AU2005263153A1 - Method and device to process, label and concentrate analytes - Google Patents

Description

WO 2006/007711 PCT/CA2005/001129 METHOD AND DEVICE TO PROCESS, LABEL AND CONCENTRATE ANALYTES Field of the Invention The present invention relates to a device and methods for the preparation, in fluid 5 samples, for the detection and quantification of an analyte that may be present in the fluid sample. Background of the Invention Micro-organisms, including toxic bacteria, yeast and mould account for several million cases of food-related illnesses and 9,000 deaths per year in the United States 10 alone. Contaminated poultry and meat products are a major cause of these deaths and illness. The four most common pathogens infecting poultry and meat products are E. coli 0157:H7, Campylobacter jejuni/coli, Salmonella species and Listeria species and L. monocytogenes. Contaminated water supplies are also a health hazard. The United States 15 Environmental Protection Agency has determined that the level of E. coli in water supply is a good indicator of health risk. Other common indicators are total coliforms, fecal coliforms, fecal streptococci and enterococci. Many disease conditions, such as bacterial and viral infections, cancers, heart attacks and strokes for example, may be detected through the testing of blood and tissues, 20 including interstitial fluid, saliva, urine, semen, feces, genetic coding and tissue sections, for markers that are associated with particular conditions. Early and rapid diagnosis and measurement of treatment efficacy is desired to enhance successful treatment. The accurate quantitation and rapid detection of micro-organisms, such as bacteria, viruses, fungi or other infectious organisms and indicators in food and water, on 25 surfaces where food is prepared and on other surfaces that should meet sanitary standards is a pressing need. There is also a serious need for the accurate and rapid identification of micro-organisms and disease markers of a patient. In a typical test assay, a fluid sample is mixed with a reagent, such as an antibody, specific to a particular analyte, such as an antigen. The reaction of the analyte with the -1- WO 2006/007711 PCT/CA2005/001129 reagent may result in a color change that may be visually observed, or in chemiluminescent, bioluminescent or fluorescent species that may be observed with a microscope or detected by a photodetecting device, such as a spectrophotometer. The reagent may also be a fluorescent or other such detectable labelling reagent that binds to 5 the analyte. Irradiation scattered, reflected, transmitted or absorbed by the fluid sample may also be indicative of the identity and type of analyte in the fluid sample. In a commonly used assay technique, two types of antibodies are used, both specific to the analyte. One type of antibody is immobilized on a solid support. The other type of antibody is labelled by conjugation with a detectable marker and mixed with 10 the sample. A complex between the first antibody, the substance being tested for and the second antibody is formed, immobilizing the marker. The label to identify the specific marker may also be an enzyme or a fluorescent or radioactive marker, which may then be detected. The reaction ingredients are all mixed in a single step for reaction in a single reaction liquid. See, for example, U.S. Patent No. 5,610,077. The reaction liquid is 15 simultaneously contacted with the immobilized capture antibody to allow for adsorption of analyte and marker antibody to the immobilized capture antibody. Competitive interference between the binding reactions typically induces instability during the testing process. The ratio of marker antibody to analyte has to be optimized depending on the analyte concentration likely to be present in the test fluid. These types of assays, known 20 in the art as "sandwich" immunoassays, typically provide only a threshold concentration for the analyte of interest and are read as qualitative, positive or negative results results. Fluid samples must often be concentrated to increase the number of analyte particles to a level that may be reliably detected. The fluid sample may then be transferred to a container, such as a test tube, for mixing with the reagents. The reacted 25 fluid sample is transferred to an assay device for analysis or to another container for mixing with other reagents. Such transfers, which are often conducted with a pipette are time consuming, labour intensive and expose the fluid sample and the people conducting the analysis to contamination. There is a need for a more convenient aseptic method to concentrate analyte, transfer fluid samples and mix fluid samples with reagents and other 30 compounds required for testing while minimizing competitive interference. -2- WO 2006/007711 PCT/CA2005/001129 Summary of the Invention The invention provides a sample processing and labelling applicator device for transferring a fluid sample, including an applicator body defining a hollow bore with an open end and an outlet, a plunger having a first end and being effective to fit within the 5 hollow bore, and a second end extending out of the open end where the first end is located within the hollow bore; and a reagent located between the piston and the outlet where the piston is located within the hollow bore. According to another aspect of the present invention, there is provided a method for concentrating an analyte present in a liquid sample, including