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Diagnostic testing process
Diagnostic testing process
Patent Drawings:
(6 images)
Marlborough, et al.
Date Issued:
January 25, 2011
December 12, 2002
Marlborough; David Ian
(Balgowlah, AU)
Sloane; Andrew John
(Balmain, AU)
Cole; Robert Alan
(Greenwich, AU)
Hunter; William Samuel
(Jan Juc, AU)
Proteome Systems Ltd
(North Ryde, AU)
Primary Examiner:
Chin; Christopher L
Assistant Examiner:
Attorney Or Agent:
Hamilton, Brook, Smith & Reynolds, P.C.
U.S. Class:
Field Of Search:
International Class:
U.S Patent Documents:
Foreign Patent Documents:
Other References:
Verheijen, R., "Development of a One Step Strip Test for the Detection of (Dihydro)streptomycin Residues in Raw Milk," Food and AgriculturalImmunology 12:31-40 (2000). cited by other.
A method and apparatus for use in a flow through assay process is disclosed. The method is characterized by a "pre-incubation step" in which the sample which is to be analysed (typically for the presence of a particular protein), and a detection analyte (typically one or more antibodies bound to colloidal gold or a fluorescent tag) which is known to bind to the particular protein may bind together for a desired period of time. This pre-incubation step occurs before the mixture of sample and detection analyte come into contact with a capture analyte bound to a membrane. The provision of the pre-incubation step has the effect of both improving the sensitivity of the assay and reducing the volume of sample required for an assay. An apparatus for carrying out the method is disclosed defining a pre-incubation chamber for receiving the sample and detection analyte having a base defined by a membrane and a second membrane to which a capture analyte is bound. In one version the pre-incubation chamber is supported above the second membrane in one position but can be pushed into contact with the membrane carrying the capture analyte thus permitting fluid transfer from the incubation chamber through the capture membrane. In another version the membrane at the base of the incubation chamber is hydrophobic and its underside contacts the capture membrane and when a wetting agent is applied to the contents of the pre-incubation chamber fluid transfer occurs.
The claims defining the invention are as follows:
1. A method for assaying for the presence of at least one reagent in a liquid sample of microbial extracts, said method comprising: a) providinga first porous membrane to which a plurality of capture analytes have been bound wherein each capture analyte of said plurality binds to a reagent in the sample, if present; b) placing a liquid sample of microbial extracts comprising particulate andnon-particulate material to be assayed and a multi-detection analyte in a chamber having a base defined by a second porous membrane through which membrane the particulate material cannot pass, wherein said multi-detection analyte comprises a plurality ofdetection compounds bound to a detectable element; c) allowing a sufficient period of time to pass for the plurality of detection compounds of the multi-detection analyte to bind to one or more reagents in the sample, if present, by virtue of anaffinity of a detection compound for a reagent, thereby forming a complex between a reagent and the multi-detection analyte; d) contacting the base of the chamber with the first porous membrane; e) causing non-particulate material in the microbialextract to flow through the first and second porous membranes, thereby separating and removing particulate material from non-particulate material by filtration through the second porous membrane and permitting non-particulate material to flow through tothe first porous membrane, under conditions sufficient for the complex at (c) to bind to a capture analyte on the first porous membrane, thereby forming a complex between the multi-detection analyte and at least one reagent in the sample and a captureanalyte; and f) detecting the detectable element in the complex formed at (e) wherein the presence of said detectable element indicates the presence of at least one reagent in the sample.
2. The method as claimed in claim 1 further comprising: removing the chamber and washing the first porous membrane with a buffer prior to detecting the detectable element.
3. The method as claimed in claim 2 further comprising: washing the first porous membrane with a buffer by addition of said buffer to the chamber before removing said chamber and detecting said detectable element.
4. The method as claimed in claim 1 further comprising removing or neutralizing unwanted analytes in the sample to be tested that may interfere with the binding of the reagent with the reagent on the first membrane in the chamber.
5. The method as claimed in claim 4, wherein the unwanted analytes are removed or neutralized by contacting the antibodies or other ligands that bind to said unwanted analytes with the unwanted analytes and wherein said antibodies or otherligands are bound to the walls or the base of the chamber.
6. The method as claimed in claim 1 further comprising swirling an absorbent swab having the sample to be tested absorbed thereto in an extraction solution in the chamber.
7. The method as claimed in claim 1 wherein the multi-detection analyte comprises a detection compound selected from the group consisting of an antibody, a ligand and an antigen.
8. The method as claimed in claim 7, wherein the antibody is an immunoglobulin G.
9. The method as claimed in claim 7 wherein the detection compound is an antibody.
10. The method as claimed in claim 7 wherein the detection compound is a ligand.
11. The method as claimed in claim 7 wherein the detection compound is an antigen.
12. The method as claimed in claim 1, wherein the multi-detection analyte comprises detection compounds consisting of two or more antibodies.
13. The method as claimed in claim 12 wherein at least one of the two or more antibodies is a control antibody.
14. The method as claimed in claim 1, wherein the multi-detection analyte comprises detection compounds consisting of three or more antibodies.
15. The method as claimed in claim 1, wherein the multi-detection analyte comprises a detectable element selected from the group consisting of colloidal gold particle, latex bead, coloured dye and colloidal carbon.
16. The method as claimed in claim 15, wherein the detectable element is a colloidal gold particle.
17. The method as claimed in claim 15, wherein the detectable element is a latex bead linked to a coloured dye.
18. The method as claimed in claim 15, wherein the detectable element is colloidal carbon.
19. The method as claimed in claim 1, wherein the multi-detection analyte consists of two or more antibodies conjugated to a colloidal gold particle.
20. The method as claimed in claim 1 wherein the second membrane is a hydrophobic membrane.
This application is the U.S. National Stage of International Application No. PCT/AU02/001684, filed 12 Dec. 2002, published in English, and claims priority under 35 U.S.C. .sctn.119 or 365 to Australian Application No. 2002950212, filed 11Jul. 2002 and Australian Application No. PR9451, filed 12 Dec. 2001.
This invention relates to a diagnostic testing process and in particular to an apparatus for use in carrying out an assay process and to a method of carrying out an assay process using that apparatus.
1. Background Art
Lateral flow and flow-through technology have been used for diagnostic assays for almost twenty years. Lateral flow technology is currently dominant because lateral flow devices are easy to produce and the assay can be performed in a simple2-step process that can be adapted for whole blood separation. This results in a simple device that can be used in the field as a rapid point-of-care diagnostic (Cole et al 1996 Tuberc. Lung. Dis. 77:363-368). However, multiple disease diagnosisusing lateral flow technology is very difficult because of differences in lateral diffusion between samples and variation in flow rates between batches of the partitioning membrane. This means that antigen or antibody signal strengths may vary bothwithin tests and between batches of tests, resulting in inconsistent results.
Existing flow-through diagnostic tests can be completed in less than two minutes compared with typical times of five to fifteen minutes for lateral flow tests. This advantage in speed however, is often at the expense of sensitivity. A furtherdisadvantage is that higher volumes of sample are required to achieve the same sensitivity as lateral flow. This may be problematic in some situations. For example, the diagnosis of analytes (reagents) in whole blood requires the separation of plasmafrom whole blood cells. The higher volumes of whole blood required for this would quickly block the membranes in the flow-through format.
The basic principal of flow-through assays is well established. The tests are designed to determine the existence of, and in some cases, the quantity of, a predetermined analyte/reagent in a sample. Often the reagent will be a protein but otherreagents can be tested for. If the assay is to test for the existence of a particular disease in a patient, the patient's body fluids may be tested for an antibody or other protein produced by the patient in response to the infection, or for a proteinwhich is expressed by the bacterium or viral agent or the like causing the disease. In a typical flow through assay a liquid sample which is believed to contain the reagent is sucked into an absorbent pad via a membrane to which is bound a captureanalyte which is known to bind to the reagent. The membrane is then typically washed with a buffer and a liquid containing a detection analyte which also binds to the reagent and which includes a tracer or marker which is detectable, is applied to themembrane. The detection analyte binds to the immobilised reagent bound to the membrane and can be seen or otherwise detected to indicate the presence of the reagent.
U.S. Pat. No. 4,246,339 discloses a test device for assaying liquid samples for the presence of a predetermined reagent. The device includes telescoping top and bottom members defining a liquid reservoir therebetween and resilient means forbiasing the members in the open position. The top member defines a series of test wells each of which has a base defined by a microporous membrane with a capture analyte immobilised on the membrane surface. Absorbent means are located in the bottommember, spaced from the membrane in the open position but in contact therewith in the closed position. U.S. Pat. No. 4,246,339 discloses adding the test serum diluted with a buffer to a test well, and incubating the device at room temperature for tenminutes prior to depressing the cassette to the closed position to pass the sample through the membranes into the absorbent material, When the membranes are dry, the membrane is washed and then covered with a solution containing a detection analyte whichbinds to the immobilised reagent followed by a subsequent step in which a stain is applied.
It will be appreciated that the process described in U.S. Pat. No. 4,246,339, is a somewhat long drawn out, time consuming and tedious process and also lacks sensitivity.
