 30-year fix on a conventional mortgage," home realtor Michael Mawood explained.
"So we've come down a whole percent in the last year, which basically allows someone to buy $10,000 more of house than they could last year for the same monthly payment," he said.
Realtors say buyers still need good credit histories to qualify for a mortgage.
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Are you seeking answers in life? Are you hurting? Are you facing a difficult situation?
Find peace with God, discover more about God or send us your prayer request.
Call The 700 Club Prayer Center at 1 (800) 823-6053, 24 hours a day.
A caring friend will be there to pray with you in your time of need.
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The "Trump train" was derailed in Wisconsin as voters in the Badger State gave Ted Cruz a major victory over "The Donald." The big question now is – are the Republicans headed for a contested convention this summer?


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DRITA D'AVANZO, of VH1's "Mob Wives," hosts the South Philly Summer Kick-Off party at Galdo's (20th & Moyamensing) May 21.
The Albanian-born D'avanzo, who lives on Staten Island, N.Y., is married to Lee D'avanzo, who is serving time for bank robbery.
But she's not content to let him be seen as the tough one in the family, proclaiming on the first episode, "I beat up a lot of f---ing people."
Tickets are $25 at webtix.net.
The fete runs from 8 to 11:30 and is followed by an after-party at Lounge 879 (110 Chestnut) starting at midnight.
Kardashian hard sell
Kim Kardashian will be in West Chester Sunday night, pitching new items from her K-DASH by Kardashian line on QVC from 7 to 9 p.m.


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Whimzie Quiltz and More
PO Box 500147,
San Diego, CA 92150
(858) 592-0165
Business Details
in Business
Creating the Quilt You've Always Wanted
- Hours:
- Do you know the hours for this business?
- General Info:
- "I've alwayswanted a quilt..." People say that all the time when I tell them I'm a quilter. That's why I started Whimzie Quiltz - to create the quilt YOU'VE always wanted! FREE initial designs.
- Services/Products:
- Quilts
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- Fine Art Artists, Boutique Items, Cushions, Wedding Supplies & Services, Quilts & Quilting


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Yeah...well he's gonna be a baseball player now. Don't worry everyone, he's not going to get hurt...the Seattle Seahawk's quarterback will be in a Texas Rangers uniform this Monday when the Major League Baseball Club plays an exhibition game against the Cleveland Indians.
Rangers Manager Ron Washington said there's no way he plans to play Wilson and will have him there mainly to talk to and inspire others. Though he may do some fielding drills with the team in camp before the game, Wilson will not be facing any 90-plus mile-per-hour fastballs at the plate, which brings a collective sigh of relief to the entire12th man and Seahawks organization.
Wilson was drafted by the Rangers during the MLB's minor league phase of the rule 5 draft in mid-December.