the step of providing an 10 applicator body defining a hollow bore with an open end and an outlet, a plunger having a first end at one end and being effective to fit within the hollow bore, and a second end extending out of the open end where the piston is located within the hollow bore; and a reagent located between the first end and the outlet where the first end is located within the hollow bore; mixing and incubating the analyte and the reagent for a period of time 15 sufficient to react the analyte with the reagent and expelling a controlled amount of the portion through the outlet. According to another aspect of the present invention, there is provided a method for reacting an analyte present in a liquid sample, including the step of providing an applicator body defining a hollow bore with an open end and an outlet, a plunger having a 20 first end at one end and being effective to fit within the hollow bore, and a second end extending out of the open end where the first end is located within the hollow bore; and a reagent located between the first end and the outlet where the piston is located within the hollow bore, mixing and incubating the portion with the reagent for a period of time sufficient to react the analyte in the portion with the reagent; and expelling an amount of 25 the portion through the outlet. Brief Description of the Drawings Figure 1 is a front perspective view of a sample applicator of the present invention; Figure 2 is a cross-sectional view of the sample applicator taken along lines 2-2 of 30 Figure 1; and -3- WO 2006/007711 PCT/CA2005/001129 Figure 3 is a perspective view of a cap of the present invention detached from the sample applicator. Detailed Description of the Invention Sample applicator 10 is shown in Figure 1. The sample applicator 10 comprises an 5 applicator body that is preferably syringe body 12, and which is preferably cylindrical in shape. The syringe body 12 has a first end 62 and a second end 64. The syringe body 12 defines a hollow bore 14 with a first open end 16. A plunger 18 has a first end that preferably defines a piston 20 at its first end within the bore 14 and a second end 21 extending out of the first open end 16 of the bore 14. The second end 21 is preferably 10 widened for engagement by a user. The syringe body 12 defines an outlet 22 at the second end 64 of the syringe body 12. A flange 24 is preferably attached to the second end 64 of the syringe body 12. The flange 24 is preferably cylindrical. The flange defines outlet 26 that is in fluid communication with the outlet 22 of the syringe body 12. The outer surface of the flange 24 may have the shape of a Luer lock or taper. Reagents 43, 15 and preferably a concentrating material 45 to facilitate the analysis of the fluid sample, are provided between the plunger 20 and the outlet 22. The concentrating material 45 is preferably super absorbent polymer particles. The reagents are preferably monoclonal antibodies that are specific for a given analyte. Most preferably the reagents are lyophilized monoclonal antibodies that rapidly reconstitute upon mixing with a sample 20 fluid. The reagents may also be frozen, in liquid form, freeze dried into a pellet or may be dried on the interior surfaces of the syringe body 12. Volume indicator graduation lines 47, 49 are preferably provided for ease of use. On a 1 ml syringe body, the first volume line 47 may correspond to drawing 200 ml from the source and the second volume line 49 may correspond to drawing an additional 100 25 150 ml into the syringe bore 14, for example. The syringe applicator 10 includes a preferably cylindrical inlet/outlet assembly 25 comprising an upper region 80 and a lower region 82. The inner diameter of the upper portion 80 is about the same as or is slightly larger than the outer diameter of the lower portion 82. The upper region 80 has a first end 46 coupled to the second end 64 of the 30 syringe body 12. The lower region has an end 48 being for receiving a fluid sample. The inlet/outlet assembly 25 defines an opening 42 at the end 48 for passage of sample fluid. -4- WO 2006/007711 PCT/CA2005/001129 The inlet/outlet assembly 25 is provided to extend the distance from the flange 24 to the source for ease of use and to avoid insertion of syringe body 12 into the source. Fluid sample on the syringe body could contaminate the user and the surroundings. A length L for the inlet/outlet assembly 25 of at least about 2 cm is preferred 5 whereas 3 cm is more preferred. A preferred range is from about 2 cm to about 7 cm. The configuration of the inlet/outlet assembly 25 may vary according to the application to be carried out using the syringe applicator 10. A cap 30 is preferably attached to the end 48 of lower portion 82 of the inlet/outlet assembly 25 thereby covering the opening 42 and sealing the syringe applicator 10 to 10 prevent contamination prior to use. The cap 30 preferably has a first outwardly tapering cone-shaped cylindrical shaped section and a flat tip 76. The cap 30 is preferably attached to the inlet/outlet assembly 25 by means of perforations 70. The cap must therefore be broken off from the inlet/outlet assembly 25. An intact attached cap indicates that the syringe applicator 10 has not been used. The cap 30 is adapted to engage the lower region 15 82 in a fluid tight fit. The cap 30 is preferably a rigid moulded plastic such as polystyrene, an acrylic or polypropylene. A semi-rigid material may also be used in