A more recent flow through device is described in U.S. Pat. No. 5,185,127 which discloses an assay device including a filter stack and an enclosure having a base portion and a lid. The filter stack has a hydrophilic membrane having a captureanalyte thereon, referred to in U.S. Pat. No. 5,185,127 as a binder. A hydrophobic membrane is located under the hydrophilic membrane and a pad of absorbent material is located under the hydrophobic membrane. The lid includes an upwardly extendingrib which defines a recess having an insert therein. In use, a sample containing the reagent (referred to in U.S. Pat. No. 5,185,127 as the analyte) is placed in the well of the assay device at which time the reagent/analyte binds to the captureanalyte/binder. Flow of the assay solution however, does not take place because the aqueous solution does not wet the hydrophobic membrane placed under the hydrophilic membrane in the filter stack. Thus as much time is necessary to complete the bindingof the detection analyte to the reagent is allowed. When binding is judged to be complete, flow may be initiated by adding a wetting agent which wets the hydrophobic membrane. After which time the aqueous liquid flows into pad of absorbent material. The membrane may then be washed and treated with a detection analyte/tracer which may be an antibody which specifically binds to the analyte, the antibody having a label covertly conjugated thereto. Again the sensitivity of U.S. Pat. No. 5,185,127 islacking and is not equivalent to that obtainable in lateral flow or ELISA formats.
2. General Information
As used herein the terms "derived from" or "derivative" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recitedintegers, steps or elements.
The embodiments of the invention described herein with respect to any single embodiment and, in particular, with respect to an apparatus or a method of assaying shall be taken to apply mutatis mutandis to any other embodiment of the inventiondescribed herein.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps orelements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations andmodifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific examples described herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of immunocytochemistry such as for example immunogold labeling and proteomics. Such procedures are described, forexample, in the following texts that are incorporated by reference: Colloidal Gold--A New Perspective For Cytochemical Marking, Beesley J (1989), Royal Microscopical Society Handbook No 17. Oxford Science Publications. Oxford University Press. (Paperback); An Introduction To Immunocytochemistry Current techniques and problems, Polak J and Van Noorden 5 (1984) Royal Microscopical Society Handbook No 11. Oxford Science Publications. Oxford University Press. (Paperback);Immunocytochemistry--Modern Methods and Applications, Polak J and Van Noorden 5 (1986) (2nd ed), Butterworth Heinemann Oxford. (Hardback); Techniques in Immunocytochemistry, Bullock G and Petrusz P (1982-1989) (4 volumes) Academic Press. Paperback);and Colloidal Gold--Principles, Methods and Applications, Hayat M, (1989-1990) (3 volumes), Academic Press. (Hardback).
All the references cited in this application are specifically incorporated by reference herein.
Because the prior art is not consistent in its terminology, for the avoidance of doubt and for the purpose of clarity, the following terms used in the specification below, are defined as follows. The term "reagent" or "target analyte" is used torefer to a macromolecule (eg. protein or enzyme) or fragment thereof, or the like which is to be detected by an assay. The term "capture analyte" is used to refer to a "capture" compound which is bound to a membrane and to which the reagent will bind. The term "detection analyte" is used to refer to an analyte which comprises a "detection" compound which will also bind to the reagent and also a "detectable" element. The detectable element is typically visually detected whether under visible light, orfluorescence.
In a first broad aspect, the present invention provides an apparatus and method for use in an assay process which is characterised by providing a "pre-incubation step" in which a reagent and detection analyte may bind together, which has theeffect of both improving the sensitivity of the assay and reducing the volume of sample required for an assay prior to reaction of the sample/analyte complex with a reaction membrane to which one or more ligands are bound.
In one embodiment, the detection analyte is a multi-detection analyte comprising more than one detection compound bound to a detectable element.
Thus, in one aspect of the present invention there is provided an apparatus for use in an assay process comprising: a first member comprising a first, porous, reaction membrane to which is bound a capture analyte for binding to a reagent to bedetected, the member having an upper surface and a lower surface; a second member being a body of absorbent material such as tissue paper or the like disposed below and touching the lower surface of the first member; a chamber spaced above the firstmember said chamber having side walls, and a base defined by a second membrane; and means for supporting the chamber above the first member in two positions, a first position in which the membrane is spaced a sufficient distance from the first member soas to not permit fluid transfer from the chamber to the body of absorbent material, and a second position in which the membrane is in contact with the first member thus permitting fluid transfer from the chamber through the first and second membranes tothe body of absorbent material.
In a related aspect the present invention provides a method for assaying for the presence of a pre-determined reagent using an apparatus of the present invention comprising the steps of: a) placing a sample to be assayed and a detection analytein the chamber, with the chamber disposed in the first position; b) allowing a sufficient period of time to pass for the detection analyte to bind to the reagent, if present; c) depressing the chamber to the second position to contact the base of thechamber with the first porous membrane; d) allowing the sample to flow through the first and second membranes to allow the reagent, if present to bind to the capture analyte carried on the first membrane, preferably wherein the detection analyte is amulti-detection analyte.
As used herein "multi-detection analyte" refers to a detection analyte which comprises more than one detection compound bound to a detectable element.
The inventors have surprisingly found that a multi-detection analyte has a number of advantages according to the present invention including, any of the following, for example, multiple-detection compounds can be bound to detectable elements atoptimum binding efficiency, detection analytes produced with multiple detection compounds have a similar size and physical characteristics for each of the detection compounds, the ratio of detection compounds on the detectable element can be alteredwithout affecting relative binding profiles of the detectable element, quantative comparisons can be performed, and the detectable element has a reduced capacity for non-specific binding.
Accordingly, in a preferred embodiment the invention provides a method for assaying for the presence of at least one pre-determined reagent comprising the steps of: a) providing a first porous membrane to which capture analytes for binding to theat least one reagent have been bound; b) placing a sample to be assayed and a multi-detection analyte in a chamber having a base defined by a second porous membrane; c) allowing a sufficient period of time to pass for the multi-detection analyte to bindto the at least one reagent, if present; d) contacting the base of the chamber with the first porous membrane; and e) causing the sample to flow through the membranes to allow the reagent to bind to the capture analyte carried on the first membrane.
Preferably, the detection compound is an antigen, antibody, or ligand.
Antibodies, antigens and ligands can be used as detection compounds as they have binding sites capable of specifically binding to the proteins of interest or fragments or epitopes thereof in preference to other molecules.
Antibodies are obtainable from a commercial source, or alternatively, produced by conventional means. Commercial sources will be well known to those skilled in the art.
As used herein the term "specifically bind" with reference to a complex-forming agent such as an antibody, refers to the agent preferentially associating with a target analyte (eg. protein of interest). With respect to antibodies in particular,it is recognised that a certain degree of non-specific interaction may occur between a molecule and a non-target protein. Nevertheless, specific binding may be distinguished as mediated through specific recognition of the antigen.
In alternate embodiments, the detection compound (antibody or ligand) is linked to an element to detect target analyte in a sample by directly or indirectly labelling the antibody or ligand, e.g with radioactive, fluorescent or enzyme labels,such as horseradish peroxidase) so that they can be detected using techniques well known in the art. Directly labelled analytes have a label (detectable element) associated with or coupled to the detection compound. Indirectly labelled detectionelements may be capable of binding to a labelled species (eg. a labelled antibody capable of binding to the developing agent) or may act on a further species to produce a detectable result.
Detectable elements can be conjugated to antibodies (or other ligands) and include macromolecular colloidal particles (e.g. colloidal gold particles) or particulate material such as latex beads that are coloured, magnetic or paramagnetic, andbiologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or changecolours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entitiesused in conjunction with biosensors. In one embodiment, the detection compound (antibody) is conjugated to a colloidal gold particle. Binding of the conjugated antibody to the sample protein produces a visual signal that can be detected by eye or readelectronically to give a quantitative result. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may also be employed.
Other labels are known to those skilled in the art.
Preferably, the detection element is selected from the group comprising colloidal gold particle, latex bead, coloured dye and colloidal carbon.
In one embodiment, the detectable element is colloidal gold. Preferably the colloidal gold provides uniform reflectance properties.
In an alternate embodiment, the detectable element is a latex bead linked to a coloured dye.
In another embodiment, the detectable element is colloidal carbon.
Preferably, a positive result is where the signal from a "test" sample in the assay is significantly higher or lower than a sample from a control sample.
Thus, the present invention provides a chamber which may serve as a pre-incubation chamber in which a pre-incubation step can occur where the sample and multi-detection analyte combine, which improves the sensitivity of the test and reduces thevolume of sample required for the assay. It has been found that the pre-incubation step increases the test sensitivity for a typical existing flow-through apparatus by approximately ten times to equivalent levels of sensitivity compared with lateralflow technology, while still allowing the assay to be completed in around two minutes compared to 10 minutes for lateral flow formats.
In one embodiment the sample is selected from or is derived from the group comprising:
agricultural product, microbial product, and biological product.
In one embodiment, the agricultural product is selected from the group comprising a seed, grain or plant extract.
In one embodiment, the sample contains particulate materials selected from the group comprising grain extract, cell extract and microbial extract.
In an alternate embodiment, the biological sample is a bodily fluid or tissue sample selected from the group comprising: blood, serum, sputum, and lung.
Other samples are not excluded.
Standard methods can be used to obtain, prepare and/or store samples for use in the present invention. For example, in one embodiment an adsorbent swab comprising a cotton matrix or similar material can be used to probe or surface or medium thatmay contain a target analyte. In one embodiment, the adsorbent swab is washed to remove the target analyte. Preferably, the target analyte and/or the particulate material comprising the target analyte is then solubilised.
