Patent Application
20140255271
The present invention relates to methods and devices useful for the collection and concentration of urine samples.
Recently there has been a shift towards non-invasive biological sample collection for research and diagnostics. Non-invasive specimen collection has a number of advantages including the fact that it is preferred by patients, requires less specialized personnel and equipment, and can be performed in various different settings, not just doctor's offices (Cook et al., 2005).
Urine represents an ideal non-invasive sample for both research and diagnostic applications. It has recently been well established that urine is a rich source of macromolecules including DNA, RNA and proteins, and the diagnosis of STI's based on the presence of DNA or microorganisms within the urine is well documented (Chapin, 2006). Urine DNA can be used in many different applications in diagnostics, including monitoring graft rejection (Zhang et al., 1999), transplantation monitoring (Botezatu et al, 2000), detecting nephrotoxicity (Le Lann et al., 1994), and even prenatal sex determination (Botezatu et al., 2000). Urine DNA is also being used in the detection of pathogenic bacteria (Shigemura et al., 2005), the Lyme disease-associated bacteria Borrelia burgdorferi (Exner et al., 2003), detection of HPV (Prusty et al., 2005), CMV (Paixo et al., 2005), BK virus and SV40 (Vanchiere et al., 2005). Furthermore, a variety of studies have shown that different types of cancer can be detected using urine derived-DNA, including bladder cancer (Pirskalaishvili et al., 1999; Su et al., 2004), and prostate cancer (Henrique et al., 2004). Also, urine-derived DNA has been used in identity testing (Marques et al., 2005). Furthermore, DNA arising from cells shed into the lumen of genitourinary tract can be used for the detection of genetic anomalies and neoplasia associated with the bladder, prostate or kidney (Cairns, 2004; Mehta et al., 2006). The use of urine as a sample for the diagnosis of non-genitourinary infections has also been successfully reported in tuberculosis (Cannas et al., 2008), leishmaniasis (Fisa et al., 2008) and malaria (Mharakurwa et al., 2006). Thus, urine can be used for point-of-care diagnosis of STI's as well as many other diseases and infections. Therefore the collection, concentration and/or preservation of urine samples for various downstream applications and tests are necessary.
Traditionally, urine samples have been collected into simple, plastic, screw-top collection containers. Shipping large volumes of urine can be problematic and costly, especially, if the urine must be shipped on ice or under cold temperature.
Another problem associated with the use of urine for diagnostic and research applications is that the analytes present within the urine are often very dilute or present in very small amounts. For example, lipoarabinomannan (LAM), a major glycolipid component of the cell wall of Mycobacterium tuberculosis, the causative agent of Tuberculosis, can be found in dilute amounts in urine. Traditional methods for the detection of LAM in urine for diagnosis of Tuberculosis require concentration and purification of the urine, which is very time-consuming (Reither et al., 2009). Thus concentration of the urine sample and concentration of the analytes present within the urine must be performed prior to analyzing the urine sample. Common methods for the concentration of urine samples involve the use of filters or membranes. These are often molecular-weight cut-off membranes and they require the use of centrifugation in order to concentrate the sample. There would be problems with using these concentration devices in the field or in resource-limited areas to concentrate the components of the urine for analysis, such as in the case of point-of-care diagnostic tests. Furthermore, these concentration steps often take 2-4 hours to complete. During such an extended period of time microorganisms present in urine would grow thus altering the composition of urine. Therefore, the combination of these drawbacks, including problems with shipping and concentration, greatly limit the use of urine for diagnostics, particularly in resource-limited settings and for point-of-care diagnostics.
The present invention provides a method that allows for the rapid concentration of urine samples that can be performed without the use of specialized equipment, such as filters or centrifuges, or the need for electricity.
In one aspect, provided is a method for concentrating one or more target analytes in a urine sample comprising the steps of:
In an embodiment, the binding agent comprises silicon carbide.
In an embodiment, the one or more analytes is DNA, RNA, microRNA, protein, bacteria, virus, yeast, exfoliated cells, polycyclic aromatic compounds and/or lipopolysaccharides.
In an embodiment, the method further comprises the step of:
In an embodiment, the preservation solution is Norgen Urine Preservative™.
In an embodiment, the method further comprises the step of:
The present invention further provides devices that allow for the rapid concentration of urine samples and which do not require the use of electricity.
In another aspect, provided is a device for the concentration of one or more target analytes contained in a urine sample, said device comprising:
In an embodiment, the collection reservoir is substantially parallelepipedal in cross-section.