alternate embodiments. To use the sample applicator 10 of the present invention, the cap 30 is torn off the lower region 82 of the inlet/outlet assembly 25 and a fluid sample is drawn from a fluid 20 source. The fluid source may be a culture medium or media, depending on the concentration of the analyte in the fluid sample and the nature of the source matrix. For example, if the source is a food sample, culturing is typically required. If the source is feces, culturing may not be necessary. The lower region 82 is inserted into the source and the plunger 18 is withdrawn to draw a fluid sample from the source through the 25 inlet/outlet assembly 25 and into the bore 14 of the syringe body 12. About 300-350 .l is an appropriate amount, for example. If the volume lines 47 and 49 are provided, the first volume 47 is preferably positioned so that an appropriate amount of the fluid sample is drawn into the syringe applicator when plunger 18 is withdrawn up to the first volume line 47. The sample applicator 10 is then removed from the source and the plunger 18 is 30 preferably further withdrawn, to the second volume line 49. The cap 30 is placed over the inlet/outlet assembly 25 to cap and seal the fluid inside the syringe body 12. The syringe -5- WO 2006/007711 PCT/CA2005/001129 applicator 10 is then inverted several times to reconstitute the reagents 43, and to adequately mix the reagents with the fluid sample. If the concentrating material 45 is present, the fluid sample is allowed to incubate for about 20 minutes. If the concentrating material 45 is not present, about 5 minutes of incubation is sufficient to reconstitute the 5 reagents which are typically lyophilized antibodies thereby enabling only the now hydrated and conjugated marker to react specifically with the analyte in a controlled environment, thereby preventing interference and maintaining excess, viable marker antibody which are required for labelling the calibration arrays also immobilized on the same solid support as the capture antibody. After sufficient incubation, the cap 30 is 10 removed and the plunger is advanced to expel a sufficient amount of the fluid sample from the bore 14 through the inlet/outlet assembly 25 and into an assay device for analysis. Instead of being transferred to an assay device, the fluid sample may be transferred to another location, such as into another container for reaction with other reagents. 15 If only the concentrating material 45 is provided in the syringe body 12, the fluid sample may be transferred into a reaction chamber for reaction with reagents. Even if reagents are present in the reaction chamber, further reaction with additional reagents may be required. The fluid sample once exposed to the reagents in the sample applicator 10 may be 20 transferred to an assay device to be analyzed for the presence of analytes. The analyte may be a microbe such as a bacterium, a virus or a fungus, for example. The analyte may also be a biological substance such as a peptide, an amino acid, a nucleic acid, or a protein, such as an enzyme, an antibody, an antigen, an immuno-globulin or a hormone, for example. The analyte may also be a chemical substance such as a chemical element, 25 chemical compound or a polymer, for example. The fluid sample may also be transferred to a reaction vessel. The source of the fluid sample may be a culture medium containing a suspect food, water or fecal matter, for example. The source may be uncultured, as well. The sample applicator 10 includes reagents for reacting with and enabling the identification of an analyte in the fluid sample. 30 The reagents may be labelled with a fluorescent chemical, colorimetric or radioactive label, for example, as is known in the art. The label can also be a latex bead, metal -6- WO 2006/007711 PCT/CA2005/001129 colloidal particle, dye, enzyme or Stoke effect transducer. Other types of reagents may also be used, as is known in the art. If the analyte is present and the reagents are antibodies, the reagent antibodies bind to the analyte and label the analyte with the detectable marker. The fluid sample and 5 the labelled analyte may then be transferred to an appropriate assay device for analysis. A preferred assay device disclosed in U.S. Patent Application Publication No. 20020019062 entitled "Assay Devices", which is incorporated by reference herein in its entirety. In one application, the analyte is E. coli 0157:H7. The sample applicator contains lyophilized monoclonal mouse-anti-E. coli 0157:H7 antibody, available from Biodesign 10 International, Saco, Maine, U.S.A., for example. The antibodies may be pre-conjugated with Alexa Fluor@ 647 fluorescent dye available from Molecular Probes Incorporated, Eugene, Ohio, U.S.A., for example. Molecular Probes also provides an Alexa Fluor@ 647 Protein Labeling Kit (A-20173) for conjugating the antibodies to the dye. The labelled antibodies are preferably lyophilized as is known in the art. Alternatively, frozen 15 labelled antibodies may be used. In another alternative, an aliquot of the labelled antibodies in a liquid suspension may be drawn into or otherwise placed within the syringe body and the sample applicator 10 may be placed in a freezer to freeze the suspension. The sample applicator 10 may be used after removal from the freezer. In another example, the reagents may be a luciferin/luciferase complex for 20 detecting ATP as an indicator of the presence of biological contamination of a sample. ATP reacts with a luciferin/luciferase complex, in the presence of oxygen and magnesium ions (Mg+2 ) to produce visible light at a wavelength of 562 nanometers. Since all living cells contain ATP, detection of light in 562 nanometers is indicative of the presence of living cells. Luciferin/luciferase is commonly extracted from fireflies. 