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Method for detection of biological particles
A monomolecular layer of first biological particles is absorbed on the surface of a non-reactive substrate. The coated substrate is exposed to a solution suspected of containing second biological particles specific to the first biological particles. Next, the coated substrate is subjected to a tagging step, following which it is exposed to a cleaving agent solution which cleaves the bond between the first and second biological particles. The used cleaving agent solution is then examined by a tag-sensing instrument following possible concentration thereof for determining whether tag-bearing second particles have been removed. The tags may, for example, be radioactive, fluorescent, etc. A supplement is described, wherein the coated substrate is studied with a tag-sensing instrument before and after exposure of the coated substrate to the cleaving agent solution to help provide said determination.
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This invention relates to a medical diagnostic method and apparatus for detecting immunologic (as well as other specific) reactions occurring on the surface of a solid, non-reactive substrate. In particular, the method is one wherein first reactive particles are attached to the substrate and after an opportunity has been provided for a specific reaction of the first particles with second particles, should they be present, to occur upon the substrate, a tagging operation is conducted. The tagging operation is followed by a cleaving operation for cleaving the tagged second particles, if present, from the first particles, the used cleaving agent being studied with a tag-sensing instrument. Alternatively, the substrate may be studied with the tag-sensing instrument before and after the cleaving operation, for initial presence then disappearance (or substantial reduction) of tags.
This application is related to my copending U.S. Patent applications Ser. No. 266,278 (now abandoned) entitled "Method and Apparatus for Detection and Purification of Proteins and Antibodies" filed June 26, 1972; continuation-in-part application Ser. No. 384,113 (now abandoned) entitled "Improved Method and Apparatus for Detection and Purification of Proteins and Antibodies" filed July 30, 1973; (now U.S. Pat. No. 3,926,564) entitled "Improved Substrate for Immunological Tests and Method of Fabrication Thereof" filed Feb. 25, 1974; Ser. No. 430,884 (now abandoned) entitled "Method and Apparatus for Surface Immunological Detection of Biological Particles by Radioimmunoassay" filed Jan. 4, 1974; and Ser. No. 503,030 (now U.S. Pat. No. 3,979,509 ) entitled "Opaque Layer Method and Apparatus for Detecting Biological Particles", filed Sept. 3, 1974 and to U.S. Pat. application Ser. No. 388,407 (now U.S. Pat. No. 3,904,367) entitled "Contrast Enhancement for Immunological Film Detection", inventor Golibersuch, filed Aug. 15, 1973, each application assigned as herein.
A related, commonly assigned patent, based on a prior application of mine is U.S. Pat. No. 3,853,467, issued Dec. 10, 1974, upon application Ser. No. 388,406, entitled "Method and Apparatus for Immunological Detection of Biological Particles", filed Aug. 15, 1973.
Other publications related to the present invention primarily as background are "The Antibody-Antigen Reaction: A Visual Observation", Ivar Giaever, The Journal of Immunology, Vol. 110, No. 4 (May 1973), pages 1424-1426 and "Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces", A. L. Adams et al., Journal of Immunological Methods, Vol. 3, (1973), pages 227-232 and U.S. Pat. No. 3,853,987, entitled "Immunological Reagent and Radioimmunoassay", issued Dec. 10, 1974 to William J. Dreyer, upon application Ser. No. 177,017, filed Sept. 1, 1971.
The term "biological particle" is intended to encompass smaller proteins (e.g. plasma proteins, antigens, antibodies, lactins) and bodies of proteinaceous material (e.g. viruses, bacteria, cells) capable of stimulating antibody production, when injected into an animal, and/or having the property of interacting specifically either immunologically or non-immunologically.
The term "tag" as used herein refers to an identifying molecule or group of atoms from which emanations occur,
a. which can be chemically integrated into an entity that is itself difficult or presently impossible to detect in dilute concentrations and
b. which, because of emanations therefrom, can be detected even in very small quantities by the use of appropriate instrumentation.
Recognition of the tag (e.g. radioactive isotope, fluorescent group) automatically identifies the presence of the entity into which it is integrated.
Immunological reactions are highly specific biophysical reactions in which a first immunologically reactive biological particle known as the antigen combines (links) with a second reactive biological particle specific to the antigen, and known as the antibody, to form an immunologically complexed protein. Immunological reactions taking place within a biological system such as an animal or human being are vital in combatting disease. In a biological system, the entry of a foreign protein, i.e., the antigen, causes the biological system to produce antibody proteins specific to the antigen in a process not fully understood at this time. However, some of the most important antibodies used in diagnostic reactions are not related to the entry into a biological system of any known foreign molecule. Some antibodies, such as anti-A and anti-B used in blood grouping, are called natural antibodies, because they are found in human sera apparently without the requirement of prior antigenic stimulation. Other antibodies seem to have the ability to react with some constituents normally present in the body. This leads to the pathological condition known as autoimmunity or autoallergy. Examples of such antibodies include the antinuclear antibodies of systemic lupus erythmatosus, the rheumatoid factors of rheumatoid arthritis, and the anti-thyroglobulin antibodies of chronic thyroiditis (Hashimoto's Disease). The antibody protein molecules have available combining or binding sites which complement those of the antigen molecule so that the antigen and antibody physically link or bond to form an immunologically complexed protein.
Most antigens are proteins or contain proteins as an essential part, whereas all antibodies are proteins. Proteins are large molecules of high molecular weight, i.e., are polymers consisting of chains of variable numbers of amino acids. The above-cited copending applications disclose that an arbitrary protein will adhere to a substrate in a monomolecular layer only, and that no other arbitrary protein will adhere to the protein layer. On the other hand, the specifically reacting protein to the first protein adsorbed onto the substrate will immunologically bond thereto. In accordance with the teachings and objectives of those applications, this phenomenon is exploited to provide medical diagnostic apparatus in which a particularly prepared slide having a monomolecular layer of one protein adsorbed thereon is used to test suspected solutions for the presence or absence of the specifically reacting protein thereto. If the specifically reacting protein is present in the solution, the slide after exposure to the solution has a bimolecular protein layer thereon. If the specifically reacting protein is absent from the solution, the slide after exposure to the solution has only the original monomolecular protein thereon. The use of various optical, electrical, chemical and radioactive means for distinguishing between bimolecular and monomolecular biological particle layers are taught in the related copending applications and have different degrees of sensitivity and economy. Examination of the protein coated slide with the unaided eye is the preferred approach for determining the number of biological particle layers on the slide due to its simplicity and some of the above-identified copending patent applications teach such an approach.
The detection of antibodies in a biological system is of medical diagnostic value in determining the antigens to which the system has been exposed. An example of diagnostic detection of antibodies is the detection of antibodies to syphilis or gonorrhea in human serum. Conversely, the detection of certain antigens in a biological system also has medical diagnostic value; examples of diagnostic detection of antigens include detection of HCG-protein molecules in urine as a test for pregnancy, and detection of hepatitis-associated-antigen (HAA) molecules in the blood of prospective blood donors.
In order to perform such diagnostic tests, the appropriate protein of the immumologically reacting pair must be obtained. At present, the source of an antibody protein must be a living biological system. More particularly, vertebrates are known to exhibit immunological reactions to the introduction of a foreign protein. For example, many antibodies are found in the blood serum of animals and human beings which have been exposed to the corresponding antigens. Invertebrates may also exhibit immunological reactions, although they probably do not have specific memory. Even some plant proteins combine with antigens and these so-called lectins may be of considerable use in diagnostic reactions. Some antigens may be controllably produced in laboratory cultures. However, most antigens, for example, hepatitis-associated-antigens, are at present, like antibodies, only obtainable from higher biological systems and thus many antigens are obtained from natural sources such as human or animal tissues.
It is known in the immunological art that antibody molecules function as antigens when introduced into the system of a vertebrate to whom they are foreign proteins. Accordingly, specifically reacting antibodies to a given antibody may be produced in such vertebrate system.