In an embodiment, the collection reservoir comprises a tapered lower portion.
In an embodiment, the collection reservoir has a volume sufficient to contain the pre-determined amount of the particulate binding agent.
In an embodiment, the binding agent comprises silicon carbide.
In an embodiment, the means for sealing the opening of the tube is a screw on cap.
In another aspect, provided is a device for the concentration of one or more target analytes contained in a urine sample, said device comprising:
In an embodiment, the binding agent comprises silicon carbide.
In an embodiment, the device further comprises means for sealing the opening of the second tube.
In an embodiment, the means for sealing the opening of the second tube is a screw on cap.
In an embodiment, the means for sealing the opening of the first tube is a screw on cap.
In an embodiment, the lower portion of the first tube and the upper portion of the second tube are provided with matching threads, whereby the lower portion of the first tube and the upper portion of the second tube can be screwed together.
In an embodiment, the collection reservoir is substantially parallelepipedal in cross-section.
In an embodiment, the collection reservoir comprises a tapered lower portion.
In an embodiment, the collection reservoir has a volume sufficient to contain the pre-determined amount of the particulate binding agent.
In a further aspect, provided is a kit for the collection and concentration of one or more target analytes from a urine sample; said kit comprising:
the concentration device according to the present invention; and
instructions for use of said concentration device.
In an embodiment, the kit further comprises a pre-aliquoted sealed ampule containing a preservation solution.
In an embodiment, the preservation solution is Norgen Urine Preservative™.
In an embodiment, the pre-aliquoted sealed ampule is a dropper, a syringe, a micro-doser, a pipette, a vial, or a pouch.
In a further embodiment, the kit further comprises a lysis solution.
Preferred embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:
Similar references are used in different figures to denote similar components.
Conventional methods for urine collection do not allow for the rapid concentration of urine samples without the requirement for special equipment (i.e. filters and centrifuges) or electricity, and as such, are not suitable for use in resource-limited settings or for point-of-care diagnostics.
In contrast, the concentration devices and methods disclosed herein rapidly and simply concentrate macromolecules, micro-organisms and cells found in urine without the necessity for additional specialized equipment or electricity and are well suited for use in resource-limited areas. The disclosed methods and devices can be used for both research and diagnostic applications, and offers considerable advantages over any other current urine collection and concentration methods and systems. The disclosed methods and devices are useful for reducing the shipping volume for urine samples as the urine samples can be concentrated as high as 50-fold to 100-fold. Furthermore, with the use of a chemical preservative, the concentrated urine analytes can be shipped at room temperature. This makes shipping urine samples much easier as refrigeration is not required and the sample volumes shipped are very small.
In a first aspect, provided is a method for concentrating one or more target analytes in a urine sample comprising the steps of: a) collecting a sample of urine into a concentration device comprising at least a container and a particulate binding agent; b) contacting the urine sample with the particulate binding agent in the presence of a binding buffer, wherein said binding agent specifically binds the one or more target analytes; c) mixing the urine sample, binding agent, and binding buffer; d) allowing the binding agent to settle within the container by gravity; and e) removing the liquid portion of the urine sample from the container, wherein the binding agent remaining in the container is bound with the one or more target analytes.
The choice of binding agent and binding buffer will depend on the desired urine analyte to be concentrated. The one or more target analytes may include macromolecules (including but not limited to DNA, RNA, microRNA, protein, polycyclic aromatic compounds, lipopolysaccharides), microorganisms (including but not limited to bacteria, fungi, yeast), viruses and cells (including but not limited to exfoliated cells). Particulate binding agents capable of binding macromolecules, microorganisms and/or cells are well known in the art and those which can be precipitated or settled by gravity are suitable for the methods disclosed herein. In one embodiment, the binding agent may be a resin wherein the surface of the resin is modified by methods known in the art in order to specifically bind the target analytes in the urine sample.
In a preferred embodiment, the binding agent comprises silicon carbide (SiC). Silicon carbide is available in a variety of grit sizes or grades, and each grade has a different average particle size. In a preferred embodiment, the binding agent will comprise silicon carbide having a grit size between 1000-2500 (diameter ca. 1-5 μm), preferably a grit size between 2000-2500 and even more preferably, a grit size of 2000.
In embodiments employing silicon carbide, the binding buffers may comprise about 0.1 to 1 mM phosphoric acid, preferably about 0.5 mM phosphoric acid, and more preferably about 0.833 mM phosphoric acid.