Accordingly, in one embodiment a ground wheat head suspension can be solubilised, and then mixed and pre-incubated in the chamber with a multi-detection analyte comprising a primary antibody against alpha-amylase linked to a colloidal goldparticle and a control secondary antibody also linked to the colloidal gold particle. The contents of the chamber are then allowed to flow through to the first membrane containing a capture analyte in the form of an immobilised anti-amylase antibody, oranti-control antibody and antibody/gold complexes will bind to the immobilised antibody forming a detectable signal. The signal can be detected by the removal of the pre-incubation unit and washing of the reaction membrane with buffer.
In another embodiment the invention can also be used for detecting reagents in whole blood since whole red blood cells can be removed in the pre-incubation chamber and the plasma allowed to flow-through to the reaction membrane containing a boundcapture analyte. In this format, the base membrane defined at the base of the pre-incubation chamber will typically be a membrane which has the correct pore size to retain the red blood cells and allow the plasma to pass through on contact with thefirst membrane. Similarly particulate samples containing grain extracts, cell or microbial extracts can be analysed with this flow-through format since particulate matter can be removed in the pre-incubation chamber and therefore cannot block thereaction area on the upper surface of the reaction membrane.
The apparatus can also be used for detecting analytes in body fluids other than blood, such as plasma, sera, urine, saliva and sputum. In this case, the sample can be retained in the pre-incubation chamber by use of a hydrophobic membrane. Toobtain efficient flow through capillary action to the second member when the pre-incubation chamber is lowered, the reaction membrane is pre-wet with a wetting agent containing a detergent or the reaction membrane is blocked with a hygroscopic solutionsuch as sucrose, trehalose, fructose, or alternatively, glycerol.
This changes the characteristics of the reaction membrane from a non-hygroscopic to a hygroscopic membrane allowing the sample to flow through to the second member upon contact of the membrane at the base of the pre-incubation chamber with thereaction membrane.
In a yet further embodiment, if a hydrophobic membrane is used as the base of the pre-incubation chamber, the apparatus may be used with the hydrophobic membrane and reaction membrane in contact, with the operator adding a wetting agent to thesample to cause flowthrough, when desired.
Thus, in a related aspect, there is provided an apparatus for use in an assay process comprising a housing including: a first member comprising a first, porous, membrane to which is bound a capture analyte for binding to a reagent to be detected,the member having an upper surface and a lower surface; a second member being a body of absorbent material such as tissue paper or the like disposed below and touching the lower surface of the first member; a chamber located above the first member saidchamber having side walls, and a base including a second, hydrophobic, membrane, having an upper and a lower surface, the pre-incubation chamber being supported above the first member with the lower surface of the hydrophobic membrane in contact with theupper surface of the first member.
The pre-incubation chamber can also be used to remove analytes that may interfere with the assay, such as human anti-mouse antibodies (HAMAS), in solution or by binding anti-analyte antibodies to the surface of the chamber. The chamber can alsobe used to extract the analyte of interest from an absorbent surface such as a swab, which has been taken from the throat of a patient, by swirling the swab in an extraction solution in the chamber. The pre-incubation chamber may be part of a pre-filterunit which acts also to pre-filter the sample prior to contact with the upper surface of the first member.
Examples of assays that can be performed by this method where two reaction steps are involved (the incubation of the analyte with the labeled anti-analyte followed by the binding of this complex to a solid-phase anti-analyte), are: Direct antigenassay 1. Ag*(analyte)+Ab*.sub.1(anti-Ag)-label 2. Solid phase-Ab.sub.2(anti-Ag)+Ag/Ab.sub.1(anti-Ag)-label complex Direct antibody assay (i) 1. Ab.sub.1(analyte=anti-Ag)+Ab.sub.2(anti-Ab.sub.1)-label 2. Solidphase-Ag+Ab.sub.1(anti-Ag)/Ab.sub.2(anti-Ab.sub.1)-label complex Direct antibody assay (ii) 1. Ab.sub.1(analyte=anti-Ag)+Ab.sub.2(anti-Ab.sub.1)-label 2. Solid-phase-Ab.sub.3(anti-Ag)/Ag+Ab.sub.1(anti-Ag)/Ab.sub.2(anti-Ab.sub.1- )-label complexIndirect antigen assay 1. Ag (analyte)+Ab.sub.1(anti-Ag)+Ab.sub.2(anti-Ab.sub.1)-label 2. Solid-phase-Ab.sub.3(anti-Ag)+Ag/Ab.sub.1(anti-Ag)/Ab.sub.2(anti-Ab.sub.1- )-label complex Indirect antibody assay (i) 1. Ab.sub.1(analyte=anti-Ag)+Ab.sub.2(anti-Ab.sub.1)+Ab.sub.3(anti-Ab.sub.2)- -label 2. Solid phase Ag+Ab.sub.1(anti-Ag)/Ab.sub.2(anti-Ab.sub.1)/Ab.sub.3(anti-Ab.sub.2)-labe- l complex Indirect antibody assay (ii) 1. Ab.sub.1(analyte=anti-Ag)+Ab.sub.2(anti-Ab.sub.1)+Ab.sub.3(anti-Ab.sub.2)- -label 2. Solid phase Ab.sub.4(anti-Ag)/Ag+Ab.sub.1(anti-Ag)/Ab.sub.2(anti-Ab.sub.1)/Ab.sub.3(a- nti-Ab.sub.2)-label complex *Ag indicates antigen *Ab indicates antibody
Alternate types of assays are not excluded.
A piezoelectric driven printer may be used to dispense precise amounts of multiple disease ligands such as antigens or antibodies or an analyte as a micro array onto a reaction membrane for use in the apparatus of the first aspect of the presentinvention. The ligands or analytes may be dispensed in particular patterns, e.g. letters for ease of recognition of results. Typically, 100 pl of fluid reagent (1 drop), or multiples thereof, is dispensed, but this will vary depending on theapplication. The resultant size of the spot on the membrane is about 55 microns or more in diameter subject to fluid diffusion on the membrane, but again this will vary depending on the application. It is possible to dispense droplets with diameters of5-10 microns, and hence lower volumes of fluid reagent (for example, 1-10 pl) can be applied. Using precise quantitative printing of micro arrays of antibodies, antigens, or other analytes means that tests using precise quantities of these reagents canbe produced for multi disease diagnosis of a single sample. This array technology can be applied to tests for drugs or other markers across all diagnostic fields.
Alternatively, an adult/neonatal syringe pump 1235 from ATOM Medical Corporation, Japan, typically used to administrator small quantities of intravenous liquids through a catheter to hospital patients can be adapted to apply single or multiplelines of a capture analyte to the first membrane eg nitrocellulose.
In one preferred embodiment, ligands for detecting tuberculosis, HIV, hepatitis, syphilis and malaria antibodies may be deposited onto a reaction membrane. This would allow the simultaneous diagnosis of tuberculosis, HIV, hepatitis, syphilis andmalaria from a single blood sample without the need for intermediate sample treatment steps.
Utilising the present invention allows the assaying of small volumes of whole blood and thus the present invention provides a very rapid diagnostic assay device that is simple to use and can be used in both laboratory and point-of-care fielddiagnostic locations. For example, a finger prick of blood would be sufficient to perform an assay. Similarly large volumes of sample can be used in this device by increasing the amount of absorbent material (second member). For instance, 10 mls ofdilute fluids like urine can be can be assayed to detect low abundance molecules.
Analytes and/or ligands (e.g. antigens or antibodies) can be printed down in titrating amounts and/or concentrations. Thus, in an individual screen, this would provide a means of quantitating analyte-ligand levels within the sample solution.
A specific embodiment of the present invention will now be described by way of example only and with reference to the accompanying drawings in which:
FIG. 1 is a schematic drawing of an apparatus embodying aspects of the present invention in a first configuration;
FIG. 2 is a schematic drawing of the apparatus of FIG. 1 in a second configuration;
FIG. 3 is a perspective view of an assay apparatus or cassette embodying aspects of the present invention;
FIG. 4 is an exploded view of the components of the cassette shown in FIG. 3;
FIGS. 5a to 5d show various stages in the use of the apparatus of FIG. 3 in carrying out an assay; and
FIG. 6 is a graph comparing test results from samples spiked with alpha amalyse undergoing no-pre-incubation with samples undergoing a one minute pre-incubation.
Preparation of Detection Analyte
The entities bound to a gold colloid to produce the immunogold conjugate (normally immunoglobulins) are passively bound to give a stable complex that retains the target activity of the antibody. The native gold colloid dispersion as produced atProteome Systems Limited (PSL) is initiated with hydroxylamine reducing agent. Based on the assumption that the oxime is partially retained as a moiety in the colloid, the immunogold conjugate is stabilised by addition of glutaraldehyde in the bindingstep to form a Schiff's base between any residual oxime in the colloid structure and free amines from the immunoglobulin.
The optimum binding profile for the immunoglobulin (concentration and pH) is obtained by titration of the antibody concentration against fixed aliquots of gold colloid at specific pH values and treatment of the mixtures with saline solution. Asufficient concentration ratio for binding between colloid and antibody prevents flocculation or aggregation of the colloid from suspension when treated with the saline solution. Hence at low protein concentrations the addition of the saline results inthe colloid aggregating out of suspension, but when the optimum level of antibody is reached the colloid remains stable in suspension (the integrity of the colloid suspension is measured spectroscopically between wavelengths of 450 nm and 600 nm). Thisvalue of antibody concentration gives the minimum protecting concentration required to form the stable immunogold conjugate complex. An antibody concentration of 0.1 to 0.2 mg/mL in 5 mM borate is used in the titration and for polyclonal antibodies a pHof 9 is chosen: this pH is normally is well above the range of isoelectric point (pI) values encountered with the range of immunoglobulins in a polyclonal serum. In the case of monoclonal antibodies from a hybridoma the immunoglobulins have a uniqueisoelectric point and the pH of the colloid is usually set at 0.5 to 1 unit above the pI of the antibody.