Although the emphasis herein will be on biological particles that immunologically react with each other, my invention also includes other forms of biological interactions between large molecules based on nonimmunological specifications such as for example, the binding of enzymes to their biological substrates, or hemoglobin to haptoglobin.
It is also known in the immunological art to attach a first biological particle such as an antigen (or antibody) to a solid surface, to expose the attached first particle to a solution suspected of containing a second biological particle to which the first particle is specific, and then to expose the system to a radioactive-tagged antibody to the second particle. The above technique is utilized to detect hepatitis by initially coating the inner bottom surface of a test tube with antibodies to HAA, then adding a blood sample suspected of containing HAA and finally adding a radioactively-tagged antibody to HAA. A radiation counter is then used to test the total bottom surface of the test tube for radioactivity.
It is known in the immunological arts to prepare antibodies to human immunoglobulins for chemical combination with fluorescent organic molecules such as the isothiocyanates. This tagged antibody is then used to render an immunological reaction visible by ultraviolet microscopy, particularly in the serological test for syphilis after the method of Coons, now known in the art as FTA-STS procedure. In the FTA-STS procedure, a quantity of Treponema pallidum (T. pallidum) is dried on a slide. The slide is then immersed in a blood specimen. The slide is subsequently immersed in a solution of tagged immunoglobulin. The anti-human immunoglobulin does not bond to the T. pallidum; accordingly, the slide will fluoresce, when observed by ultraviolet microscopy only if the specimen contained antibodies to T. pallidum.
In my above-mentioned co-pending application, Ser. No. 430,884, the detection of immunologically reactive biological particles such as viruses, bacteria and other cells is obtained by determining whether an immunological reaction occurs on a substrate between the particle to be detected and its radioactive-tagged antibody. A mono-molecular layer of first immunologically reactive biological particles is adsorbed onto the surface of the substrate in a distinct pattern, and the substrate is then exposed to a solution suspected of containing the particles to be detected, which particles are specific to the first particle. The substrate is exposed to the radioactive-tagged antibody to the particle to be detected and, finally, the substrate surface is monitored for radioactivity. If the particle to be detected was present, the distinct pattern will emit a significantly higher radiation level than the surrounding substrate.
In my above-mentioned co-pending application, Ser. No. 503,030, a method is described in which a monomolecular layer of first biological particles is adsorbed on the surface of a substrate fabricated of virtually any nonreactive solid material; the coated substrate is then exposed to a solution suspected of containing second biological particles specific to the first biological particles; next a porous, opaque layer of nonreactive third particles is formed on the coated substrate, and then the coated substrate is exposed to a cleaving agent solution which cleaves the bond between the first and second biological particles. A visual examination of the coated substrate surface or examination thereof by suitable instrumentation is made, to determine whether the suspect solution contains the second biological particles by discerning whether the opaque layer is complete or whether a portion common to the second biological particles, has been removed.
Still, there are instances in which the reaction proceeds so slowly, or wherein so few of the second particles are present in the specimen that the second layer, while present, is sparse and difficult to detect by optical means.
Thus, a method which can improve the contrast of a search by tag-sensing without requiring a prolonged reaction time is much sought and such is an object of this invention.
In contrast to prior teachings, wherein the tag-sensing instruments are used to examine a protein-coated surface for the presence of a pre-tagged second protein, secured to the first protein, the present invention provides a process wherein second biological particles (untagged) are permitted, if present in the specimen, to complex with first biological particles attached to a substrate and thereafter tagging is accomplished. Next, the system is contacted with a solution of cleaving agent to sever the bond securing the two layers of biological particles to one another. The used cleaving agent solution is collected and investigated for presence of tags therein, which would establish the presence of the second biological particles.
The presently preferred tags are radioactive ones, for instance, iodine 125 (designated I - 125 or .sup.125 I).
The tags can be applied in at least these ways:
a. Second biological particles may be tagged directly, after a layer thereof has become secured to the first layer taking care not to prematurely separate the first layer from the substrate. (Also, if some of the first layer becomes tagged during performance of the tagging operation, a consistent result may still be obtained from a tag sensing study of the solution of used cleaving agent, because the cleaving agent does not remove the first layer and its tags would still be attached to the substrate.)
This preferred mode of tagging can reduce expenses of operation while improving contrast. Pre-tagged biological particles are expensive and have a short half-life. They may also be less reactive towards the first biological particles than the untagged biological particles from which they were made would have been. One currently marketed test kit for Hepatitis Associated Antigen which includes .sup.125 iodine pre-tagged "third layer" Hepatitis Associated Antibody bears a shelf life of 28 days.
b. The second biological particles may be tagged after the securing thereof to the first layer, by the application of any pre-tagged non-biological material which will adhere to the second protein sufficiently to form a uniformly distributed coating thereover sufficiently porous to permit the cleaving agent solution to readily diffuse therethrough. While the cleaving agent is to sever the bond between the second and first layers of biological particles it must not significantly disturb the adherence of the pre-tagged adhering material to the first layer. The pre-tagged adhering material, which is carried from the surface in the cleaving agent solution provides a reliable indicator of the presence or absence of the second biological particles.
Tagging methods (a) and (b) are clearly preferred, because the drawbacks associated with pre-tagged biological particles are avoided. These drawbacks are a shortened operating life span (depending on the length of time the tag is effective), reducing the number of active sites on the biological particles and actual loss of the biological particles (before complexing) from radiation or other adverse effects.
c. Second biological particles may be tagged indirectly by contacting the coated substrate, following its exposure to a specimen possibly containing the second protein, to a solution of pre-tagged third biological particles, specific to the second but not to the first wherein, as in the instance of hepatitis, such a system exists. Then, by subjecting the coated surface to a cleaving agent, the bond between the first and second and/or the second and third layers is severed and the used cleaving agent solution is subjected to the tag-sensing operation.
In any of the above-identified ways of tagging, the coated surface may also be studied with the tag-sensing means before and/or after the cleaving step has been conducted, in order to provide checks, measures of background and the like.
It is an important object of the invention to provide a method of the type described, wherein a used cleavage agent solution containing tags in a lower concentration than could be readily or reliably detected, may be concentrated prior to being subjected to the tag-sensing step. For conducting the process of the invention, the substrate surface need not be flat, and may be of as great an area as economical use of the process will permit in order to increase the probability that more second particles will become secured to the first layer within a reasonably brief test period.
(Tests which may be conducted and the results determined (a) while an operative procedure is in progress or (b) over night, are most desired.)
The features of my invention which I desire to protect herein are pointed out with particularity in the appended claims.
The invention itself, however, both as to its organization and method of operation together with further objects and advantages thereof, may best be understood by reference to the following description taken in connection with the accompanying drawings wherein:
FIG. 1 is a schematic elevation view in cross-section through a portion of a substrate having adsorbed thereon a monomolecular layer of first biological particles;
FIG. 2 is a schematic elevation view in cross-section of the coated substrate of FIG. 1 following contact thereof with a specimen containing a second biological particle, which is specific to the first and has formed a second monomolecular layer thereupon;
FIGS. 3a, 3b, and 3c schematically show individual second biological particles tagged in three respectively different ways following securement thereof in the untagged state to the first biological particles of the first layer;
FIG. 4 is an elevation view in section depicting the detection step applying tag-sensing means to the cleaving agent solution and rinsing liquid collected following the cleaving step; and
FIG. 5 is an elevation view, similar to FIG. 4 depicting the detection step after concentration, as by evaporation, of the cleaving agent solution and rinsing liquid of FIG. 4.