The amount of binding agent and binding buffer used will depend on the volume of the urine sample and the predicted amount of the target analyte contained in the urine sample and can be determined by the person skilled in the art using conventional methods. In embodiments employing silicon carbide, typically, for a urine sample of about 20-30 ml, the amount of silicon carbide will be about 0.3-0.5 g and the amount of binding buffer will be about 0.5-1 ml.
The urine sample, binding agent, and binding buffer can be mixed by sealing a container containing the urine sample, binding agent, and binding buffer and shaking the container. After mixing, the binding agent settles rapidly by gravity to the bottom of the container. In embodiments where the binding agent comprises silicon carbide, the settling time is typically about 10 minutes. It will be appreciated however, that the binding agent may take more or less time to settle depending on the amount of binding agent used and the volume of sample. After the binding agent has settled, the liquid can be removed from the container by decanting. Alternatively, the liquid can be removed through the use of a pipette, syringe, dropper or any other device capable of transferring liquid. The binding agent remaining in the container is bound with the one or more target analytes.
The container containing the binding agent and target analyte(s) can be sealed for storage. It is generally recommended that urine samples be tested within two hours of collection. If testing is not performed within 2 hours, the urine samples may be placed in a refrigerator for short term preservation of the urine sample. However, during field studies and in resource-limited areas a refrigerator may not be available to allow for storage of the urine sample. Thus, chemical preservation of urine specimens may be utilized if testing or refrigeration within that two hour window is not possible. In a further embodiment, prior to storage and/or further processing of the target analytes, a urine preservation solution is added to the binding agent bound with the one or more target analytes.
A variety of urine preservatives (e.g. tartaric and boric acids) are available that allow urine to be kept at room temperature. These preservatives are designed to maintain the specimen in a state equivalent to refrigeration by inhibiting the proliferation of organisms that could result in a false positive culture or bacterial overgrowth. Generally, the length of preservation capacity ranges from 24 to 72 hours. A preservation solution can also be used to preserve the integrity of the target analytes (such as for example, DNA, RNA and proteins) at room temperature. The preservation solution may also kill concentrated microorganisms bound to the binding agent thereby resulting in the microorganism being non-infectious.
In some embodiments, it may be desirable to provide a preservation solution which also allows for preservation of the nucleic acids and proteins at room temperature for storage or shipping. While it will be appreciated that the time period for preservation may be as short as the time necessary to transfer a sample from the point of collection to the point of analysis, extended periods of preservation may be also be desirable (i.e. days, months or greater). In a preferred embodiment, the method disclosed herein employs NORGEN URINE PRESERVATIVE™ (Norgen's Urine Preservative Single Dose Ampules, Thorold, Canada, Cat#18124) as the preservative. The use of NORGEN URINE PRESERVATIVE™ allows for the preservation of the concentrated analytes for about 1 year at room temperature. While this preservation solution can be used to preserve samples for room temperature storage and shipping, which is generally from 15° C. to 40° C., in other embodiments the samples may be stored in cool environments, such as −20° C. or 4° C., or in warm environments including up to about 55° C.
In another embodiment, the method disclosed herein further comprises the step of adding a lysis solution to the binding agent bound with the one or more target analytes. The method can be practiced using conventional lysis solutions known in the art such as, but not limited to lysis solutions comprising lytic enzymes, detergents and/or chaotropes. The lysis solution will lyse the concentrated microorganisms and/or cells such that the nucleic acids and proteins are released. Once lysed the released nucleic acids and proteins can be isolated using methods known in the art for research or diagnostic purposes. The isolated nucleic acids can then be analyzed by any technique known in the art which can be used for analyzing nucleic acids, including but not limited to gel electrophoresis or PCR amplification techniques while the isolated proteins can be analyzed by any technique known in the art which can be used for analyzing proteins, including but not limited to immunoassays.
As shown in
As shown in
The device 10 may be constructed using polypropylene or other suitable materials. The size of device 10 will depend on the amount of urine to be collected. In a preferred embodiment, the opening of the tube 12 can have an outer diameter of about 26.5 mm and an inner diameter of about 24.5 mm. The tapered lower portion 18 of the tube 12 can have an outer diameter of about 5.0 mm and an inner diameter of about 3.0 mm. The tube 12 can be about 110 mm in length wherein, the upper portion 14 is about 70 mm in length, the lower tapered portion 18 is about 15 mm in length, and an upper portion 21 of the collection reservoir 20 is about 12 mm in length and a lower tapered portion 22 of the collection reservoir 20 is about 14 mm in length. The volume of the tube 12 can be about 25 ml. The volume of the collection reservoir 20 can be about 1.1 ml.