The concentration of antibody used for the conjugation is 110% of the minimum `protecting` concentration determined from the titration procedure described herein.
Conjugation Protocol (for Single Antibody)
The required volume of colloid is measured out (assuming a 90% yield the volume is nearly equivalent to that of the final conjugate at the optical density measured for the colloid).
A volume of 5% glutaraldehyde solution is added to the rapidly-stirred colloid to give a final glutaraldehyde concentration of 0.002%.
Five minutes after addition of the glutaraldehyde the calculated amount of antibody solution is added to the rapidly-stirred suspension. If a significant volume of antibody is to be added (>10 mL) it is added in a steady stream of drops(preferably through a dropping funnel).
After 30 to 90 minutes depending upon the volume of colloid (from 200 mL to 5 L) the pH of the suspension is taken down from pH 9 to pH 7 (unless the conjugation is carried out between pH 7.5 to 6) with 0.2M phosphoric acid. A calculated volumeof 10% (w/v) bovine serum albumin is added to the suspension to give a final concentration of bovine serum albumin in the suspension of 0.5%.
The suspension is left stirring for 3 to 4 hours or overnight when the volume is >1 L and then centrifuged at 10,000 rpm for between 35 minutes and 60 minutes (depending upon volume and colloid size).
The supernatant is removed from the centrifuged suspension and the concentrated liquid centrifugate taken up in 2 mM borate at pH 7.2 containing 0.2% bovine serum albumin and 0.1% sodium azide (as preservative).
Conjugation Protocol (for Multiple Antibodies)
For more than one antibody bound to the colloid, the protecting concentration titrations (probed with saline for aggregation) are carried out for each of the antibodies. The volumes of antibodies used are those found for the individualantibodies (+10%) from the titrations.
If equivalent binding levels of the antibodies at their individual optimum binding levels are required, the antibodies are pre-mixed and added together to the colloid (after glutaraldehyde addition). The processing follows the procedure thendescribed above for single conjugations. If the antibodies are required to be at a particular ratio to each the primary antibody is added first and the secondary antibodies added at intervals later. The time course of the binding levels to the colloidhave been determined for some combinations of antibodies and the levels of the second and subsequent antibodies decrease in a regular pattern with the time interval between addition of the first antibody and subsequent additions. After 30 to 40 minutesinterval a constant level of the second antibody bound at approximately 10% of the primary reactant level is found. In some cases an interval of 5 minutes between test antibody and procedural control antibody at the required concentration ratios isfound to be optimal.
In one example, the primary antibody is mouse anti .alpha. amylase conjugated at pH 8.3, and the secondary antibody (control) is goat immunoglobulin G. In another example, two primary antibodies (eg. anti-Human IgG1 and anti-Human IgG2) can beadded simultaneously and a control antibody added at a suitable interval afterwards.
Preparation of Membrane with Capture Analyte
Capture analytes in the form of ligands such as antigens or antibodies (e.g. TB, HIV-1) are printed onto a protein-capture membrane matrix (e.g. a nitrocellulose membrane) in an appropriately sized array using piezoelectric chemical printingtechnology. A suitable chemical printing system for use in the present invention involves the use of piezoelectric drop-on-demand ink jet printing technology for micro-dispensing fluids in DNA diagnostics or the Combion Inc synthesis process called"CHEM-JET". To explore drop on demand fluid dispensing for DNA diagnostics, an eight fluid printer has been developed as part of the Genosensor Technology Development (GTD) project funded by the Institute of Standards and Technology (USA). Research todate, is focused on printing oligonucleotide micro-spots onto solid supports. In the CHEM-JET technique, which was developed at the California Institute of Technology, tiny volumes of reagent bearing liquid are squirted onto specific spots or addressesof a solid substrate much as an ink-jet printer squirts ink onto a page. By repeatedly returning to each address with one or another of a small set of building blocks, in this case, nucleotides modified for the process, huge two-dimensional libraries ofshort DNA chains (oligonucleotides) can be assembled. Such a device including an imaging means is described in the applicant's co-pending International patent application No. PCT/AU98/00265, the entire contents of which are incorporated herein byreference. In the described embodiment, antigen is printed onto a reaction membrane in 100 pl droplets, or multiples thereof (eg. 10 nl), with each aliquot being 1 mm apart. However, these volumes and distances can be increased/decreased accordinglydepending on the chosen antigen titre and array size. For example, it is possible to dispense droplets with volumes as low as 1-10 pl.
In a particularly preferred embodiment, antigens or antibodies can be printed down in a matrix of dots or lines or in the shape of letters so that quantitative multiple analyte analysis of a single sample is possible.
After the dispensed antigen has dried, non-specific protein-binding sites on the (nitrocellulose) membrane are blocked using 0.5% (v/v) casein in phosphate buffered saline (PBS)+0.05% (w/v) sodium azide+0.1% (v/v) Tween-20 (PBSA wash buffer). Itis however an option to leave the membrane unblocked following the printing of the antigen (or antibody) or other ligand.
In another preferred embodiment syringe pump technology used for the administration of liquids intravenously to patients can be adapted to lay down single or multiple lines on nitrocellulose membranes.
Use of Immunoconjugates in Flow-Through Format
The optical density of the immunoconjugate suspension is usually measured at a wavelength of 520 nm. This value gives a relative measure of the concentration of the colloid. Also from knowledge of the conjugation conditions the level ofantibody bound to the colloid at an optical density=1 is known and this can be used to determine the optimum volume to use in the flow-through test (antibody concentration per mL of colloid.times.volume of conjugate.times.optical density of conjugate).
The immunoconjugate is added to the sample that is pre-incubated in the filter assembly prior to contact with the active membrane in the housing. This enables the maximum level of binding of the analyte in the sample to bind to the activeantibody conjugated to the colloid. The conjugate: analyte complex is then captured by the immobilised test antibody on the membrane as the mixture filters through the active membrane exposed to the sample fluid at the same time as the proceduralcontrol antigen binds to the secondary antibody linked to the conjugate.
For multi-analyte samples the multiple antibodies on the conjugate (detectable element) would bind at the same level to the sample analytes as the conjugated antibodies would be at their optimum binding levels on the colloid particle.
Description of the Figures
Turning to the drawings, FIG. 1 shows a flow-through assay device 10, which utilises the nitrocellulose membrane described above. The device is in the form of a cassette 12 and an associated removable filter frame 14. Inside the cassette thereis the membrane (typically nitrocellulose) 16 on which capture analytes in the form of ligands are printed, as described above, which is located on top of an absorbent matrix 18. The absorbent matrix preferably comprises multiple layers of absorbenttissue or an absorbent pad such as blotting paper, in the specific embodiment twenty-four layers (double ply), which have been found to possess an ideal porosity that permits the most rapid flow-through of various solutions. This rapid flow-through isimportant as it results in lower backgrounds with higher reaction specificity and higher signal resolution.
As shown in FIG. 1 the top of the cassette defines an opening in its upper face and a depending generally frusto-conical well whose sides depend down as far as the membrane 16, to define a chamber having sloping sides and a base defined by themembrane 16.
The filter unit frame 14 is spaced above the upper surface of the cassette 12. It also defines a depending conical well in the form of a chamber 21 also referred to as a "pre-incubation chamber" having sloping sides and a base 22 formed from a 5.mu.m Whatman grade 1 membrane or a 0.22 .mu.m hydrophilic Durapore membrane filter (Millipore, North Ryde, Australia). However, other types of filter/membrane and pore size would be suitable depending on the application. The function of the membraneis to retain a sample to be assayed in the well or pre-incubation chamber 21 long enough for a "pre-incubation step" to take place. When membrane 22 is lowered to contact the membrane 16, capillary attraction draws the sample from the chamber 20 throughmembranes 22 and 16 and into the tissue 18.
For ease of use, two pins 24 are provided which support the filter frame 14 at an appropriate distance above the cassette 12 during the pre-incubation step but which allow the filter frame to be pushed down so that the membranes 22 and 16 are incontact for the second stage of the process shown in FIG. 2. The frame 14 is also removable so that the membrane 16 can be viewed to determine the results of the assay.
FIGS. 3 to 5d illustrate one commercial assay device design embodying the aspects of FIGS. 1 and 2.
In those Figures, the components which are equivalent to components shown in FIGS. 1 and 2 carry the same reference numerals. The cassette 12 comprises an upper moulding 12a and a lower moulding 12b. The porous membrane 22 is defined by thebase of a pressed filter paper frustro cone 22a held in place by a filter retainer 23. The filter unit frame 14 defines two dimples 14a on which an operator's thumbs may press when depressing the filter frame to contact the membranes 22 and 16.
FIGS. 5a to 5d illustrate the stages of operation of the apparatus. FIG. 5a illustrates the filter frame separate from the cassette 12. FIG. 5b illustrates the pre-incubation positioned with the base of the chamber/well 21 spaced from membrane16. FIGS. 5c and 5d illustrate the device after the filter unit has been pressed down to bring the membranes 22 and 16 into contact to allow the sample to flow through to the blotting paper 18.