Referring now to FIG. 1, there is shown schematically in cross-section at the surface a typical substrate 10 that may be utilized in my method for detecting an immunologic or other specific reaction involving biological particles. The substrate 10 may be fabricated of virtually any solid material that is nonreactive with the biological particles, cleaving agent solution and other materials used with the substrate in the conduct of the method. The surface 11 of substrate 10 must be impermeable and non-absorbent to the solutions of biological particles, tags and cleaving agent used in the method and must permit the adsorption of a monomolecular layer of the first biological particles thereon. The surface 11 need be neither light transmitting nor flat. Although the substrate could be constituted by a plain glass slide, this is not the preferred material, due to a tendency for cleaving agent solution to desorb too many first biological particles therefrom. Preferred and useful materials include a plurality of small spheres (e.g. about 25 mm. total surface area) presenting a thin tenacious, non-porous surface of gold, chromium, tantalum or molybdenum, by way of example. Slides, rods, glass container, internal surfaces and the like are alternatives for use as the substrate.
The size of the substrate 10 is governed primarily by the cost of materials consumed in the test, compared to the cost of time for conducting the test. Enlarging the area of the surface may increase the cost of the materials, but decrease the contact time needed to produce a collection of tags that is sufficiently concentrated to be clearly distinguished by the tag-sensing means.
The surface 11 must be free of any contaminants, such as cleaning solution residue, which might prevent or impede adsorption of the first monolayer of biological particles thereon.
Having selected a suitable substrate 10 having a thoroughly clean surface 11, a monomolecular layer 12 of first biological particles is adsorbed thereon. The identity of the material 12 will vary with the particular test being conducted. Bovine serum albumin is exemplary of a material comprising first biological particles.
While the emphasis herein will be on immunologically reactive biological particles (the simplest case being the antigen-antibody pair), for the purposes of simplicity and exemplification, it should be understood as explained at the outset that my invention is equally useful with sets of biological particles that undergo forms of biological interaction other than an immunologic reaction, the only criterion being that the particles are mutually specific.
The adherence of the first biological particles as a monomolecular layer may be accomplished by depositing one or more drops of a solution of the first biological particles on the surface 11 using a dropper or other means. Alternatively, the substrate 10 may be dipped into a solution of the first biological particles. The first biological particles are selected on the basis of being specifically reactive to particular second biological particles, which will form a second layer on the substrate surface, if they are present in a second solution, or sample thereof, to be tested.
The first biological particles may be produced in laboratory cultures or obtained from the higher living biological systems as described hrereinabove, and are generally commercially available in a relatively highly purified form, and if not available commercially, may be purified chemically. The solution of the first biological particles may be a salt solution of water or other liquid appropriate to, and not reactive with, the first biological particles and the substrate material. A more detailed description of the formation of the first (and second) particle monolayers in all the embodiments of my present invention is included in the teachings of the aforementioned copending U.S. patent applications of Giaever, which are herein incorporated by reference.
The time interval (generally up to one hour) for the formation of the first monomolecular layer 12 on substrate surface 11 is an inverse function of the concentration of the first biological particles in the first solution. The first monomolecular layer 12 covers substantially the entire surface 11 of the substrate 10 and substrate 10 is, therefore, made as small as practical in order to conserve the amount of biological material used in the process, yet sufficiently large to permit the test to be conducted reliably within a short time. A rinsing of the monomolecular layer coated surface of substrate 10 with tap water or distilled water is recommended in order to remove excess first biological particles (and possibly others existing in the first solution) that may accumulate on top of monolayer 12.
The monolayer-coated substrate 10 is then dried, if the substrate is to be shipped commercially or stored, preferably by blowing air at room temperature across the substrate in order to speed the drying process. If the substrate is to be used immediately, there is no need to dry it after the rinsing. The first monomolecular layer 12 is generally invisible to the unaided eye since the thickness of such monomolecular layer may be anywhere from 20 to 100 Angstroms, depending on the particular biological particle forming such layer.
Referring now to FIG. 2, the monolayer-coated substrate is next exposed to a second solution, or sample thereof, suspected of containing the second immunologically reactive biological particles that are specific to the first biological particles in a direct test for such second particles. This exposure is generally accomplished by immersing the monolayer-coated substrate in the second solution for a time interval, which is again an inverse function of the concentration (usually unknown) of the second biological particles in the second solution. Since the concentration of the second particles in the second solution is generally much less than the concentration of first particles in the first solution, the immersion step generally takes much longer than the time interval for forming the first biological particle monolayer 12, and may take up to 24 hours. The time can be shortened if a larger-area surface 11 is used.
Presence of the second biological particles in the second (test or suspect) solution will result in the formation of a second substantially complete monomolecular layer 13 over layer 12 as a result of the biophysical reaction between the second biological particles and the first biological particles. After the monolayer-coated substrate has been sufficiently exposed to the second solution, the coated substrate is removed therefrom and is rinsed with tap water, distilled water or a dilute salt solution depending upon the nature of the solution in which the second biological particles were contained. This rinsing step is utilized to prevent an excess build-up of the second biological particles on the substrate as well as to minimize non-specific absorption (i.e. the second solution is typically a complex, unseparated serum containing a great number of other type biological particles. The coated substrate may then be dried in a manner described hereinabove, if required for optional subsequent treatment described hereinbelow. If second biological particles are not present in the second solution, no deposition of a second monomolecular layer occurs over layer 12.
After the second monomolecular layer 13 has formed on the coated substrate, the second biological particles may be directly tagged as illustrated at 14, FIG. 3a. Radioactive tagging is preferred. A method for radioiodination of particles is described by W. M. Hunter and F. C. Greenwood (Nature, 194 (1962) page 495).
The tagging should be carried out in neutral solution in order to avoid premature separation of the second layer, if it exists, from the first layer. Suitable methods for radioactively tagging an anchored layer of biological particles are thoroughly described in Handbook of Experimental Immunology [Ed. D. M. Weir, Blackwell Scientific Publications (1973)] Chapter 17 "Radioimmunoassay" by W. M. Hunter pp. 17.1- 17.36 and in references cited therein. Direction to the technician is also provided in Principles of Competitive Protein-Binding Assays [Ed. W. D. Odell & W. H. Daughaday. Philadelphia: Lippincott (1971)] in Chapter X "Radioiodination of Peptide Hormones: Procedure and Problems" by F. C. Greenwood pp. 288-296. Another reference work which describes suitable radioactive tagging methods is in Radioimmunoassay Methods [Ed. K. E. Kirkham & W. M. Hunter, Churchill: Livingstone (1971)] "The Preparation and Assessment of Iodinated Antigens" by W. M. Hunter pp. 3-23 and "The Immuradiometric Assay" by G. M. Addison and C. N. Hales pp. 447-461.
Fluorescent tagging is an alternative to radioactive tagging and others will occur to those skilled in the art. The procedure for fluorescent tagging as well as radioactive tagging are generally understood by those skilled in the art. Suitable methods for fluorescent tagging of an anchored layer of biological particles are thoroughly described in the aforementioned Handbook of Experimental Immunology in Chapter 18 "Immunofluorescence" by G. D. Johnson and E. J. Holborow.
The aforementioned publications describing tagging techniques are incorporated by reference.
As an alternative to direct tagging of the second biological particles, as depicted in FIG. 3a, the second biological particles may be indirectly tagged, by pretagging third particles 15r bearing tag 14, particles 15r being known to be specific to the second biological particles, but not specific to the first biological particles. These tagged particles are used to form a third layer upon the monomolecular layer of second biological particles. This alternative is depicted in FIG. 3b. The third particles are first tagged as described above for the embodiment of FIG. 3a. The tagging of the third particles is also preferably radioactive, being exemplified by being iodinized with .sup.125 I.