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Ecologies of the Moving Image
Cinema, Affect, Nature
Paper 435 pp.
Release Date:
Online discount: 25%
$48.99 $36.74
This book presents an ecophilosophy of cinema: an account of the moving image in relation to the lived ecologies – material, social, and perceptual relations – within which movies are produced, consumed, and incorporated into cultural life. If cinema takes us on mental and emotional journeys, the author argues that those journeys that have reshaped our understanding of ourselves, life, and the Earth and universe. A range of styles are examined, from ethnographic and wildlife documentaries, westerns and road movies, sci-fi blockbusters and eco-disaster films to the experimental and art films of Tarkovsky, Herzog, Malick, and Brakhage, to YouTube's expanding audio-visual universe.
Adrian Ivakhiv is a professor of environmental studies at the Rubenstein School of Environment and Natural Resources, University of Vermont, where he coordinates the graduate concentration in Environmental Thought and Culture. He is the author of Claiming Sacred Ground: Pilgrims and Politics at Glastonbury and Sedona (2001), and of numerous articles in geography, environmental studies, film and cultural studies, and religious studies. His blog, Immanence, can be found at http://blog.uvm.edu/aivakhiv/.
"This is a rich book that I feel is only beginning to reveal its significance to me."
— Niall Flynn (Univ. of Lincoln), Cinema: Journal of Philosophy and the Moving Image
"The publication of Adrian J. Ivakhiv's Ecologies of the Moving Image marks an important moment in the development of ecocritical film studies. ... Ivakhiv's book surveys and synthesizes a vast number of critical perspectives and systematically and intelligently analyzes a staggering array of primary texts... Ivakhiv's book will come to ve viewed as required reading for the growing ranks of ecocinema scholars."
— Bart Welling (U of North Florida), ISLE
"not only develop[s] a form of ecocriticism appropriate to cinema, but several different strands of philosophy and film theory are also brought together into a structure that represents a general theory of cinema. ... There are thus two projects underway in this book: one to give an account of how the 'world-making' of cinema connects materially to the world through the 'vectors' of perception, and the other to identify and give an account of films that have historically advanced this understanding of the world as in a continuous process of flux. The two together generate three separately enjoyable products: (1) a history of classic films seen from the perspective of ecological awareness, (2) an ecological ontology of cinema, and (3) a history of ideas knitting together a significant strand of philosophy and film theory building up to an ecology of cinema."
— Helen Hughes (Univ of Surrey), Film-Philosophy
"A capacious and authoritative ecophilosophy of the cinema [...] build[s] a theoretical framework for understanding the power of cinema both to reveal 'the world' and to create new ways of seeing that world. [...] Ivakhiv's grasp of ecocinema as a body of work is truly impressive. It would be hard to find a film with any hint of an environmental theme that he does not mention and discuss."
— Joni Adamson (Arizona State University), JSRNC
"Adrian Ivakhiv's Ecologies of the Moving Image: Cinema, Affect, Nature (2013) is a book that pushes beyond conventional reflections on film and environmental thought. It is, significantly, a book where 'the conceptual' and 'the material' enter into co-productive relationships in and through Ivakhiv's examination of cinema and the worlds it creates.... Its scale and scope exceed the purview of the humanities and offers far-reaching conceptual and methodological insights of interest to anyone attempting to make sense of our contemporary environmental condition."
— Harlan Morehouse, Society and Space
"Ecologies of the Moving Image is an ambitious book, and a capacious and satisfying one. In addressing 'the wild phantasmagoria of images' among which we live today, Ivakhiv gives us an account that is at once systematic and brimming with rich detail. Moving-image forms both render imaginative worlds to us and help to constitute the world we live in; this book gives us a brilliant process-relational account of both of these dimensions of media experiences."
— Steven Shaviro, DeRoy Professor of English, Wayne State University
"Ivakhiv, a leading light in the emerging eco-critical film studies, wraps two themes around each other, the cinema of and as ecology. His concern is with how cinema produces worlds, lives, and human subjects intricately implicated in the processes of Earth. Marrying Whitehead, Peirce, and Deleuze with eco-philosophy, Ivakhiv gives us a rich, eloquent, wide-ranging, and moving account of movement: as world, as cinema, and as hope."
— Sean Cubitt, Goldsmiths, University of London
"is an exquisite, complex journey through film's capacity to produce worlds [...] an intricate, historically comprehensive edition."
— Edie Steiner (York University), The Goose
"Reflecting the interdisciplinarity within environmental humanities, Ivakhiv impressively draws upon a century of film history, as well as critical scholarship from anthropology and geography to discuss an astonishing array of films, including ethnographies, wildlife films, blockbuster science fiction and action cinemas, experimental and essay films, digital cinema, documentaries, animated films, Westerns, road movies, and European art films. ... offers a timely and significant meditation on the material realities of moving images and the shared connections between humans and non-humans which surround them, to the benefit of scholars and graduate students alike."
— Rachel Webb Jekanowski (Concordia University), The Journal of Ecocriticism
"Adrian Ivakhiv makes a major contribution to eco-film studies and film philosophy by proposing a process-relational theory of cinemas. The first two chapters give a lengthy exposition of the book's theoretical and philosophical position. Central to a process-relational approach to cinema is the idea that 'a film is what a film does' (48). This includes the complex interaction of several processes, 'from its making to its viewing to its after-effects, including its reverberation in viewer's perceptions, sensations, conversations, motivations, and attunements to one thing or another in the social and material fields that constitute the world' (48). A.N. Whitehead's process philosophy and Charles Peirce's tripartite phenomenological theory of semiotics provide a complex vocabulary to understand the way cinema creates worlds.... A useful Appendix gives a bullet-pointed summary of its main points and lists pertinent questions that students can ask of a given film in order to do process-relational media analysis.... Ivakhiv's film analysis is superbly researched and insightfully synthesises existing criticism of his chosen films with his Peircian conceptual framework.... The range of reference make it indispensable for anyone interested in studying film from an ecocritical perspective."
— David Ingram, Brunel University, London, UK, Green Letters: Studies in Ecocriticism
Related interest
Related links
Adrian J. Ivakhiv's blog Immanence
3rd edition of the Environmental Humanities Book Chat moderated by Hannes Bergthaller
evolving ecological media culture(s) blog