As discussed above, choice of binding agent and binding buffer will depend on the desired urine analyte to be concentrated. In embodiments, wherein the analyte specific resin is silicon carbide, the binding buffer may comprise about 0.1 to 1 mM phosphoric acid, preferably about 0.5 mM phosphoric acid, and more preferably about 0.833 mM phosphoric acid. In another embodiment, the binding buffer may comprise about 10% to 30% ethanol. In further embodiment, the binding buffer may comprise about 0.5 M to about 2 M guanidine hydrochloride. In a still further embodiment, the binding buffer may comprise about 0.1M to about 1M sodium citrate. Typically, the device 10 will comprise about 0.3-0.5 g of silicon carbide resin and about 0.5-1 ml of binding buffer.
In use, the device 10 can be used to rapidly concentrate a urine sample without the use of specialized equipment such as centrifuges, and accordingly, without the need for electricity. To concentrate a urine sample, the device 10 is opened by removing any lids or seals and urine is collected into the tube 12 either through urinating directly into the tube 12 or by transferring from an initial collection container. The device 10 is then closed, and the urine is mixed well with the analyte specific binding agent 20 and the binding buffer 22 contained in the tube 12 by shaking the device 10 and its contents. Once the binding agent 20 has settled into the collection reservoir 20, the device 10 is opened and the remaining liquid is removed by decanting. The liquid may also be removed through the use of a pipette, syringe, dropper or any other device capable of transferring liquid. The target analyte is now in concentrated form and bound to the binding agent and may be subjected to further processing including the addition of a preservation solution or a lysis solution.
As seen in
In a further embodiment, the second tube 80 can be substituted with the device 10 as described above (not shown). In such embodiments, the lower portion 76 of the first tube 32 and the upper portion 14 of the device 10 can be provided with matching threads, whereby the lower portion 76 of the first tube 70 and the upper portion 14 of the device 10 can be screwed together to form a liquid tight seal.
The size of device 60 will depend on the amount of urine to be collected. In a preferred embodiment, the opening of the first tube 70 can have an outer diameter of about 29.5 mm and an inner diameter of about 27 mm. The lower portion 76 of the first tube 70 tapers at the bottom and defines a bottom opening having outer diameter of about 13 mm and an inner diameter of about 9.9 mm. The first tube 70 can be about 149 mm in length. The second tube 80 can be about 47.5 mm in length. The opening of the second tube 80 can have an outer diameter of about 9.6 mm and an inner diameter of about 8.4 min. The inner tapered portion 86 may be about 14.5 mm in length with the tapered portion 86 having an upper diameter of about 8.1 mm and the collection reservoir 88 having a diameter of about 4.6 mm. As discussed above, the choice of binding agent and binding buffer will depend on the desired urine analyte to be concentrated. In a preferred embodiment, the binding agent comprises silicon carbide and the binding buffer may comprise about 0.1 to 1 mM phosphoric acid, preferably about 0.5 mM phosphoric acid, and more preferably about 0.833 mM phosphoric acid. In another embodiment, the binding buffer may comprise about 10% to 30% ethanol. In further embodiment, the binding buffer may comprise about 0.5 M to about 2 M guanidine hydrochloride. In a still further embodiment, the binding buffer may comprise about 0.1M to about 1M sodium citrate. Typically, the concentration device may comprise about 0.3-0.5 g of silicon carbide and about 0.5-1 mL of binding buffer.
In use, the first and second tubes 70, 80 are attached together and urine is collected in the larger first tube 70 by either urinating directly into the tube or transferring the urine from another collection container. The first tube 70 is closed and the urine, binding agent 100 and binding buffer 102 are mixed well by shaking. The binding agent 100 with the bound analytes then settles rapidly by gravity into the smaller second tube. Once the binding agent 100 has settled, the cap 78 is removed and the liquid is decanted with the binding agent 100 with bound target analytes remaining at the lower tapered portion 86 of the smaller second tube 80. The liquid can also be removed through the use of a pipette, syringe, dropper or any other device capable of transferring liquid. The smaller second tube 80 can then be removed from the first tube 70 for further processing of the urine analytes, including adding a preservation solution or adding a lysis solution. The resulting preserved and/or lysed sample contained in the smaller tube can then be stored at room temperature and shipped at room temperature.