Thus, in greater detail, after the coated substrate 10 of FIG. 3b has been sufficiently exposed to the second biological particles in solution, the substrate is removed from the second solution suspected of containing such particles and is washed by a suitable solution (e.g. water or a dilute aqueous salt solution) in order to remove most of the non-specific particles stuck to the substrate. The coated substrate is subsequently exposed to a solution of third biological particles 15r which are radioactively-tagged and are biophysically reactive with the second biological particle, for example, an antibody to the second biological particle. In any event, the radioactive-tagged third biological particle is specific to the second biological particle so that presence of the latter on substrate 10 causes the third particles to complex therewith and form a third monomolecular layer on substrate 10 over the previously deposited monomolecular layers, as illustrated in FIG. 3b. The exposure of coated substrate 10 to the third biological particles can be accomplished by immersion in a third solution of such particles or by being coated with the third solution in the case of a sufficiently high concentration thereof. Again, the length of the exposure of the substrate to the third solution required in order to obtain a substantially complete layer of third particles 15r upon the layer of second biological particles, is primarily a function of the concentration of such particles in the third solution. The concentration of the third particles can be controlled, as in the case of the first particles, since these particles are produced in laboratory cultures or in the higher living biological systems. The concentration of these biological particles is generally much higher than the concentration of the second particles in the second solution so that the time interval of exposure to the third particles is generally much less than the immersion in the second solution.
A third alternative is depicted in FIG. 3c wherein the second biological particles of the second layer are tagged, after the layer of second biological particles has become affixed, by the deposition thereon (and on any still exposed part of the first layer) of a layer or coating of third particles 15n. The layer of third particles 15n is characterized by being nonreactive with substrate 10 and with the biological particles forming monolayers 11 and 12, by being pre-tagged (for instance, radioactively-tagged with .sup.125 I) and by being deposited over the entire coated surface of the substrate to form the third and outermost layer. In the case in which the second solution did not contain second biological particles, layer 15n forms only the second layer on substrate 10. Nonreactive layer 15n must be sufficiently porous so that subsequent exposure of the coated substrate to a cleaving agent solution permits the cleaving agent to penetrate layer 13 as will be described hereinafter. Because of the subsequent exposure to the cleaving agent solution, particles 15n of the third layer must also be nonreactive with the cleaving agent.
The layer of particles 15n may be formed of virtually any material that exists in discrete small particle form and can be dispersed in a porous continuous layer, that can be pre-tagged using known techniques, that will adhere to the biological particle layers already deposited and that are nonreactive with the first and second biological particles, substrate and cleaving agent solution.
Thus, the third layer may be formed by dipping the coated substrate into an agitated third solution containing a relatively high concentration of such pre-tagged nonreactive particles so as to form a layer of the particles 15n in contact with each other or in slightly spaced apart relationship. Metal, glass or plastic pre-tagged particles in a suitable agitated solution may also be utilized to obtain layer 15n. The solution in which the metal, glass or plastic particles are contained may simply be water. In the case wherein the coated substrate is dipped into a solution of the small particles, there is no need to dry the coated substrate after the exposure of the monolayer-coated substrate to the solution suspected of containing the second biological particles and rinsing thereof, because the solution containing particles 15n will moisten the coated substrate. The size of the generally spherical third particles 15n, whether they be of glass or plastic, can be over a wide range of values (diameters of 0.1 to 100 micrometers) since the function of layer 15n is merely to present a tagged porous layer to the cleaving agent solution to be utilized hereinafter.
An alternative method for applying the third layer of particles 15n on the substrate is to utilize a conventional aerosol spray can filled with virtually any material nonreactive with the substrate, biological particles and cleaving agent, properly tagged and formative of a porous layer, preferably opaque. The coated substrate should be in a dry condition prior to this spraying process in order to obtain more efficient sticking of particles 15n to layer 13 (or 12). The particles suspended in a gas to constitute the aerosol may either be finely-divided solid or liquid. As a typical example, the dried, coated surface of the substrate may be lightly sprayed with modified MS-122 FLUORCARBON, a release agent and dry lubricant product of Miller-Stephenson Chemical Company. This product normally consists of an aerosol preparation of a low molecular weight synthetic resin polymer wax. For use in the present invention, prior to being packaged, the polymer particles are uniformly coated with a hydrocarbon, oil-based (water-insoluble) solution of e.g., a radioactively iodinated inorganic iodine salt. The tags may be physically trapped in, dispersed in, adsorbed on or chemically bound to the particulate carriers therefore.
If the layer 15n is made of material of high contrast, for instance, the tagged fluorocarbon wax aerosol, the extent of homogeneity of the distribution of tags can also be optically checked. If optical checking is not feasible or is not comtemplated, the layer 15n need not comprise a highly visible carrier material such as tagged fluorocarbon wax, but may consist of e.g., a hydrocarbon oil-based (water insoluble) solution of a radioactively iodinated inorganic iodine salt.
The thickness of the sprayed-on layer 15n may be substantially greater than the thickness of monomolecular layers 12 and 13, and is typically in the range of 1 to 10 micrometers, but can be in a range as great as monomolecular to 100 micrometers. The ranges for the discrete particle sizes or sprayed-on layer thicknesses may be different for different materials due to the different porosity and opaqueness thereof.
After the tagging step has been performed, e.g., in accordance with the discussion of FIGS. 3a-3c above, the coated surface of substrate 10 is exposed e.g. drops to a cleaving agent solution such as a weak acid solution, alkaline solution, or a solution containing a concentration of salt. Since the layer 13, if present, and any subsequently deposited layer are porous, the cleaving agent penetrates to the monomolecular layer of first biological particles, and, if the second biological particles are present, cleaves the bond between the first and second biological particles, but not the bond between the first biological particles and the substrate. Monomolecular layer 13 is removed from the region in which cleavage occurs being carried in the cleaving agent solution 16 (and in the rinsing fluid used). The detached second biological particles and the liquid are collected in a container along with (in the case of the alternatives shown in FIGS. 3b and 3c) the particles 15r or 15n attached thereto.
In the event that the specimen being tested for the presence or absence of second biological particles is free of second biological particle content, application of the cleaving agent solution to coating on the surface 11 has no effect on the coated surface of the substrate and tags are not collected in the cleaving agent solution beyond a small, background amount. Further, in this event when the alternate method illustrated in FIG. 3c is used, the layer consisting of particles 15n will remain complete during conduct of the cleaving, rinsing and collecting steps.
In the case of the cleaving agent being an acid solution, the acid solution utilized is generally a weak one, that is, it is sufficiently strong enough to cleave the bond between layers of first and second biological particles, but not strong enough to cleave or otherwise affect the bond between the first layer 12 of biological particles and the solid surface of substrate 10 to which it is adsorbed. A 0.1 normal (N) citric acid solution is suitable for the purposes herein described. The range of concentrations of the citric acid for obtaining the desired results are approximately 0.01 to 1.0 N. Other suitable weak acids that may be utilized are 0.1 N malic acid and 0.1 N formic acid. Stronger acids such as hydrochloric acid and sulfuric acid may also be utilized, but in a much smaller concentration (i.e., approximately 0.01 N). The weak acid solution that may be utilized in my invention may be generally described as being any acid solution that does not attack the substrate or any third layer material to be applied and, preferably has a pH in a range between 2 and 5, although a pH as low as 1.0 was found to be satisfactory (using 0.1 N hydrochloric acid).
As noted hereinabove, alkaline and high salt concentration solutions may also be used as cleaving agents. The alkaline solution useful herein has a pH in the range 9-13, and typically, a 0.2 N sodium hydroxide solution has been used to cleave the bond between egg albumin and its antigen. Various salt solutions of elevated salt concentration, such as NaCl and NaI are known to function as cleaving agents. Thus, a 1.79 molar NaCl solution cleaves pneumococcal polysaccharide from its antibody.
When, for example, radiation tagging is employed, the autoradiography technique may be used to detect radiation emission by the use of special photographic film sensitive to gamma rays, this film being placed in proximity to the used cleaving agent solution. The presence of gamma radiating tags in the solution becomes readily apparent on such film in view of the much higher level of exposure as compared to the background exposure level. In the case radioactive-tagged third biological particles have been used (FIG. 3b) emitting primarily gamma rays, the radiation counter 17 may be a conventional gamma ray counter comprising a tubing having a scintillator crystal at the aperture end thereof and a photocathode at the output end. The tubing, crystal and voltage bias connections in the counter, being conventional, are not shown. The scintillator crystal, which may be fabricated of sodium iodide as one example, is responsive to gamma rays impinging thereon, and emits light photons which are detected by a photocathode (not shown) and are converted to voltage pulses corresponding to the number of counts detected. The output voltage from the photocathode is conducted to a suitable electronic amplifier (not shown) for amplifying the voltage count pulses. The output of the amplifier is applied to the input of a suitable real-time read-out device (not shown) to provide a visual (oscilloscope) or audible (loudspeaker) output or may be a memory storage device such as magnetic tape for permitting a delayed read-out.