If the membrane 22 is replaced with a hydrophobic membrane, it is possible to operate the device with a pre-incubation step solely in the position shown in FIGS. 3 and 4 with the membranes 22 and 16 always in contact. The hydrophobic membrane 22will prevent flow of the sample in the incubation chamber 21 to the reaction membrane 16. After a sufficient period of time has past for detection analyte in the chamber 21 to bind to the reagent, a suitable wetting agent is added to the sample in thechamber which allows the sample to flow through the hydrophobic membrane past the reaction membrane 16 and into an absorbent matrix 20.
Application of the Pre-Filter Chamber
Whatman membrane (paper) or Reemay filters (polyester; 1 cm.sup.2) are inserted into the chamber 21 in the filter frame to form a conical retaining vessel (pre-filter unit).
The sample is pipetted into the plastic pre-filter chamber (50-100 ul) along with a detection analyte in the form of a detecting antibody (50-100 ul) bound to colloidal gold (particle size 20-50 nm). The sample is pre-incubated with thegold-conjugate (O.D.4) within the pre-incubation chamber for thirty seconds after gentle pippetting to ensure adequate mixing. After thirty seconds the chamber is pressed into the well 20 of the test cassette 12. Upon contact with the membrane 16containing the detection zone, the solution filters through to the absorbent layer 18 beneath. The pre-filter 14 is discarded when the solution has filtered through and two drops of PBSA wash buffer are then added to the reaction membrane to wash awayexcess gold-conjugate revealing the results of the assay on membrane 16.
The use of the pre-incubation of the sample with the detection analyte increases sensitivity by approximately ten fold. Further, any particulate matter is retained in the pre-incubation chamber all of which can be removed to provide a clearsignal. The use of the preincubation chamber with the dual roles of permitting a pre-incubation step and a pre-filtering step, also allows multi-analyte detection on the reaction membrane by pre-incubating with a multi-analyte probe, e.g. colloidal goldbound to different detecting analytes. In addition, interfering analytes or substances that could cause false positives or negatives in the assay can be removed or absorbed out in the pre-incubation step, e.g. human antibodies to mouse antigens can beabsorbed out by anti-HAMA antibodies.
Although the above described example relates to the antigens relating to disease, the immunoassay apparatus could be used, for example, as an allergy test kit, as a test kit for drugs of abuse or for analysing non-human derived samples e.g.bovine, porcine, veterinary tests, and tests in agriculture such as grain quality evaluation, etc.
The method and apparatus of the present invention is particularly suited to use with swabs which can be simply placed into the chamber 21, swirled around in liquid containing a detecting antibody (50-100 ul) bound to colloidal gold for 30 secondsbefore the pre-filter unit is depressed to contact the membranes 22 and 16 together.
Any combination of ligands and analytes can be applied to the system of the present invention. The choice of ligands could be tailored to detect prevalent diseases in a particular country or population. For example, analytes from the followingcombination of diseases could be used for diagnosis using this array. 1. TB and HIV 2. Hepatitis-B & C, HIV 3. Chagas, HIV, TB, Syphilis and Hepatitis-B & C 4. Malaria, Dengue, TB, Chagas.
Alternatively antigens representing different varieties of wheat or other agricultural products could be printed on the reaction membrane enabling detection of multiple strains with a single test.
The assay device can also be used for detecting analytes in body fluids other than blood such as plasma, sera, urine, saliva and sputum. In this system the sample can be retained in the pre-incubation chamber 22 by use of a hydrophobic membranesuch as Reemay or Hollingsworth and Vose 7303 instead of the Whatman grade 1 membrane or a 0.22 .mu.m hydrophilic Durapore membrane filter described above. The sample is mixed with the detection analyte for the required pre-incubation period. To obtainefficient flow through capillary action to the absorbent layer 18 when the pre-incubation chamber 22 is lowered onto the cassette 12, one of two procedures can be followed: 1. The membrane 16 containing the capture analyte is pre-wet with at least onedrop of wash buffer containing 0.01 M phosphate, 0.15 M NaCl, 0.0% Azide, 0.5% Tween 20 or any wetting agent containing a detergent; 2. The membrane 16 containing the capture analyte is blocked with a hygroscopic solution such as sucrose, trehalose,fructose, or alternatively, glycerol. This changes the characteristics of the membrane 16 from a non-hygroscopic to a hygroscopic membrane allowing the sample to flow through to the absorbent layer 18 upon contact of the membrane at the base of thepre-incubation chamber 22 with membrane 16.
Comparison of no pre-incubation and 1 minute pre-incubation of a sample spiked with alpha amylase in the above described format.
A 6% solution of bovine sera albumin was spiked with 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml and 1000 ng/ml and applied to the above format according to the following procedure.
I. The pre-incubation chamber was pressed down so that the base of the chamber comes into contact with the first member containing the capture antibody against alpha amylase.
II. Sixty microliters of 0.5% tween in saline was added to the pre-incubation chamber and allowed to filter through to the absorbent material beneath the first membrane.
III. One hundred microliters of spiked alpha amylase sample was added to the chamber and allowed to filter through to the absorbent material beneath the first membrane.
IV. Sixty microliters of 0.5% tween in saline was added to the pre-incubation chamber and allowed to filter through to the absorbent material beneath the first membrane.
V. Sixty microliters of anti-alpha amylase antibody linked to colloidal gold (particle size 20-50 nm) was added to the pre-incubation chamber and allowed to filter through to the absorbent material beneath the first membrane.
VI. Sixty microliters of 0.5% tween in saline was added to the pre-incubation chamber and allowed to filter through to the absorbent material beneath the first membrane.
VII. The pre-incubation chamber was removed and the result on the reaction membrane scanned with a densitometer. Signal strength was measured in pixel intensity.
One Minute Pre-Incubation
I. Sixty microliters of 0.5% tween in saline was added to first membrane and allowed to filter through to the absorbent material underneath.
II. The pre-incubation chamber was suspended over the first membrane so that there was a space between the chamber and the membrane.
III. One hundred microliters of spiked alpha amylase sample and 60 microliters of anti-alpha amylase antibody linked to colloidal gold (particle size 20-50 nm) were incubated in the pre-incubation chamber for 1 minute.
IV. The chamber was lowered until it came in contact with the first membrane and the mixture of sample and antibody-gold conjugate allowed to filter through to the absorbent material.
V. Sixty microliters of 0.5% tween in saline was added to the pre-incubation chamber and allowed to filter through to the absorbent material.
VI. The pre-incubation chamber was removed and the result on the reaction membrane was scanned with a densitometer. Signal strength was measured in pixel intensity.
Each data point on the graph is the average of two experiments using the apparatus described above. The results show that pre-incubation of the sample with the detection analyte has a minimal detection limit defined in pixel density of around500 pg/ml of alpha amylase. This is compared to a minimum detection limit without the pre-incubation of about 50 ng/ml and indicates the pre-cubation increases the sensitive by around 10 fold.
Demonstration of increased sensitivity with increased pre-incubation of the sample with the detection analyte.
Samples of amylase diluted in 0.5% saline to 400 ng/mL were treated with immunogold conjugate against amylase and aliquotted onto the flow-through format in different protocols as shown below.
A. The sample was added to the format (without a filter present) and allowed to filter through prior to adding conjugate, followed by an aliquot of conjugate immediately the sample had passed through the membrane.
B. The sample was mixed in the correct proportions with gold conjugate and aliquotted immediately onto the flow-through format.
C. The sample was mixed as with protocol B but added to the flow through format after a 60 second interval.
The results presented in pixel intensity are shown in the tables below (for 2 experiments)
TABLE-US-00001 Sample Control Sample Control Protocol peak peak area area S/PC ratio A 82 286 657 2120 317 B 288 758 2062 5509 383 C 823 949 5843 6765 884 A 89 516 588 3890 588 B 482 830 3736 6345 602 C 708 829 4506 5822 792
Clearly there is a significant increase in the sample signal when the analyte is preincubated with the conjugate probe, as distinct to sequential detection on the flow-through format. The difference in detection levels (for the 400 ng/mL sample)equated to between a 7.5-fold to 10-fold increase in detectable amylase in the flow through format when the sample is preincubated separately to the detecting capture antibody.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
* * * * *
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text:
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text:
The top 20 games of 2017?
Subject: General Tech | January 3, 2018 - 02:15 PM | Jeremy Hellstrom
Tagged: gaming
Ars Technica have assembled an eclectic list of games that they consider the best from last year. Their picks include a variety of platforms, from the Switch to PC, with games ranging the full gamut. VR games are not well represented, interesting considering the number of new games which were released this year or ported, in the case of Skyrim and others. This could be due to the high price to be able to play, or it could represent the somewhat lacklustre uptake of VR gaming. Take a read through the list and then post the games that they completely ignored, that deserve top billing.
"While single-player adventures dominated our rankings this year, the full list encompasses everything from traditional shooters and throwback platformers to indie puzzles and narratives, plus a few VR experiences for good measure."
Here is some more Tech News from around the web:
- Max & Maya: Cat Simulator might not be the most authentic thing ever @ Rock, Paper, SHOTGUN
- 15-game 'Premium' VR Review – Pascal vs. Vega @ BabelTechReviews
- Life is Strange: Before the Storm review: The path to tragedy @ Ars Technica
- The Most Disappointing PC Games of 2017 @ Techspot
- Humble Staff Picks Bundle
- Scanner Sombre Review @ OCC
- PUBG Graphics Performance Revisited: 44 GPUs Tested @ Techspot
Destiny 2 was given an award for being one of the shittiest games of 2017
no jim sterling.
having Destiny 2 in that list of "top" 20 games automatically disqualifies this entire list
That list was awful IMO, but I guess everyone's taste are different. They're games on the list that were nowhere near one of the best games of the year and others that are higher than they should be.
for more best pc games you can check it........