In a further aspect, disclosed is a kit for the collection and concentration of urine. The kit can be used to practice the methods disclosed herein. The kit may comprise either the single tube or double tube embodiments of the disclosed concentration devices along with printed instructions for use of the concentration device for concentrating urine analytes. The choice of the binding agent and the binding buffer comprising the concentration device will depend on the target analyte.
The kit may further comprise a pre-aliquoted, sealed ampule containing a preservation solution. In a preferred embodiment, the preservation solution is the NORGEN URINE PRESERVATIVE™ (Norgen's Urine Preservative Single Dose Ampules, Thorold, Canada, Cat#18124). The pre-aliquoted, sealed ampules may include, but are not limited to droppers, syringes, micro-dosers, pipettes, vials or pouches. The kit may further comprise a lysis solution.
Although the invention has been described with reference to illustrative embodiments, it is to be understood that the invention is not limited to these precise embodiments, and that various changes and modification are to be intended to be encompassed in the appended claims.
A 50 mL sample of mid-stream urine was collected into a urine collection container, and 30 mL was then transferred into a urine concentration/shipping device (Norgen's Urine Concentration and Preservation Device, Thorold, Canada, Cat#38056) containing silicon carbide and Binding Buffer. The device was closed and was inverted by hand several times in order to mix. The device was then placed upright and the silicon carbide is allowed to settle by gravity for 10 minutes. After the silicon carbide was settled, the top of the device was removed and the supernatant was decanted into a second 50 cc tube, ensuring that none of the silicon carbide was transferred with the supernatant. Next, 1 mL of Norgen Urine Preservative (Norgen's Urine Preservative Single Dose Ampules, Thorold, Canada, Cat#18124) was added to the silicon carbide and again the silicon carbide was mixed well by hand. The urine was then stored at room temperature for 1 hour prior to processing. After 1 hour, proteins were isolated from both the preserved urine sample and the decanted urine supernatant. Proteins were isolated from the preserved urine sample using Norgen's ProteoSpin Urine Protein Concentration Kit (Thorold, Canada, Cat#17400) and a modified procedure. Briefly, 500 μL of Protein Wash Solution is added to the preserved urine sample, mixed well by hand, and settled by gravity for 10 minutes. The Wash Solution was removed using a 1 mL micropipette and discarded. This wash step was then completed a second time. Next, 300 μL of Elution Buffer was added, mixed by inversion, and the silicon carbide settled by gravity. The elution (E1) was then removed using a 1 mL micropipette and transferred to an eppendorf tube containing 30 μL of Protein Neutralizer. The elution step was then repeated to obtain E2. To isolate proteins from the urine supernatant, 1 mL of the supernatant was processed as per Norgen's ProteoSpin Urine Protein Concentration Kit (Thorold, Canada, Cat#17400). To analyze the proteins present in both E1 and E2 from the concentrated and preserved urine sample, as well as from the urine supernatant, a Bradford Assay was performed and the results were graphed. As shown in
A 30 mL human urine sample was spiked with HEK 293 (10,000 cells/mL) and DH5α (10,000 cells/mL). The spiked urine sample was transferred into a urine concentration/shipping device (Norgen's Urine Concentration and Preservation Device, Thorold, Canada, Cat#38056) which contains silicon carbide and Binding Buffer. The device was closed and was inverted by hand several times in order to mix. The device was then placed upright and the silicon carbide was allowed to settle by gravity for 10 minutes. After the silicon carbide was settled, the top of the device was removed and the supernatant was decanted into a second 50 cc tube, ensuring that none of the silicon carbide was transferred with the supernatant. Next, 1 mL of Norgen Urine Preservative (Norgen's Urine Preservative Single Dose Ampules, Thorold, Canada, Cat#18124) was added to the silicon carbide and again the silicon carbide was mixed well by hand. The urine was then stored at room temperature for 1 hour prior to processing. After 1 hour, DNA was isolated from both the preserved urine sample and the decanted urine supernatant. To isolate DNA from the preserved sample, Norgen's Urine DNA Isolation Mini Kit (Thorold, Canada, Cat #27000) was used with a modified protocol. Briefly, Pronase and Proteinase K were added and the sample mixed well by hand. Next, 500 μL of Binding Solution was added, the sample mixed well by hand, and allowed to settle by gravity for 10 minutes. After settling