25 Luciferin/luciferase reagents are commercially available. An Adenosine 5' Triphosphate (ATP) Bioluminescent Assay Kit, FL-AAM, available from the Sigma-Aldrich Corporation, Milwaukee, Wisconsin, U.S.A., may be used, for example. FL-AAM is a lyophilized powder containing firefly luciferase, luciferin, MgSO 4 , EDTA, dithiothreitol ("DTT") and bovine serum albumin ("BSA") in a tricine buffer. After mixing with the 30 reagents, the fluid sample can be transferred to an appropriate assay device for detection -7- WO 2006/007711 PCT/CA2005/001129 of luminescent species resulting from the reaction. A device containing a photomultiplier tube may be used, for example. In addition to the labelled antibodies or other such reagents specific to the analyte, a second type of labelled antibody or other such reagent may be provided specific to a 5 control spot in an assay device. Application of the fluid sample to the assay device results in the second type of antibody binding to the control spot. This provides an indication that the assay device has been used, even if an insufficient amount of analyte is present in the fluid sample for detection of the analyte. In addition to the labelled antibodies or other such reagents specific to the analyte 10 and a second type of labelled antibody or other such reagent, a fluid dye may be added to the pre-lyophilization mix to confirm the presence and successful transfer of the dyed fluid sample to another vessel or assay device. A typical dye is Bromophenol Blue, Sigma Chemicals, St. Louis, Missouri 63178. The concentrating material 45 absorbs liquid from the fluid sample, increasing the 15 concentration of the analyte, such as bacteria, in the fluid sample. The concentrating material may be any material that absorbs fluid and does not react with the analyte in the fluid sample. Super-absorbent polymers, such as polyacrylates, cellulose derivatives and hydrogels, for example, are preferred. Super-absorbent polymers are discussed in Absorbent Polymer Technology, Studies in Polymer Science 8, edited by Lisa Brannon 20 Peppes and Ronald S. Harland, Elsevier Sciences, 1990, which is incorporated by reference herein, in its entirety. A suitable commercially available super-absorbent polymer is Favor@-Pac 100, from Stockausen Inc., Greensborough, North Caroline, U.S.A. Favor@-Pac 100 is said to be a salt of crosslinked polyacrylic acid and grafted copolymer. According to the manufacturer, the carboxylic groups of the polymer are 25 solvated when brought into contact with water or water based liquid. As a result, the groups partially dissociate into negatively charged carboxylic ions. In this state, the polymer chain contains a large number of similarly charged ionic groups that repel each other. The polymer coils become more bulky and thus extend their propensity to absorb aqueous fluid. Due to the cross-linking between the polymer chains, a gel is formed. 30 Water is strongly bonded by hydrogen bonds in the gel. The physical characteristics of the Favor@-Pac 100, as provided by the manufacturer, are: -8- WO 2006/007711 PCT/CA2005/001129 Physical Form White granules Particle Size 100-850 microns Product Density 540g/ +/-30 Sifting Properties Free Flowing 5 Moisture Content 5% +/-2 pH value (1% gel in 0.9% NaC1) 6.0 +/- 0.5 Storage >1 year under dry conditions Thirty (30) milligrams of Favor@-Pac 100 has been found to be an appropriate amount of Favor@-Pac 100 to use with a fluid sample of about 300-350 microliters, to 10 increase analyte concentration by a factor of three (3). Excessive concentration, such as 10 times or more, is generally not desirable. If the concentrating material is being provided with the lyophilized antibodies, it may be desirable to add additional antibodies to compensate for antibodies that may be absorbed by the concentrating material along with the fluid and for the initial high level of fluid in the sample. As mentioned above, 15 the super-absorbent polymer particles have a size preferably greater than about 0.80 millimeters. Smaller particles may be removed using a mechanical separation process, such as a sieve. Another example of a super-absorbent polymer is carboxymethyl cellulose. Depending on the sensitivity of the detection system and the concentration of the 20 analyte in the fluid sample, concentration of the fluid sample may not be required. The concentrating material 45 may be provided without the reagents. For example, concentration may be advantageous but it may be desired to label the analyte afterwards. The analyte could be labelled in the assay device, for example. In some applications, it is necessary to concentrate the fluid sample prior to labelling. 25 The present invention is not intended to be limited in scope by the specific embodiments described herein. Although the present invention has been described in detail for the purpose of illustration, various modifications of the invention as disclosed, in addition to those described herein, will become apparent to those of skill in the art for the foregoing description. Such modifications are intended to be encompassed within the 30 scope of the present claims. -9-