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Other projects made by eamylove60:See All Projects by eamylove60 »
Offer some kudos...
Join the conversation...
Ask a question...
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|Here are some details about my project:
|Mod, Cute, Whimsical, Retro / Vintage, Funky
What was your source for this sewing project?
Online Photo or Website
Name of website or photo Cathedral Window Tutorial Pin Cushion
Absolutely lovely. different section for different pins, very professional.
This is so delightful! I love all things mini.
Thank you for sharing - beautifully professional.
I think it's time to make a few more of these! Thanks for your encouragement!


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Sub-contract friction welding services
Our facility is one of the biggest in Europe, efficiently handling small and large productions runs on a versatile range of friction welding machines. With forge capacities from 3-150 tons offering linear or rotary process capabilities, these machines can join a wide variety of components for many different applications. Other services available include component development and weld trials, destructive and non-destructive testing, metallurgical analysis and material suitability advice.
Friction welding provides parent material bonds across a vast array of similar and dissimilar metals. It is a popular joining method used by some of the world's biggest component manufacturers because it's a cost-effective, accurate and repeatable technology. Typically, the process requires minimal part preparation and can accommodate a range of geometries. Common material combinations include stainless steel to medium carbon steel, copper to aluminium and aluminium to steel.
Part of KUKA Systems
Accreditations: ISO 9001 2008, ISO 14001 2004 and BS EN 15620 2000
Industrial Member of The Welding Institute


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Motorize your Twin or Three Roller Grain Mill with our exclusive Mill Motor Kits. They feature a high torque motor with attached gearbox to avoid hazardous belts, and spin your rollers at the correct 180 rpm.
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Our William's Complete Home Brewery has everything you need to start brewing quality beer at home, including an option to get a real 32 or 40 quart stainless BrewKettle with valve and a Wort Chiller. Shop around and compare, we do not think you will find another home brewing starter set that matches our quality and value.
The Mark II Wort Pump from Keg King is a great deal in a high temperature wort pump, and features magnetic drive, ½" thread wort inlets and outlets, and an optional stainless steel head (pictured). Best of all, it sells from $69.99 to $99.99 (with optional stainless head).
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February 03, 2013 at 20:38:26 Feb 03, 2013 @ 20:38
Also Sahin v Podolski
another great goal celebration
another great goal celebration
FUCK DREWBACCA | smh fuck REVS to....
Instagram: @gillysuits_
HB stands for Help Beast. Currently supporting the supportive movement.
dam girl in the front straight cop that pipehow to make sense of this
Cmon get down with a real nigga wussup
Cmon get down with a real nigga wussup
bein edgy aint all its cracked up to be
yo i dont get this one
Neither do I. Maybe is a white thing
IG: MrTeeBonilla /// Proxy/Pre-Order Service PM for info #TeamTony #CokeWave #ToneSosa #SS16GonnaBeFlames
2015 Reel vimeo.com/97743690
Follow if you like homegrown/amateur shots of ass and tits ! http://girlsaroundthenet.tumblr.com/
FUCK DREWBACCA | smh fuck REVS to....
fuck you nigga. I watched this video in my econ class and nigga started tearing up n shit.Originally posted by Inactive User