the supernatant was removed using a 1 mL micropipette and discarded. Next, 500 μL of Wash Solution I is added to the preserved urine sample, mixed well by hand, and settled by gravity for 10 minutes. The Wash Solution was removed using a 1 mL micropipette and discarded. Next, 500 μL of Wash Solution II is added to the preserved urine sample, mixed well by hand, and settled by gravity for 10 minutes. The Wash Solution was again removed using a 1 mL micropipette and discarded. Next 300 μL of Elution Buffer was added, mixed by inversion, and the silicon carbide settled by gravity. To isolate DNA from the urine supernatant, 1 mL of the supernatant was processed as per Norgen's Urine DNA Isolation Micro Kit (Thorold, Canada, Cat#18100). The purified DNA was then analyzed by running aliquots on a 1.5% agarose gel. As shown in
Four different 1 mL urine samples were spiked with 5 pg/mL of lipoarabinomannan (LAM) and mixed well. Next, 100 mg of silicon carbide (grit size 2500) was added to the tubes containing the urine sample. Ethanol was then added to 3 of the tubes in order to allow the LAM to bind to the silicon carbide resin. The ethanol was added such that the final concentration of ethanol was 10% in the first tube, 20% in the second tube, and 30% in the third tube. The fourth tube was used as control and no ethanol was added. All the tubes were closed and mixed by inverting for 30 seconds. After mixing, the resin settled through gravity to the bottom of the tubes. The urine supernatant was then removed using a pipette and transferred to a clean tube. Next, 200 μL of water was added to the resin and mixed by inversion for 30 seconds in order to elute the bound LAM from the silicon carbide resin.
To test the effect of increasing ethanol concentration on binding LAM to silicon carbide, 100 μL of the urine supernatant that was removed from each tube after binding was subjected to the Clearview TB ELISA test, which uses antibodies specific to LAM.
As seen in
Four different 1 mL urine samples were spiked with 10 pg/mL of lipoarabinomannan (LAM) and mixed well. Next, 100 mg of silicon carbide (grit size 2500) was added to the tubes containing the urine sample. Guanidine hydrochloride was then added to 3 of the tubes in order to allow the LAM to bind to the silicon carbide resin. The guanidine hydrochloride was added such that the final concentration of guanidine hydrochloride was 0.5M in the first tube, 1.0M in the second tube, and 2.0M in the third tube. The fourth tube was used as control and no guanidine hydrochloride was added. All the tubes were closed and mixed by inverting for 30 seconds. After mixing, the resin settled through gravity to the bottom of the tubes. The urine supernatant was then removed using a pipette and transferred to a clean tube. Next, 200 μL of water was added to the resin and mixed by inversion for 30 seconds in order to elute the bound LAM from the silicon carbide resin.
To test the effect of increasing guanidine hydrochloride concentration on binding LAM to silicon carbide, 100 μL of the urine supernatant that was removed from each tube after binding was subjected to the Clearview TB ELISA test, which uses antibodies specific to LAM.
As seen in
Three different 1 mL urine samples were spiked with 5 pg/mL of lipoarabinomannan (LAM) and mixed well. Next, 100 mg of silicon carbide (grit size 2500) was added to the tubes containing the urine sample. Sodium citrate was then added to the 3 tubes in order to allow the LAM to bind to the silicon carbide resin. The sodium citrate was added such that the final concentration of sodium citrate was 0.2M in the first tube, 0.4M in the second tube, and 0.6M in the third tube. All the tubes were closed and mixed by inverting for 30 seconds. After mixing, the resin settled through gravity to the bottom of the tubes. The urine supernatant was then removed using a pipette and transferred to a clean tube. Next, 200 μL of water was added to the resin and mixed by inversion for 30 seconds in order to elute the bound LAM from the silicon carbide resin.
To test the effect of increasing sodium citrate concentration on binding LAM to silicon carbide, 100 μL of the urine supernatant that was removed from each tube after binding was subjected to the Clearview TB ELISA test, which uses antibodies specific to LAM.
As seen in
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2710904 | Jul 2010 | CA | national |
This application claims priority under 35 U.S.C. §119 to Canadian patent application No. 2,710,904 filed Jul. 23, 2010, the contents of which are incorporated by reference. This application is a divisional of U.S. Ser. No. 13/188,583, filed Jul. 22, 2011 under 37 CFR 1.53(b), the contents of which are incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13188583 | Jul 2011 | US |
| Child | 14265450 | US |