text:
This great little 2-story, 3-bedroom, 1-bath cabin may be just what the doctor ordered! The 1st floor includes a cozy living/dining area with a gas fireplace, a VCR-DVD/monitor, a full kitchen, 1-full bath, and a laundry cubby with washer/dryer. The 2nd floor features one bedroom with 1-full bed, a 2nd bedroom with 1-full bed and a 3rd bedroom with 1-twin bed. Outside, there is a gazebo covered gas grill/picnic area, a private outdoor HOT TUB, and a deck leading to a walking trail down to Silver Lake! This pet-friendly vacation property will sleep up to 5-persons. (Enforced Maximum Occupancy!). HOT TUB/ PET FRIENDLY/ NON SMOKING/ NO WI-FI
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text:
Death Grips - The Powers That B [Hip Hop / Experimental]
Never thought I would kick off the reviews this year in April, but the team has been swamped with school work on other projects. Thought I would like to jot down some thoughts on the "final" Death Grips album:
niggas on the moon
The first half of the double album technically "leaked" last year, but I will review it here because (i)it's part of a double album and (ii) it has only been physically released this month. The album uses samples of Björk for background, with barely any actual drums from drum lord Zach Hill. The album isn't as aggressive as Death Grips' earlier work, and barely shows any semblance to the second half of The Powers That B. The eight songs flow together as one large song with barely any segue moments between the tracks. As far as the songs go, some manage to stick in your mind, eg. Up My Sleeves and Black Quarterback, but for the most part, the album plays more like background music. It isn't actually chill background music either because MC Ride's vocals are anything but chill. Björk's vocals are employed as trippy atmospherics, so the album has a bit of a trance like feel, wildly punctuated by manic shouts. Overall, not one the Grips' strongest albums, perhaps even their worst.
Jenny Death
The much better half of the album, almost every song on the album is gold. The soundscape has changed quite a bit with distorted guitars, live drums and more manic vocals (almost like punk rock). The lyrics are also absolutely quotable ("I like my iPod more than fucking!!"). Jenny Death is surprisingly not only accessible in terms of sound, it is also probably the first time I have been able to emotionally connect with the band. The album's final actual song is 'On GP', quite possibly my favourite Death Grips song of all time. It has elements of shoegaze and sticks out sorely from the rest of the album, almost coming off like a ballad. It's a grand swansong for the band, with its foreboding lyrics "It's been a pleasure, Stefan (the vocalist's real name)" or "All these nights I don't die for you". Zach Hill is also on fire on this album when he finally lets loose with his neo-jazz-metal assault on the drums. Seriously, check out his solo work, his drumming is on par with the best in the world.
The album ends on a rather bizarre note, with 'Death Grips 2.0'. Is their break up just a PR stunt? Are they hinting of another return? Or is their way of saying goodbye? It's a mostly instrumental, futuristic sounding piece that does break new ground for the artistes' direction, but only time will tell if we will ever get more from this talented duo.


text: The official edit from this years Winterclash has just gone live. Featuring all the action from Area 51 in Eindhoven has been brought to you through a team of videographers and brought together in the artistic vision of Benjamin Büttner. The result is an edit that fully sums up just what this event is to every single person who attended it and to the mass of people as a collective. Sit back, get comfortable and enjoy the biggest blade event of the year of which Valo international pro David Sizemore managed to take first place at. Im sure once you have taken it all in we shall see you there next year!


text:
Performance Details
Event 3200 Meter Run
Mark 10:16.01
Meet Friday Night Invite
Place 4th
Race/Results Page Fresh/Soph Results
Round Finals
Heat 1
Native Country USA
Season Outdoor 2017
Level HS
Meet Date Mar 3, 2017
Date Added Mar 5, 2017
Grade 2020
Timing FAT
Personal Best? No
Season Best? No
Venue San Jacinto High School, San Jacinto, CA, USA
No comments on this performance. Add a comment


text:
The NNT is used to assess the gain/risk equation of the therapy. In this particular instance, fifty nine% in the team are non-responders, and therefore they are actually subjected to the adverse results of neuroleptics with none advantage.
Nevertheless, Regardless that they did not present the longitudinal information, their overview from the brain-analysis continue to led them into an intellectual quagmire. How could it's the reduction in brain volumes noticed in psychotic patients was related fully into the disorder and by no means into the drugs, when the shrinkage has also showed up in animals addressed While using the prescription drugs?
In the next section in their study, Lieberman and colleagues concentrate on the "prolonged-expression results of antipsychotic medication on clinical course in schizophrenia.
That was the investigate which was posted within the early nineteen eighties, and the trendy longitudinal research, which observed better remission costs in unmedicated clients in excess of the long run, supply new evidence to support the summary that drug-induced dopamine supersensitivity raises the chance that somebody will become chronically ill (see Harrow, Moilanen, and Wils).
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As Sandy Steingard wrote in an MIA weblog various decades back, there isn't a transparent picture, from these scientific studies, of no matter whether shortening the time of psychosis right before treatment by having an antipsychotic produces a meaningful difference over extended periods of time.
I had been in clinic in Ireland for two months but I never ever completely recovered within the large duty drug battering of that time period.
From the early nineteen sixties, the NIMH executed a study of 229 acutely sick inpatients with schizophrenia who have been randomized to at least one of a few antipsychotics or to placebo. Fifty per cent have been 1st-episode people.
Of their evaluation, Lieberman and colleagues did not point out this. They present it as a protracted-expression study that compares medicated to unmedicated folks, when in actual fact it is a study that compares a pick out team of older Long-term people to some fewer sick team that, at some point of their life, have been hop over to these guys "taken care of.
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The psychiatrist functions particularly just like a Avenue dealer as well as point out, in place of repressing the dealer, forces the drug addict to consume his harmful.
"The critics who gave rise to this notion that antipsychotic cure adversely impacts long-term results were being sowing seeds of untruth and, in their pursuit of some ideological intention or need to have for self-serving acclamations, were being disregarding entirely the damage which they have been producing A lot of people who unwittingly would settle for these as credible statements and observe their assistance. As I movie this movie, the contents of The brand new posting are embargoed, so I are unable to reveal the contents just still.
Guild pursuits needed shrinks pose as Physicians from the beginning, as well as their attitude toward psychotics has always been disdainful and punitive–their "treatment options" have normally been destructive.
I remained in medical center for under two days after which you can I discharged my self. This hospitalization was classified like a "Pre Psychotic Episode".
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The NNT is used to assess the gain/risk equation of the therapy. In this particular instance, fifty nine% in the team are non-responders, and therefore they are actually subjected to the adverse results of neuroleptics with none advantage.


text:
Worldwide Epidemiology of Atrial Fibrillation: A Global Burden of Disease 2010 Study
Study Questions:
What is the global burden of atrial fibrillation (AF)?
The investigators systematically reviewed population-based studies of AF published between 1980 and 2010, from the 21 Global Burden of Disease (GBD) regions to estimate global/regional prevalence, incidence, as well as morbidity and mortality related to AF (DisModMR software).
Of 377 potential studies identified, 184 met prespecified eligibility criteria. The estimated number of individuals with AF globally in 2010 was 33.5 million (20.9 million males [uncertainty interval (UI), 19.5-22.2 million) and 12.6 million females [UI, 12.0-13.7 million]). Burden associated with AF, measured as disability adjusted life-years, increased by 18.8% (UI, 15.8-19.3) in males and 18.9% (UI, 15.8-23.5) in females, from 1990 to 2010. In 1990, the estimated age-adjusted prevalence rates of AF (per 100,000 population) were 569.5 in males (95% UI, 532.8-612.7) and 359.9 in females (UI, 334.7-392.6); the estimated age-adjusted incidence rates were 60.7/100,000 person-years in males (UI, 49.2-78.5) and 43.8 in females (UI, 35.9-55.0). In 2010, the prevalence rate increased to 596.2 (UI, 558.4-636.7) in males and 373.1 (UI, 347.9-402.2) in females; incidence rate increased to 77.5 (UI, 65.2-95.4) in males and 59.5 (UI, 49.9-74.9) in females. Mortality associated with AF was higher in females, and increased by two-fold (UI, 2.0-2.2) and 1.9-fold (UI, 1.8-2.0) in males and females, respectively, from 1990 to 2010.
The authors concluded that these findings provide evidence of progressive increases in overall burden, incidence, prevalence, and AF-associated mortality between 1990 and 2010.
This systematic review of the current worldwide epidemiological data on AF confirms the emergence of AF as a global epidemic, with significant and progressive effects on estimated disability and mortality. Furthermore, there were specific differences identified based on age and GBD region that are likely to have significant implications for global public health. The evidence for geographic variation in AF burden, especially in developing versus developed nations, suggests that systematic, global surveillance of AF is required to formulate effective region-specific prevention and treatment strategies.
Keywords: Uncertainty, Prevalence, Incidence, Morbidity, Developed Countries, Software, Cost of Illness, Quality-Adjusted Life Years
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...This is the story of Kyle (as told by his wife). Kyle is fighting Stage 4 Cholangiocarcinoma (terminal Bile Duct Cancer of the Liver)-He is a father, a husband, a friend, a son, a fighter and a hero. Join him on his journey.
Wednesday, December 3, 2014
Here's the scoop, for now.
has come our way since the devastating news (that we always
knew was coming--but still....).