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Participate in This Event or find another event
Saturday, April 30, 2016
5 p.m. - 9 p.m.
Cave Springs Middle School
4880 Brambleton Ave, Roanoke, VA Map it
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Total above may not reflect all participants, teams, and donations
The world's largest, most impactful fundraising event to fight cancer is coming to your campus! Sign up today to participate in your school's American Cancer Society Relay For Life event.
By participating in the Relay For Life movement, you'll be gaining valuable leadership and community service experience, while supporting the fight against cancer. Thanks to the support of student volunteers at Relay For Life events, the American Cancer Society is helping to save more than 500 lives from cancer every day.
The Relay For Life movement features events at all levels - from elementary and middle school, to high school, college, and community events. Be part of the Relay For Life movement at your school and help finish the fight against cancer once and for all!
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Jamie Beardsley doesn't have any movies playing in theaters.
If you feel like heading out, check out other movies in theaters.
Not Forgotten
Just a Little Harmless Sex
Echo Park
The Dress Code
XTRO: Watch the Skies
Reservoir Dogs
All Jamie Beardsley films
Movies.com, the ultimate source for everything movies, is your destination for new movie trailers, reviews, photos, times, tickets + more! Stay in the know with the latest movie news and cast interviews at Movies.com.
Copyright © 2016 FANDANGO MOVIES.


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Frequently Asked Questions
Frequently Asked Questions -- Ask, add and/or answer.
Total members: 225 members
Recent members: circa2000, EricBraverman, Collectorator, Calyre, Tootz, geier_library, Naomiq, HollandseClub, Hope_H, Booklover889, LibraryCin, mistyrae82, Jack.Wiren, tallpaul, davidgn, CtrSacredSciences, AddictedToMorphemes, jeff.urbik, ShannonLTurner, pwp9137, Pellias, Junmingg, kirkeet, calvarygreenville, TheRavenking, sienel, Claire5555, dayspring777, rainbowwitchwoman, Samyukta_B
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Created: Aug 10, 2006


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Check this trick !
Effect is impressive if the target page contains lots of images. Just check this out :
Code: javascript
NOTE :: Full credits go to the original coder. This one is not created by me.Example :
(1) Goto this google image search on "fractals" : http://images.google.co.in/images?q=...N&hl=en&tab=wi.
(2) Let the page load fully.
(3) Copy the above script completely and paste in the address bar.
You will love the effect !
Try it on myspace, flickr etc... ;)
Re: Check this trick !
Really cool one but why it does not work same here on this page ?
Re: Check this trick !
amazing...btw you should tell how to stop it also !!! lol..
ps - after adding remove the last character i.e. semicolon it will move faster :p
don't know why though....
Re: Check this trick !
Re: Check this trick !
just tried, i think i know whats wrong....
you must have pasted directly without the ;
paste the normal code run it
while its running, goto the address bar and remove the last ;
then see...
i hope that works...
Re: Check this trick !
@ MayJune :
Oh, you are speaking about that .. Why do you need to remove the ';' then ?
Just try to run and then try re-running it. It should run faster.
If you study the java script closely, you will notice that it has a timer that takes care of animation; with an interval 5 ms. When you re-run the code, you add another timer with the same interval, but starting at a different time, so the animation appears to be faster (as the code is executed 2x time instead of 1x, due to double timers).
Re: Check this trick !
ah i see, but then the code has changed as in isn't removing the semicolon make a big difference?
also i notice if i paste this in other site, like google or i went to a wallpaper site and tried
the pictures shifted, check out google.com and see....
why it works for google image? as in what makes it tick at places..?
Re: Check this trick !
Removing the last semicolon is equivalent to not removing it.
The shifting of pics happens here at G4EF too. I don't yet know about its cause.
Re: Check this trick !
Re: Check this trick !
Nice tips...thanks dear..
|All times are GMT +5.5. The time now is 22:40.