In case the radioactive-tagging employed is such that beta rays are emitted from the tagged entities, the tag-sensing means 17, e.g. a radiation counter may be a conventional beta ray counter comprising a sealed tubing which is gas-filled and has an electrode (not shown) therein connected to a source (not shown) of relatively high voltage such that the beta rays ionize the gas and each gas ionization event is detected by an electronic amplifier (not shown) and read-out device (not shown).
One advantage provided by the method of the invention is that when the tags 14, no matter what their type or affiliation, are in such low concentration in the bulk of the used cleaving agent solution 16 that the tag-sensing device 17 cannot provide a reliable reading, the solution 16 may be concentrated to a smaller bulk 18, thereby concentrating the tags 14 for more reliable detection by the tag-sensing device 17.
If desired, as a check upon the measurement made in FIGS. 4 or 5, the coated substrate may be subjected to study by the tag-sensing means 17 (using substantially the same instrumentation and techniques as have been described above) both prior to and after conduct of the cleaving and rinsing operations to determine any differential read-out.
In the description of my invention hereinabove, reference has been made to first and second immunologically reactive biological particles wherein the second biological particle is specific to the first. The use of this language is deliberate and the immunologically reactive antigen biological particles that may be utilized, and detected, with my invention are meant to include hormones, viruses, bacteria, enzymes and other particles which can be readily grown or otherwise isolated and collected or are present in human serum or other solution being tested. However, my invention is further useful with virtually any pair of biological particles that will physically react (combine) with each other. That is, in addition to being applicable to immunologically reactive antigen-antibody systems, my invention also includes other forms of biological interactions between large molecules based on non-immunological specificities, such as for example, the binding of enzymes to their biological substrates, or the binding of hemoglobin to haptoglobin.
The solution suspected of containing second particles is generally a human serum sample, although it can be another type of solution appropriate to the particular biological particle being investigated. In direct testing wherein it is sought to detect an antibody, such as in the case of syphilis or gonorrhea, the antibody is detected in the human serum of a patient. Alternatively, in the inhibition test to detect an antigen, e.g. hepatitis HAA, the antibody may be developed in a goat, rabbit, or other suitable animal and the appropriate quantity of this animal serum is mixed with the human serum of the patient to be tested.
All of the steps in the various examples herein can be conducted at room temperature, that is, in a temperature range of approximately 65.degree.-75.degree. F. However, temperature is not critical to my invention.
It should be understood, as has been implied hereinabove, that the description of the substrate 10 as being "nonreactive" with the biological particles and tagging layer applies to its chemical and biological inertness to the extent that it will not attack or destroy the biological particles, the tags, or the cleaving agent employed. The substrate must, of course, have the capability of having the first biological particles adsorb thereon as a monomolecular layer. In like manner, the material of particles 15n is nonreactive with the biological particles of layers 12 and 13 (and with substrate 10), but has the capability of binding or adhering to the layer of second biological particles 13 (or layer 12 of first particles in the absence of the second layer).
It should be apparent that the steps of the method, including the concentrating and tag-sensing steps conducted upon the cleaving agent solution collected after use are suited for use on automated, large scale testing systems.
Having described my invention with reference to particular embodiments and examples, it is believed obvious that modification and variation of my invention is possible in the light of the above teachings. Thus, a blood serum sample can be quantitatively tested for a particular biological particle in a simple variation of my invention in the following procedure. A plurality of containers of equal size are used, the size being sufficient to contain the cleaving agent solution (and rinse liquid) to be applied to one of the coated substrates to be used. Four containers and four substrates comprise a convenient number. Each substrate is exposed in the same manner to receive the same quantity of a particular first biological particle in purified form, for example, a hormone, for which the quantitative test is to be conducted. This results in the establishment of the same area monomolecular layer of the hormone on each substrate.
The blood serum sample to be assayed for this hormone is divided into four equal parts, and various known concentrations (i.e., a, 2a, 4a, 8a) of antibody to this given hormone are respectively added to the four blood samples for complexing with some or all of the hormone in the blood serum in the separate parts of the blood sample to the extent of the concentration of antibody added. Assuming the concentrations of antibody to the hormone were properly selected, one or more of the separate parts of blood sample will now contain excess antibodies. Each of the separate parts of blood sample is then placed in contact (e.g. by immersion) with the hormone layer on a separate substrate for the same period of time. The substrates are then removed and rinsed.
The coated substrate area is next exposed to the tagging medium e.g. a method selected from the three general categories described herein. Next each substrate is contacted with an equal amount of cleaving solution (and, if necessary, rinse water). The cleaving solution and rinse used for each substrate is collected in a separate one of the containers. The four containers are then tested for radioactivity. The hormone concentration is thereby quantitatively determined as having a value between the concentrations (a, 2a, 4a, 8a) at which substantial radiation is first detected and the next lower concentration.
It is, therefore, to be understood that changes may be made in a particular embodiment of my invention as described which are within the full intended scope of the invention as defined by the following claims.
1. A diagnostic method for determining the presence or absence of select biological particles in a liquid sample comprising the steps of:
- contacting surface area of a substrate with first biological particles, said first biological particles being specific to the select biological particles and being dispersed as a first layer coating said surface area;
- contacting the coated surface area of said substrate with the sample liquid for a preselected period of time;
- applying a tagging medium to the coated surface area resulting from the preceding step, said tagging medium making tags available to said coated surface area;
- contacting the coated surface area resulting from the preceding step with a cleaving agent solution, said cleaving agent solution being capable of selectively destroying such bonding as may exist between said first biological particles and select biological particles;
- separating from said substrate the cleaving agent solution used in the preceding step together with any matter contained therein released from the coated surface area and
- examining said separated cleaving agent and contents thereof for the presence of emanations from the tags to indicate the presence of the select biological particles in said liquid sample.
2. The diagnostic method recited in claim 1 wherein the substrate is substantially planar.
3. The diagnostic method recited in claim 1 wherein the substrate is non-planar.
4. The diagnostic method recited in claim 1 wherein the surface area of the substrate contacted with the first biological particles is metallic.
5. The diagnostic method recited in claim 1 wherein the surface area of the substrate contacted with the first biological particles is non-metallic.
6. The diagnostic method recited in claim 1 wherein the first biological particles are protein.
7. The diagnostic method recited in claim 6 wherein the first biological particles are antigen.
8. The diagnostic method recited in claim 6 wherein the first biological particles are antibody to the select biological particles.
9. The diagnostic method recited in claim 1 wherein the select biological particles are cellular.
10. The diagnostic method recited in claim 1 wherein the select biological particles are viruses.
11. The diagnostic method recited in claim 1 wherein the select biological particles are bacteria.
12. The diagnostic method recited in claim 1 wherein the tagging medium contains a composition chemically reactive with biological particles for direct tagging thereof.
13. The diagnostic method recited in claim 1 wherein the tagging medium contains third biological particles specifically reactive with the select biological particles, said third biological particles having been directly tagged prior to being applied to the coated surface.
14. The diagnostic method recited in claim 1 wherein the tagging medium contains adherent, nonreactive non-biological pre-tagged particles.
15. The diagnostic method recited in claim 14 wherein the nonreactive pre-tagged particles are applied as a layer by lightly spraying an aerosol thereof over the coated surface.