We cannot begin to express enough thanks ever to everyone
who has loved us in big and little ways.
Seriously floored and grateful beyond words.
I loved watching Mark in his maid outfit steam clean our
and Uncle Dave bend over and give Kyle a kiss on the
and Amy listen to me swear and gnash my teeth.
and for treats that have shown up on our doorstep.
and for people hugging and loving my children.
and for dinners that have been brought in.
and for podiatrists that have made house calls.
and for so so so so so many acts of love and kindness.
and for social workers that let me text anytime
and cry into the phone.
and for everyone who is anyone at the Huntsman Institute
who has touched our lives.
and for people who say F cancer for us and with us.
I am so so grateful for a boss who went above and
beyond to make sure I could be home with my husband now.
I am so so grateful for co-workers in the "business
closet" that have truly been my friend and cared
about me and my life and my husband.
We are crushed and our hearts are filled with a sadness
so deep and heavy that it consumes us at all the worst
My kids are the hardest to watch.
Digesting this is a blow, a punch in the stomach.
I have a feeling that will be the case for a long time
to come.
Some good news is that I had a boss that worked and
worked until she could find a way to let me have
time off with my husband (since I did NOT qualify for
FMLA until December 23)
Long story short is that *I* can take a leave of absence
for *MYSELF* and be home to take care of Kyle.
That starts on Monday after I finish up some lists
and things to do at work.
We know that there are many people who want to visit,
and as I said on Facebook, without being rude, there
need to be parameters for Kyle at this point in time.
Kyle is tired and visiting makes him even more
tired. It is emotionally and physically taxing on him.
That is the honest truth.
Wise words from people who have been through this before
are as follows:
**Please keep visits short (1/2 hour or less)
That's what he has in him for now.
**Please call or text ahead of time and make sure
he's up for it. (If you need a cell number message
one of us on Facebook) This varies from day to
day and hour to hour honestly.
**He is home during the day and also available then.
Again text beforehand to see how he is and if
he's up to it.
**Nighttime is when his pain seems to come on strong
and visits after about 8 PM are too late (I know
we are worse than 90 year old people).
**He would love a text or email as well if there's
something you want to share with him, or want him
to know, or want to say.
**This is also a sacred time for OUR family.
Please be respectful of that time. (No one has
been disrespectful so far in case anyone is worried!)
Thanks again for an outpouring of love and acts of
kindness. I don't even know who to thank for half
of them.
This is our update for today.
2 comments:
Thinking of you all.ReplyDelete
All my thoughts and prayers are with you and your family, Dorien. I'm sorry for what you are facing. As someone who has been there with my own father, I can both sympathize and empathize with what you and your kids are facing right now. Hold him close, savour every moment, and know you have people around the world praying for you and sending strength, even if they aren't commenting. If the kids ever need someone to vent to who understands, I'm here. I may be a complete stranger, but I'm a stranger who understands all to well what they are feeling.ReplyDelete
Oh, and a huge F CANCER coming your way from Calgary, Canada.
Sending strength,
Jen Sadler


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Kamis, 13 September 2012
The History of France
Stone tools indicate that early man was present in France at least 1.57 million years ago. The first modern humans appeared in the area 40,000 years ago. The first written records for the History of France appear in the Iron Age. What is now France made up the bulk of the region known to the Romans as Gaul. Roman writers noted the presence of three main ethno-linguistic groups in the area: the Gauls, the Aquitani, and the Belgae. The Gauls, the largest and best attested group, were a Celtic people speaking what is known as the Gaulish language.
Over the course of the first millennium BC the Greeks, Romans, and Carthaginians established colonies on the Mediterranean coast and the offshore islands. The Roman Republic annexed southern Gaul as the province of Gallia Narbonensis in the late 2nd century BC, and Roman forces under Julius Caesar conquered the rest of Gaul in the Gallic Wars of 58–51 BC. Afterwards a Gallo-Roman culture emerged and Gaul was increasingly integrated into the Roman Empire.
In the later stages of the Roman Empire, Gaul was subject to barbarian raids and migration, most importantly by the Germanic Franks. The Frankish king Clovis I united most of Gaul under his rule in the late 5th century, setting the stage for Frankish dominance in the region for hundreds of years. Frankish power reached its fullest extent under Charlemagne. The medieval Kingdom of France emerged out of the western part of Charlemagne's Carolingian Empire, known as West Francia, and achieved increasing prominence under the rule of the House of Capet, founded by Hugh Capet in 987.
A succession crisis following the death of the last Capetian monarch in 1337 led to the series of conflicts known as the Hundred Years' War between the House of Valois and the House of Plantagenet. The wars ended with a Valois victory in 1453, solidifying the power of the Ancien Régime as a highly centralized absolute monarchy. During the next centuries, France experienced the Renaissance and the Protestant Reformation, as well as recurring religious conflicts and wars with other powers. A burgeoning worldwide colonial empire was established from the 16th century.
In the late 18th century the monarchy and associated institutions were overthrown in the French Revolution, which forever changed French and world history. The country was governed for a period as a Republic, until the French Empire was declared by Napoleon Bonaparte. Following Napoleon's defeat in the Napoleonic Wars France went through several further regime changes, being ruled as a monarchy, then briefly as a Second Republic, and then as a Second Empire, until a more lasting French Third Republic was established in 1870.
France was one of the Triple Entente powers in World War I, fighting alongside the United Kingdom, Russia, and their allies against the Central Powers.
France was one of the Allied Powers in World War II, but was conquered by Nazi Germany within two months. The Third Republic was dismantled, and most of the country was controlled directly by the Axis Powers, while the south was controlled by the collaborationist Vichy government. Following liberation, a Fourth Republic was established.
This was succeeded in 1958 by the French Fifth Republic, the country's current government. After the war decolonization saw most of the French colonial empire become independent, while other parts were incorporated into the French state as overseas departments and collectivities. Since World War II France has been a leading member in the UN, the European Union and NATO, and remains a strong economic, cultural, military and political influence in the 21st century.
Author: Giska Puspita Label: French


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The Definitive Guide to calgary garage door repair
IMHO a single must prevent non-vented furnaces. They insert to Considerably CO2 and humidity to the garage building for complications and rust challenges.
Otherwise yourself do it on your loved ones-carry out semiannual routine maintenance checks. I know we're all busy but 30 minutes is all it will take.
Which is fundamentally the technique also adopted by Nick T. in Minnesota. He just lately additional insulation during the garage attic, and at the very least in mid-November was having fun with temperatures 30 levels above outdoors ambient air.
We are going to examine 4 most important components that contribute to the price of basement development in Calgary. This tends to also allow you to to raised define your necessities and needs.
For temporary heating for your weekend undertaking, Charlie Sullivan implies an electric infrared (IR) heater, a straightforward unit that has a quartz aspect and reflector but no enthusiast.
Your garage door repair routine maintenance guidelines are very useful with illustrations or photos. I found a great number of fascinating recommendations within your blog, Specially its discussion about repair and routine maintenance.
Be certain to not spray any nylon roller wheels with lubricant and when you receive any about the track clear away it as this could trigger the garage door to slip.
You're so welcome Jerry. I really feel the same way about my garage doors. Let me know if you have any more queries soon after lubricating:)
My partner will get Extremely upset Once i make use of the overhead door for entry within the entrance in lieu of the entrance door. I use it soon after staying outside the house and do not choose to enter the front.
Spot & Format – The location and specifically the height of your deck performs an important function when seeking to find out the expense of a deck. The better the deck the more structural guidance goes into it.
But that small air temperature and superior necessarily mean radiant temperature can't be sustained over extensive amounts of time for the reason that shipped radiant warmth is inevitably re-expressed as conductionMovement of warmth through a material as kinetic energy is transferred from molecule to molecule; the handle of an iron skillet anonymous on the stove will get sizzling as a result of heat conduction.
Insulating a garage door is usually low-priced and simple (either DIY or with one of numerous commercially offered remedies, for example this one), but air-sealing a door is admittedly challenging. I checked in with neighborhood builder users on the Sustainable Power Outreach Network and they all agreed that when gasketing among person door panels and also the concrete ground can be achieved, air sealing at The pinnacle on the door and along the aspect tracks is damn near difficult.
Terrific suggestion about putting in two new garage door springs if you must change one of these. I didn't recognize that only changing one can toss the door off equilibrium and trigger it to operate significantly less view it efficiently. Now that I'm sure this, I will be sure to understand that just in case I at any time want to interchange a spring.
I absolutely need to do A few of these matters for getting my garage Doing work far better. I are going to be Specifically certain to try out cleansing the rollers Learn More Here and using the spray lubricant.


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Contrary to popular belief, the dog paddle may not come naturally to all dogs. While some dogs are natural-born swimmers, others may not be. And even the best swimmers can use some training when starting out. The American Kennel Club offers the following tips to help dog owners keep their four-legged friends safe in and around the pool.
First and foremost, never leave your dog unsupervised while in a lake or pool. Your dog may need your help and be unable to bark loudly enough to get your attention.
Build confidence
It's important to build confidence in your dog around water. Even the breeds that are natural swimmers tend to be afraid the first time they enter a lake or pool, so be sure to take it slowly and praise your dog every step of the way.
Don't let your dog overexert itself. Swimming is great exercise, but, as with any workout, it should be increased in small increments. Your dog is using new muscles to swim and may get worn out quickly. Also keep in mind that smaller breeds tend to get tired faster because of their short limbs.