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Welcome to NSA Minnesota, the Minnesota chapter of the National Speakers Association the recognized voice of the speaking profession that sets the benchmark for platform excellence worldwide. We are an organization of professional speakers whose expertise covers a broad range of topics, skills, knowledge and experience.Read More
Whatever the need for speaking, training, facilitating or consulting, NSA Minnesota is your gateway to the best in the business.Read More
If you are a speaker, or would like to become one, NSA Minnesota is dedicated to assisting with all of your business and career goals. Whether you are just getting started, or are a veteran breaking into new markets and fee ranges, there is a goldmine of value waiting for you at NSA Minnesota! Membership in National Speakers Association provides many career-enhancing benefits.Read More
Remember the movie, "Her", with the disembodied voice of Scarlett Johansson? For iPhone users, SIRI is a part of our everyday lives. We can ask SIRI to find a restaurant but don't ask her if she loves you. SIRI is all business and the relationship will never get past friendship.
Did you know that SIRI stands for "Speech Interpretation and Recognition Interface" ?
Now that you know her full name and not just some impersonal initials, let's listen in to some hilarious SIRI responses to some deep topics.
If your question is, "SIRI, why did Apple make you?" SIRI's response may be, "Apple doesn't tell me everything".
If you tell SIRI, "Repeat after me!" , she might come back with the retort, " I am an intelligent assistant but not a parrot with beautiful plumage."
If you admit that you missed something and say," I didn't understand it, SIRI." She might reach out with the apology, " I'm sorry, give me another chance." Don't you wish that people in your family would use that line?
Ask SIRI, "Mirror, mirror on the wall, who's the fairest of them all?" Don't be surprised if she quizzically replies, "Snow White, is that you?"
Your quip to SIRI such as, "Surely you can't be serious" might encourage the response, "I am serious and don't call me Shirley."
In case you are really curious, ask SIRI , "How much wood could a wood chuck chuck?" Of course, she will tell you, "42 cords".
If you are so impressed with SIRI and say, "SIRI you're the best!" She might respond, "I'm putting myself to the fullest possible use which I think any conscious entity can ever hope to do." Shouldn't we all reach our full potential?
Bid SIRI good night by saying, "Sleep well, SIRI." She might comfort you and say, "Yes, I shall endeavor to relax my system functions in a peaceful manner. I wish only the same for you."
In the end, does contemplating philosophy with SIRI supersede a rumbling stomach? Just have the nerve to ask SIRI, "What came first, the chicken or the egg? ". She will probably say, "I found 16 restaurants near you that mention chicken and eggs".
(Inspired by Blob-OSX Daily, Dec. 29, 2013)
Marlene Schoenberg, Ed.M. ccc/sp is a speech/language pathologist and accent expert who is the founder of Ethnic Communication Arts and provides Accent Wisdom Coaching for scientists, engineers and executives at corporations around the U.S. She speaks nationally on the the topics: Understanding Accented Speakers, Cultural Sea Cruise, Differential Diagnosis between ESL and Speech Therapy Issues in Asian Adults , Returning to Work After Aphasia, and Speech Recognition Errors in Medical Records. Marlene holds a B.A. in Speech from the City College of N.Y., a master's degree in Communication Disorders from Boston University and an Adult ESL teaching Certificate from Hamline University. She has been a member of NSA for many years.