16. The diagnostic method recited in claim 1 wherein the tags emit radiation.
17. The diagnostic method recited in claim 16 wherein the tags are radioactive.
18. The diagnostic method recited in claim 16 wherein the tags are fluorescent.
19. The diagnostic method recited in claim 1 wherein the cleaving agent solution is a weak acid solution.
20. The diagnostic method recited in claim 19 wherein the pH of the weak acid solution is in the range of 1-5.
21. The diagnostic method recited in claim 1 wherein the cleaving agent solution is an alkaline solution.
22. The diagnostic method of claim 21 wherein the pH of the alkaline solution is in the range of 9-13.
23. The diagnostic method recited in claim 1 wherein the cleaving agent solution is a concentrated salt solution.
24. The diagnostic method of claim 1 further comprising the steps in which after the coated surface area is contacted with cleaving agent solution, said coated surface area is rinsed and both the cleaving agent solution and the rinse are collected and used for the subsequent examining step.
25. The diagnostic method of claim 1 further comprising the step of concentrating the cleaving agent solution used together with any matter contained therein after separation from the substrate and before the examining step.
26. The diagnostic method of claim 1 further comprising the step of examining the coated surface area of the substrate for tag emanations prior to contacting said area with cleaving agent solution.
27. The diagnostic method of claim 14 wherein the nonreactive pre-tagged particles are applied over the coated surface area and form a porous, opaque layer.
28. The diagnostic method recited in claim 15 wherein the layer is opaque.
29. The diagnostic method of claim 12 wherein the composition is radioactive.
30. The diagnostic method of claim 29 wherein the composition contains.sup.125 I.
31. The diagnostic method of claim 12 wherein the composition is fluorescent.
Referenced Cited
U.S. Patent Documents
|November 1966
|Sutherland et al.
|January 1971
|Axen et al.
|July 1971
|February 1972
|March 1973
|Bennich et al.
|July 1974
|Bratu, Jr. et al.
|July 1975
|September 7, 1976
Other references
- Chidlow et al, Biochem. Journal (Great Britian), vol. 117, 1970, pp. 49-55.
Patent History
Type: Grant
Filed: May 1, 1975
Date of Patent: Aug 9, 1977
Assignee: General Electric Company (Schenectady, NY)
Inventor: Ivar Giaever (Schenectady, NY)
Primary Examiner: Benjamin R. Padgett
Assistant Examiner: Christine M. Nucker
Attorneys: Leo I. MaLossi, Joseph T. Cohen, Jerome C. Squillaro
Application Number: 5/573,610
International Classification: G01N 3300; G01N 3316; A61K 4300;


text:
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Bellamy's Organic Ready To Serve Baby Food Banana Apple Porridge
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text:
Favorite Vegan Dinner Recipe!
So first off, I am not a vegan but definitely am open to vegan recipes and the lifestyle! It has been an interesting journey for me to realize that despite what I grew up thinking, you don't need to have meat in a meal to make it a real meal. And to be honest, it was probably this recipe that really made me agree and get excited to start trying more!
I have recommended the Oh, She Glows cookbook before but wanted to share with you my go to dinner recipe from the book just so you can see how amazing they really are!
Sweet Potato, Chickpea, and Spinach Coconut Curry
(By: Oh, She Glows)
- 4 teaspoons (20 mL) virgin coconut oil
- 1 tablespoon (15 mL) cumin seeds
- 1 medium onion, finely chopped (about 2 cups/500 mL)
- 3/4 to 1 teaspoon (4 to 5 mL) fine sea salt, to taste, plus a pinch
- 3 large cloves garlic, minced
- 4 teaspoons (20 mL) grated fresh ginger
- 1 teaspoon (5 mL) ground turmeric
- 1 teaspoon (5 mL) ground coriander
- 1/4 teaspoon (1 mL) red pepper flakes, or to taste
- 1 medium/large sweet potato, peeled and cut into 1/4- to 1/2-inch (5 mm to 1 cm dice (about 3 cups/750 mL)
- 1 (14-ounce/398 mL) can chickpeas, drained and rinsed, or 1 1/2 cups cook chickpeas
- 1 (14-ounce/398 mL) can diced tomatoes, with juices
- 1 (14-ounce/400 mL) can light coconut milk
- 1 (5-ounce/142 g) package baby spinach
- Freshly ground black pepper
1. In a large saucepan, heat the oil over medium heat. The oil is hot enough when a cumin seed sizzles when tossed into the pan. Add the cumin seeds and toast for about a minute, until fragrant and lightly darkened in color (be careful not to burn them). Immediately stir in the onion, season with a pinch of salt, and cook for 3 to 5 minutes, or until the onion is soft and translucent.
2. Add the garlic, ginger, turmeric, coriander, and red pepper flakes. Stir to combine and sauté for a couple of minutes, until the garlic softens.
3. Add the sweet potato, chickpeas, tomatoes with their juices, and coconut milk. Stir to combine, cover, and simmer over medium heat for 20 to 30 minutes, until the potatoes are fork-tender. At this point, I always mash one-third of the mixture to thicken the sauce (using a potato masher), but this step is optional.
4. Stir in the spinach and cook until wilted. Season with the salt and black pepper to taste.
5. Serve on a bed of cooked grains, garnished with cilantro and coconut. If desired, offer lime wedges for squeezing over the curry. Store the cooled curry in an airtight container in the fridge for 4 to 5 days, or in the freezer for up to 1 month.
For Serving:
- Cooked basmati rice, quinoa, millet, or sorghum
- Chopped fresh cilantro leaves
- Unsweetened shredded or large-flake coconut
- Lime wedges (optional)
If you want her cookbook you can purchase it below! Let me know if you try this recipe or another from the book!!


text:
Migrating DNS from DO to Route53
We'd like to move our DNS over to AWS Route53 but instead of doing a full transfer, pointing the nameservers to AWS.
After setting the NS records for the domain, it seems like it hasn't yet propagated. My experience has been that DNS changes typically take a couple of minutes, unless I've made a mistake somewhere (albeit often it's said that it can take between 24-48 hours).
After running some dig commands, it seems like the NS records are still pointing to digital ocean.
Should I have made additional changes to get this to work?


text:
I first noticed this house many years ago because the front garden was part of the "Dunbar in Bloom" tour during Salmonberry Days. At that time, there were not many walled front gardens. This one was full of Cranes-bill geraniums waving in the breeze. I was struck by the beauty of the small garden. The mailbox sitting on the fence was a permanent contribution to "Dunbar in Bloom." Let's hope the owners took it with them. The house, with those large French doors which you can see in the background beyond the mailbox, was built in 1925 and demolished in November 2015. These photos were taken in March 2011 when it was sold.
The housing stock in the Dunbar area of Vancouver has undergone significant change in the past five years. Originally a working class neighbourhood with many quite modest homes surrounded by lovely gardens, it is now a neighbourhood that 99% of the people working in Vancouver cannot afford because the replacement homes are built to the maximum footprint and cost millions. Greenspace has been reduced. Included on this website are photos of many (not all) of the disappeared houses.
View Teardowns in the Dunbar area of Vancouver, BC in a larger map
View Teardowns in the Dunbar area of Vancouver, BC in a larger map
Saturday, December 19, 2015
Dunbar Not in Bloom
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Oregon Offense Gives Reason to Hope
The Virginia Cavaliers found their way to Autzen Stadium Saturday night, making it the first ever visit by an ACC team to the stadium.
They may never want to come back.
Though Oregon still has a lot of work to do on defense, especially before heading to Nebraska for game 3, the Offense was outstanding in the 44-26 win. There were so many play makers, it'd be hard to highlight just one.
Royce Freeman was the work horse- at one point in the first half, Freeman was the leading rusher and receiver- and game MVP after racking up 239 yards all purpose yards and 2 touchdowns, including an 85 yard scoring run.
"Last week, I fell short of my standards," Freeman said after the game. "I always want to get a hundred plus off carries.
This week, Freeman said he felt good and was getting a lot of "love" from Offensive Coordinator, Matt Lubick's play calling.