The dog paddle
If your dog begins to dog paddle with just the front legs, lift your dog's hind legs to help it float. Your dog should quickly catch on and will then keep its back end up.
Learn the ladder
Helping your dog learn to use the ladder to exit the pool is an important skill to ensure your dog's safety, but it can cause your dog to panic. Dogs are not used to climbing a ladder and will need to be taught how to use it. Direct your dog toward the ladder and help it climb out of the pool.
Rinse off
The chemicals from the pool can irritate your dog's skin. Spray off your dog with a hose after it gets out of the pool. You might want to take the same precaution after your dog has gone swimming in a lake.
Clean the ears
Most ear infections in dogs with floppy ears are caused by too much water and dampness. Dab your dog's ears with a dry towel. If your dog has thick fur on its ears, consider using a blow dryer set to cool to get rid of excess moisture.
StarTribune.com welcomes and encourages readers to comment and engage in substantive, mutually respectful exchanges over news topics. Commenters must follow our Terms of Use.
- Keep it civil and stay on topic.
- No profanity, vulgarity, racial slurs or personal attacks.
- Comments with web links are not permitted.
- Comments that violate the above will be removed. Repeat violators may lose their commenting privileges on StarTribune.com.
Comments will be reviewed before being published.


text:
This is one of the most intersting and useful courses I've ever taken. The course book was very well structured and helps trainees approach new skills for teaching later. The materials also brought clear insights of how to prepare lesson plan as well as to manage class professionally.
My observed practice teaching sessions was where my knowledge was turned into skill. It started with a first day so full of anxiety and thinking I could never do this to my last session where I felt supremely confident. That was also a sad day in that my practicum was over and I wouldn't be with my students anymore.
Vantage really stand out in their quality of their trainers. They are incredible in terms of knowledge, and teaching experience. He guided trainees with his heart and never said no when trainees asked for help. You couldn't ask for anything more.
I will strongly recommend this course to my friends. I strongly believe that they will benefit from all the knowledge I gained during the course.


text:
colour-block shorts
Since 1978, Versace has been synonymous with luxury fashion. For their SS18 collection, their iconic prints and glamorous designs sit alongside sleek, modern ideas. Crafted in Italy from blue and yellow cotton blend, these colour-block shorts from Versace feature a waistband with belt loops, a concealed front fastening, side pockets, a panelled colour block design, a short length and front slits.
Made in Italy
Designer Style ID: A78806A220955
Farfetch ID: 12654958
Measurement Information
- Height:
- 1.78 m
- Bust:
- 81 cm
- Hips:
- 88 cm
- Waist:
- 63 cm
Model's wearing: 38
- Height:
- 5 ft 10 in
- Bust:
- 31.9 in
- Hips:
- 34.6 in
- Waist:
- 24.8 in
Model's wearing: 38
All measurements are made by Farfetch
Still need some help? Try our Size Guide
- Composition
- Spandex/Elastane 6%
- Composition
- Cotton 53%
- Composition
- Polyamide 41%
- Washing Instructions:
- Machine Wash
No one does opulent Italian fashion quite like Versace. Bold patterns and clashing colours made Gianni Versace famous; today it's his sister Donatella's impeccable tailoring and glamorous evening-wear that keeps the brand in the spotlight. Since its establishment in 1978, Versace has come to be one of the most recognisable brands in the world.}
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Dalat or Nha Trang?
Friday, May 18, 2018 2:29 AM
Labels: tour, beach reddit , 0 comments
Labels: tour, beach reddit , 0 comments
I'm in Vietnam heading south. I'm am currently in Hoi An and can't decide between the two cities before head to HCMC and flying out. I'm looking for more scenic nature and less partying.
Submitted May 18, 2018 at 10:10AM by SuperFunk3000 https://ift.tt/2GsKYfq


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May 6, 2018, Sunday - Fancy Frilly Cakes Class at Cooks of Crocus Hill
Time: 1 PM - 3 PM
Location: Cooks of Crocus Hill, 208 N. 1st Street
There are few things more alluring in the culinary world than a beautifully decorated cake. Join us and learn the tips, tricks and techniques for successful, and stunning, decorating. Soon you'll be on your way to creating that custom cake that will be photo worthy!
Menu: Mini Mirror Glaze Cakes; Free-form Letter Cake with Berries; Chocolate Flower Cupcakes.


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Reacting to the weekend hit-and-run crash that left one dead and 16 injured, the Los Angeles City Council voted Tuesday to install temporary traffic barriers at the Venice Boardwalk.
Saying the Venice neighborhood is "shaken" by the incident, Councilman Mike Bonin asked his colleagues to instruct various city agencies to place the temporary barriers at the most hazardous intersections along Ocean Front Walk until a permanent strategy for keeping cars away from pedestrians is developed.
Bonin said the city should treat the boardwalk, which receives millions of visitors per year, less like a neighborhood park and more like a global tourist destination. Although four pylons were on Dudley Avenue separating cars from Ocean Front Walk, the suspect in Saturday's crash swerved around them by driving on the sidewalk and then into the crowd.
Alice Gruppioni, a 32-year-old Italian woman who was on her honeymoon, was killed in Saturday's crash. Police arrested Nathan Louis Campbell, 38, on suspicion of murder. He is scheduled to be charged Tuesday.


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Megan Gann, SHRM-CP
HR manager and solo HR
practitioner at
Camp Gladiator, a fitness startup
Susan Smith, SHRM-SCP
Director of HR at the
People's Community Clinic, a
nonprofit health facility
Why did you seek a mentor?
Gann: I was new to the HR manager role. Susan is in health care
and I'm in fitness, so it was a good
Smith: Talking to an HR professional outside of your company
gives you a wider perspective.
Megan's energy and enthusiasm
are contagious. I gave her some
advice on documenting a performance issue, and she showed me
a cool presentation tool she found
for an employee handbook.
Gann: We meet in person at a
Starbucks once a month, and I
bring the agenda. It's nothing formal, just things I'm dealing with or
working on. She shares best practices and the lessons she's learned.
She helps me navigate all the gray
areas within HR.
Smith: Be a reflective listener. And
you must be trustworthy because
your mentee may be sharing sensitive information. You need to take
the role seriously and honor your
commitment to meet regularly. As
HR professionals, we have the
responsibility to give back.
Gann: As with any relationship, ifyou want it to work, you make thetime. Most of us can find an houror so once a month to devote tothis. I try to respect her time, too,by saving my questions until wemeet. It's important for every HRprofessional to have someone likethis in their lives. Susan Smith, SHRM-SCP, andMegan Gann, SHRM-CP
Austin, Texas-based organizational
consultancy. Experts say a few actions
facilitate a productive pairing:
Dedicate the time. Both parties
need to commit. "One common mistake [among mentors and mentees] is
not connecting as quickly or as often as
they should. We recommend pairs meet
at least once a month," Templeton says.
Be available in between as well.
"Respond in a timely manner to all
communications from your mentee or
mentor. If one person doesn't make the
relationship a priority, it won't work,"
Kochanski says.
Establish your objectives. "Menteesshould come into the relationship withclear goals and understand the areaswhere they'd like a mentor's feedback,"recommends Natalie Morgan, HR andcommunications manager for Career-Plug, an applicant tracking softwarecompany in Austin, Texas.Moreover, those goals should berealistic and achievable. "As a mentee,don't expect your mentor to be a searchfrm for your next job," Kochanski says."Mentors can help you be more suc-cessful in your current job and plan forthe future, but you have to make yourown way."
Give sound advice, and give it selec-tively. "Don't use this as a chance totell old war stories," Kochanski advisesmentors. "Ask lots of questions andhelp the mentee to develop options,but [let the mentees] make their owndecisions."And don't toss out glib advice. "Youcan't give of-the-cuf opinions. You needto give substantiated proof—examplesand experiences—as to why the adviceyou're giving works," Oliverio says.
Regardless of which side of the desk
you sit on, mentoring can advance
your career. In a comprehensive study
that followed 1,000 employees of Sun
Microsystems Inc. over fve years, 25
percent of mentees in the company's
mentoring program, and 28 percent
of mentors, received raises, compared
with 5 percent of those who did not
Click to subscribe to this magazine


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Events for May 27, 2018
Events Search and Views Navigation
Boxed Lunches
May 16 to September 2, 2018 If you're interested in refreshments but prefer a more casual meal than our Tea on the Lawn service, boxed lunches are available and can be enjoyed while picnicking on the Museum grounds. Boxed lunches include one of the sandwiches being prepared for the day's tea service, a dessert, a […]Find out more »
10:00 am
Heritage Power
Sunday, May 27 – 10 am to 4 pm Hosted by Vintage Iron and Traditions of Eastern Ontario, this popular event showcases the machines, tools, and techniques that made early rural life in this region possible. Come check out exhibitor displays and demonstrations from farm equipment to steam engines. The popularization of new technologies like […]Find out more »
History Hikes
Sundays from May 20 to September 2 – 10 am to 12 pm* Are you planning to spend some time exploring the scenic parkland, shoreline, and farmers' fields at Pinhey's Point? Complement your visit with a short introduction (approximately 30 minutes) to the natural heritage of the area. Every week we'll touch on a […]Find out more »
10:30 am
Tea on the lawn
Wednesdays, Thursdays, Fridays, and Sundays from May 16 to September 2. Tea on the lawn is served overlooking our beautiful flower gardens and is a perfect way to relax and enjoy the grounds of this national historic site. Specializing in refreshments and baked goods made from scratch, our food service is a unique addition to the […]Find out more »
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