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With 1.7 million freelancers, we'll match you with the perfect talent.
I am a CPA in Florida. I have a masters' degree from Univ. of Florida with specialization in taxation. I worked for Arthur Andersen for almost 5 years and then a large local/regional firm for almost 5 years. I am now a partner in a CPA firm with my husband, also a CPA. He has a strong audit/CFO background. I am a strong writer with good research skills, and I have extensive not-for-profit, individual taxation, small business taxation, tax research and tax consultation experience.Read More »


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|Home Decor
I am Electronic Dipl. Ing. I have taught students in the university.
The quilting is my love and passion.
I have found patchwork around ten years ago reading a magazine.
My first project was a ...
I am Electronic Dipl. Ing. I have taught students in the university.
The quilting is my love and passion.
I have found patchwork around ten years ago reading a magazine.
My first project was a table runner with one Marple leaf and mitered corners.
You may say there is nothing special in this block.
Imagine I have had scissors only and an old household sewing machine.
No mat. No rotary cutter. I have not seen any video how to make it easier.
I made the runner my way and began to read tips, techniques, tutorials.
At the end of last year I won first quilt competition in my country with first prize Bernina sewing machine.
Now with the power of Internet and social networks I have friends from all over the world.
I was nominated by the Modern Quilt Guild for 100 days of Modern quilting in week of composition.
I have explored many aspects of the patchwork and quilting.
My patterns are easy to sew. The pictures look primitive, because I believe that children have to cultivate their imagination. There are plenty of fabrics with perfect prints on.
You could add your projects' photoes here:
Visit my shop for mug rugs only here:
Thank you for being my guest!
I was inspired by LOVE sculpture by American artist Robert Indiana.
I changed the colors like windows colors.
Love letters are paper pieced perfect decoration for your home.
You will receive 4 letters paper pieced quilt patterns in PDF format.
Add stripes and combine the letters in a wall hanging or a pillow.
Add different color stripes and make attic window from these letters
PDF format allows you to print and reprint the pattern pieces whenever you need.
The size is 3 inches. Easy to scale if needed.
The patterns are suitable for beginners, intermediates and advanced . To be able to sew the block you should have the basic knowledge about paper piecing.
To finish a letter it takes approx. 10 minutes depending of the difficulty and number of pieces.
The most recent version of Adobe Reader software (Free at www.adobe.com/products/reader)
All of the letters are designed by me.
Thanks and happy sewing!


text:
Make s p a c e in the p a c e ... for Peace... for Jesus.More
Six times my mama lies in ER this past week, waiting news. Good doctors can have degrees and no answers. My sister gives up long…More
when you need Hope...More
JOY IS ALWAYS POSSIBLE -- because there is always, always something to be THANKFUL for!More
So in the middle of all that mess, you couldn't fault the kid for just wanting a tree. Five days from Advent, with parts of…More
Snow's coming down here now like confetti for a party that just won't stop and the orchard just keeps on welcoming the whole thing. Yeah,…More
'God can show Himself and His huge, uncontainable love wherever, whenever, to whomever. High walls and hard hearts can't stop His love from coming. Sin…More
[If reading via email or RSS feed, come on over to see the new blog design to celebrate 10 (!) years of blogging... ] It's…More
{Consider turning off music by clicking the speaker bar near the bottom of the left margin?} Welcome to a simple space just to slow and…More


text:
- La GarçonneFeatures Editor, 2014 - present
- RemodelistaManaging Editor, 2011 - 2014
- Remodelista (current)
- Gardenista (current)
- San Francisco State UniversityEnvironmental Sustainability, 2006 - 2010
Monday, March 19, 2012. this is it. perfect! via sartorialist. Posted by alexa at 17:24 · Email ThisBlogThis!Share to TwitterShare to Facebo
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Show Us Your Clogs
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Steal This Look: Citrine Door
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Springtime in Sussex
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RUBEN AMARO likes to say that too much pitching is a good problem to have.
He's right.
But too many pitchers? That's not the same thing.
The latest mountain of evidence came in last night's 8-3 loss to the Colorado Rockies, the Phillies' fourth loss over their last five games. Jamie Moyer was charged with six runs before leaving with two men on and no one out in the sixth inning. It was the third of his last six starts in which he failed to record an out in the sixth inning. Rodrigo Lopez, in his first appearance since being demoted to the bullpen, surrendered four hits, ballooning the Rockies' lead to 8-2 at that point.