With his performance last night, Freeman is well on his way to breaking several Oregon records this season and remaining in the Heisman Trophy race.
Wide receiver, Devon Allen, showed he is in full football mode, after returning from the Rio Olympics just a few weeks ago.
Well, kind of.
After catching a 77 yard pass (yes, you read that right) from quarterback, Dakota Prukop, and taking it in for a touchdown, Allen gave a nod to his Rio hurdles by leaping over imagine ones in the end zone.
It turns out Allen had planned the move for over a week, all in good fun of course.
"I wanted to do something that sort of incorporated the whole Olympic hurdle thing," Allen said after the game. ". . .My teammates got a big kick out of it."
Making the night even more special for Oregon fans was Tight end, Pharaoh Brown. Not only did Brown return to the game for a second time this season (a miracle in and of itself), but he started at his position and scored a touchdown, making the crowd erupt with pride. Brown's story has been one of a kind and closely followed, making him one of College Football's favorite "comeback kid".
After the game, Brown was all smiles as he talked through the scoring play and hearing the cheers afterward.
"It felt good to score," Brown said. "It's been a long time."
Perhaps one of the best takeaways of the night was quarterback, Dakota Prukop's, performance. After playing a decent, yet safe, first game with the Ducks, Prukop stepped it up a notch and gave Oregon fans a reason to be hopeful in his ability to lead the team into Pac 12 play.
Prukop finished the night 21 of 31, 337 passing yards and 3 touch downs as he spread the ball all over the field to the many skill players on the field.
If all that weren't enough, Prukop made good decisions and secured the ball all night, ending the game with zero interceptions or fumbles.
But it may not be his solid stat line that meant the most.
After a pretty safe first game, many fans wondered if Prukop had that big play-making ability that will inevitably be needed moving forward into the meat of the Ducks' schedule.
Prukop answered that question last night as he made plays out of very little at times and showed he has a cannon of an arm, especially with that 77 yard pass to Devon Allen.
After the game, Prukop said he had total faith in his offensive line and that it was easy to play "when you have the skill players around you who just make the play no matter what," Prukop said.
When asked if he ever feels like he wants to show off his own skills, so-to-speak, Prukop said he definitely prefers to play "point guard" and get the ball to the guys around him. He talked about having to be the guy to carry his previous teams, at times, while still losing the game.
"Nothing replaces a win," Prukop said.
Looking ahead to Nebraska and Pac 12 play, Prukop said the team still has room to grow and that for him, personally, he is working on loosening up a bit.
"One thing that gets me sometimes is I get a little wound too tight and I got out there and I'm too calm," Prukop said. "One big takeaway is that it's ok if I get a little excited and I'm sure I will next week."
Speaking of next week. . .
The Ducks will head to Nebraska this week to face the Huskers who are now coached by former Oregon State Head Coach, Mike Riley, who Oregon fans should be very familiar with. It will prove to be a big stage and will be the Ducks' first true test of the season.
There are more questions to be answered, but one thing is abundantly clear: Oregon's offense will need some help from the defense, but they are definitely doing their part to move the Ducks up the polls.
After last night's game, Oregon fans have a lot of reasons to be hopeful.


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Boards Get Brains, Chalk Vanishes: "Schools across the country dump dusty chalkboards for touch-sensitive whiteboards connected to computers. Kids can solve problems, surf the web and even edit video with their fingertips..."
Today Wired reports on the rise in popularity of interactive whiteboards. At Lewis Elementary we currently have 6 classrooms equipped with these and with plans to add one more over the summer. The boards aren't cheap and the ideal installation involves ceiling mounts which can be rather expensive. At Lewis we choose to use projectors on carts, but would love to have them installed with ceiling mounts. The Wired article also has a slide show with examples of classroom use.


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We apologise for any inconvenience if you are wanting to place an order. The hopefish shop will be reactivated when the hopefish team are back in action. If you have any inquiries please contact us at
Code: Par108
Make your Pirate Party awash with young Long John Silvers with a T shirt painting pack for six children. Pack contains six quality T shirts and all you need to decorate them with shark, treasure, surfboard, palm tree and skull and crossbone motifs.
Price: £31.99
six good quality, white T shirts
six paint brushes
one foam stamp of a treasure chest (11.5x10.5cm)
one stencil of a flag and skull and crossbones with insert for blocking out skull and crossbones (8.6x11.8cm)
one stencil of a shark with insert to locate eye (12.5x7cm)
one palm tree stencil (7.7x13cm)
one surfboard stencil (4.4x11.7cm)
four DecoArt 'dry to fix' paints: glitter charcoal, bronze, grape purple and glorious gold and a bright green glitter writer
one pot of DecoArt gold Fabric Beadz for treasure
six disposable mixing trays
six disposable aprons
full 'how to' instructions and six 'T shirt care' instructions for taking home
a pot or two of water
a table with a wipe clean surface or disposable tablecloth
cardboard inserts to place inside each T shirt
because of the drying time for the Fabric Beadz or glue some children may need to take their T shirts home to dry on the cardboard supports
children aged 6 and upwards with adult help
paint and glue are non toxic but may stain. Children should be supervised throughout
|Dinosaur T Shirt Painting Party - Kit for 6
|Rain Forest T Shirt Painting Party Pack
|Mermaid, Fishes and Turtle T Shirt Painting Party Kit
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5 Safe Cosmetics Tips for Women of Color
Five Safe Cosmetics Tips for Women of Color
1. Skip toxic hair products.
2. Go natural! Bring your own neutralizing shampoo to the salon.
3. Avoid nail polishes that include any of the toxic trio: dibutyl phthalate, formaldehyde, and toluene.
4. Reduce your use of products with added fragrance.
5. Read labels closely and find safer alternatives using tools like the Think Dirty app and GuideGuide
Author: Sumana Rao | Posted on: April 28, 2017


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What happens when you combine the creative vision of a GoTriangle marketing guy, the reach of the United Way network, the powerhouse media outlets of WRAL and Capitol Broadcasting, and the generosity of the Triangle community? Within hours you surpass your original goal of filling one bus to help our neighbors impacted by Hurricane Matthew.
It can be tough for some young people growing up in Wake County. You might be struggling to graduate, or you might just be the one in five high school students who is a victim of bullying. You might even feel hopeless and be thinking about suicide. Everyone agrees we should be there for our
Since I was young, I knew I wanted to be a part of something bigger, something life changing, something that would make me feel like I made a difference at the end of the day. I had heard of United Way, but it wasn't until I started work as a Community Engagement Fellow that I
If it's autumn, it must be campaign season. Although we are working to engage donors and potential donors year-round, this is the time of year when, true to our history, workplace campaigns go into full force. What's the focus of the Campaign? This year, our approach is a little different — and it was inspired
It's the most wonderful time of the year The holiday season is just around the corner, and with it comes many volunteer opportunities in our community. Agencies across the Triangle area are working to bring holiday cheer to their clients, and you can help. Whether you're looking to volunteer for a few hours, find a
UWGT's goal is to be a catalyst for long term solutions as we address the short-term needs of our community. So, we continue to meet basic needs for healthcare, food, housing and safety for the most vulnerable people in our area, but we are also mobilizing the community effort to create better pathways for low-income


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Right now I'm in Kenya for the first time since my third grade heart got set on coming since my teacher, a former missionary here, described buffalo gorings and snakes being cut open to reveal live babies. Obviously, I had to go. Kenya is my 30th country. I'm super excited to be here working on photo stories for The Adventure Project and Africa New Life as well as touring schools with The Nobelity Project. Ostensibly, this trip is for work, but really, I was eager to come and explore on my own.
Last week I traveled briefly with a person who, I can only imagine, carries a lot of white guilt or self delusion or something, because he just didn't get it. When we passed by schools, he lobbed soccer balls into hordes of children and videotaped it. Kind of like throwing feed to koi, it caused thrashing and fights. Hopefully the balls didn't end up deflated and none of the kids were injured. Hopefully the kids don't grow up expecting this Santa aberration whenever they see cars of white people pass by. Later that evening, the guy walked up to a perfectly normal looking person in the parking lot outside a grocery store and handed them 500 Kenyan shillings and wished them the best. Because, well, I don't know why -- they were Kenyan? they were black? Somehow, he had decided that they deserved his charity. I was gobsmacked.
The most stomach churning part came when we passed an accident and there was a dead guy face down in the road (which I've seen more times than I would like while traveling abroad) and the guy in our van shutter-clicked the whole way through like the medical examiner. What the hell is he going to do with those photos? How is he going to explain it when those come up on his screen saver? Not everything is a photo opportunity. Around this point, I began to wonder if maybe he's just a bad person all-around and not simply a bad tourist.
The kicker (oh yeah, this was all within about 36 hours) came when our team was working on a photo shoot, taking beautiful pictures of a family in their garden that had become more green and productive with the purchase of a water pump we are promoting. The guy took one of the producers aside and asked if the photo subject should change clothes because she didn't look poor enough. Forcryingout... I didn't ask him to change out of his fully ventilated REI safari outfit because he didn't look cool enough. Since when is it appropriate to tell people how they should dress in their own homes? And poor doesn't mean undignified, for goodness' sake.
I say all of this not really to dump on the guy (but while we're at it!), but to explain why I didn't come to Africa. I didn't come to save anyone, offer anyone money, to take pictures of terrible things or to convince myself that I am different from anyone. I didn't come with the expectation of poor people looking poor or commanding my sympathy. I came because Kenya is freaking awesome. So often I tell people that I'm headed to (some developing country), and they say "Oh that's so nice of you," automatically assuming that I'm making myself miserable or doing someone a solid by visiting their shitty country. I truly just love places that some people assume are only mission-trip worthy. There's beautiful hiking in Haiti. Rwandan bird watching is top-notch. Some of the best, most luxurious (and, okay, the worst) experiences of my life have been in India. I go to Cuba for the beaches, Nepal for the scenery and Malaysia has the best street food you could hope for.
If anything, while traveling I rely on the mercies of others -- endless cups of tea, directions, translations, borrowed phone minutes. All of this from people who might have less money than I do, but they share of their country and themselves with me. I didn't come here for them, I came here for me. And that's okay, I think. There's a glorification of selfless travel, of the typical three days of "building" a school or "digging" a well with a day of beach and shopping at the end, that is truly misguided. Travel is enough. Travel responsibly, sustainably, lightly, respectfully, humbly, generously. That is what other countries want from us as Americans, I think. They don't want our charity; they want our tourism and our respect as equals. Leave the soccer balls and the vented safari hats at home and just be yourself. Unless you truly can't help yourself. Then you should go to Europe and a French person can look down on you for being tasteless. What goes around comes around.


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27 Jul The Noteworthy Benefits of Multi-Disciplinary Designers and Its Future
Multi-Disciplinary Design links a curious and empathetic method for seeing the world with strategies for perception, ideation, and generation. Designers morally react to the necessities of individuals and nature using an inventive procedure based methodology. Thoughts are executed in an important way and dependably address the status norm.
We as a whole practice design when to take any decisions. It may incorporate when we settle on which dress for purchasing, how we ought to serve our guest or which gift to give to a friend on his birthday. We design all the more intentionally in an expert way while in regards to the way toward molding of what we see and how we connect with it. Designing regularly requires considering the visual, useful, financial and sociopolitical measurements of both the design process and design object. It might include significant thought, research, demonstrating, intuitive conformity, and re- design.
Since all design work is established in a recognizable language, honing one, hence, implies showing signs of improvement at another. This is the worth of a multidisciplinary foundation. Your development in a particular order can expand your general configuration instinct at the same time.
Regarding aesthetics, design is firmly identified with artistic work. But, design's definitive objective is to give the client ease of use inside the boundaries of a selected experience. Plan changes over feeling into an experience.
It's additionally significant that since all configuration work has the same social connections and conditions at all the time, any design is intrinsically influenced by another. The designed sphere will dependably impact the designers which possess it. In a perfect world, designs of different orders ought to cooperate immaculately to shape a solitary curated experience. Knowing these associations could be crucial.
Benefits of a Multi-Disciplinary Designers:
- You see how to do things quick and effectively by comprehending what programming can be utilized to do what.
- You can assists individuals from various offices accomplish better or speedier results or by giving tips
- You are one stage nearer towards being a designer director, venture lead or design director
- You have a strong information of all features of design that you've accumulated over the years, making you an important individual that can apply learning from various corners to the current one
- You can simply move commercial ventures if a niche is as of now struggling
- You can make automated revenue from an industry while working in a totally distinctive one.
When you make new things or think of the new idea, there is a likelihood that it has been either totally or mostly done by another person. Searching for, and finding illustrations can help your basic process and furnish you with conductive experiences that you can execute. Understanding from others' oversights can likewise be useful. Designing something alluring and systematic doesn't generally grant achievement.
Therefore, thinking about designs is as old as human perception. You can explore and understand it, however, it will continuously be capable of reinventing itself and giving you with thrilling new things.


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I was just about to pull the trigger and order a mf733cdw but I read the scanner is limited to native scanning at 300dpi and anything above this is achieved through image processing. Has this been addressed through a firmware upgrade or is this correct, in which case I'm likely to go for a single purpose color laser instead and look elsewhere for image scanning.
This is the comment I found online, "I had both the MF634Cdw and MF733Cdw and returned both. The scanning specs on both printers are wrong. I spent many hours with Canon reps, and finally their engineer confirmed that both the MF634Cdw and MF733Cdw scan at native 300 dpi optical only via the ADF. Anything higher than 300 dpi has to be done through interpolation with the software via a PC, which is why you can't scan more than 300 dpi optical from the printers' control panel.
Canon uses the same scanning hardware as HP, and there is a defect on the scanning hardware, where the scanning element is slightly off the vertical axis that causes the scanned document to be crooked. You have to look hard and scan vertical parallel lines to notice this defect. The only fix is to use the software to correct it."
thanks for any clarification.
Solved! Go to Solution.
I've been doing more research on this and I found some more details pertinent to this matter. I understand the HP and Canon share the same scanning hardware and this reduction to 300x300 only occurs when using the automatic document feed (ADF) unit and scanning, copying or scanning with the glass bed doesnt restrict the scanning resolution. Theres some discussion on the HP forum this was a decision made to balance seed, cost and quality. This was rebuked becuase copying via the ADF does not suffer the same limitation and therefore appearsto be a marketing based decision to prevent cannabalisation of their scanner lines. They've come under some criticism for not being clear in their communications around this matter.
I've taken a closer look at the Canon specs and they still aren't 100% clear, i.e
Copier Specifications
Send Specifications (at the bottom of the page)
Sending Method: SMB, E-mail, FTP, iFAX Simple
Color Mode: Full Color, Monochrome
Scan Resolution: 300 x 300 dpi, 200 x 200 dpi
Its look like scanning is 600dpi but why is scan resolution under send specifications listed as 300dpi?
Hi canianon.
I can provide more information about the sending resolution.
Sending is limited to 300 dpi because of the limitations of sending emails and the receiving units on the other end of faxing. Most email providers have an upper limit of 10 MB. Scanning pages in 600 dpi will cause that limit to be reached quickly with only a few pages.
Regarding the previous post, the ADF only supports a maximum of 300 dpi, while the platen supports a maximum of 600 dpi. To scan a document to the computer from the platen, in 600 dpi, will require using the scanning software. Anything higher than 600 dpi will be interpolated by the the software. You can view more about the resolution guidelines at this [LINK].
I hope this information is helpful. If you have any further questions, please call us at 1-800-OK-CANON (652-2666), Monday - Friday 10:00 a.m. to 6:30 p.m. ET (excluding holidays).