Claims (11)

1. An applicator for transferring a fluid sample, comprising: a body defining a hollow bore with a open end and an outlet; a plunger having a first end, said plunger being effective to fit within said hollow bore, and a second end extending out of said open end when said first end is located within said hallow bore; and a reagent located between said first end and said outlet when said first end is located within said hollow bore.

2. The applicator of claim 1 wherein the applicator is a syringe.

3. The applicator according to claim 1 wherein the first end of the plunger defines a piston.

4. The applicator of claim 1, further comprising: an inlet/outlet assembly, said assembly having being attached to said body at said outlet.

5. The applicator of claim 1, further comprising a seal between said outlet and said hollow bore.

6. The applicator of claim 1, wherein said reagent is a member selected from the group consisting of an antibody linked to a detectable marker and a luciferin/luciferase complex.

7. The applicator of claim 6, wherein said marker is a member selected from the group consisting of a heavy metal or a fluorescent, chemical, colorimetric or radioactive label.

8. The applicator of claim 1, wherein said applicator contains a concentrating material, and said concentrating material is a superabsorbent polymer.

9. The applicator of claim 6, wherein said superabsorbent material is selected from the group consisting of a polyacrylate, a cellulose derivative, Favor®-Pac 100, and a hydrogel. -10- WO 2006/007711 PCT/CA2005/001129

10. The applicator of claim 8, wherein said superabsorbent material is Favor@-Pac

100. 11. The applicator of claim 8, wherein said superabsorbent material is carboxymethyl cellulose. 12. The applicator or claim 1, further comprising a concentrating material located between said first end and said outlet when said first end is located within said hollow bore. 13. The applicator of claim 12, wherein said superabsorbent material is selected from the group consisting of a polyacrylate, a cellulose derivative, Favor®-Pac 100, and a hydrogel, and said reagent is a member selected from the group consisting of an antibody linked to a detectable marker and a luciferin/luciferase complex. 14. The applicator of claim 6, further comprising an antibody effective to bind to a control spot on an assay device. 15. The applicator of claim 4, wherein said inlet/outlet assembly has a first end coupled to a flange that defines said outlet, and a second end capable of being inserted into a source of fluid material to withdraw a sample of said fluid material. 16. The applicator of claim 15, wherein said reagent is a concentrating material, and said concentrating material includes particles of a superabsorbent polymer; and said inlet/outlet assembly includes a first tube having an inner diameter small than the size of the particles of said superabsorbent polymer; and said inlet/outlet assembly includes a second tube made of heat shrink tubing and capable of coupling said first tube to said flange. 17. The applicator of claim 1 further comprising a cap attached to said applicator body for sealing said oulet. 18. The applicator of claim 15 wherein the cap is attached to said applicator body by perforations. 19. A method of concentrating an analyte present in a liquid sample, comprising the steps of providing an applicator comprising an applicator body defining a hollow bore with a -11- WO 2006/007711 PCT/CA2005/001129 open end and an outlet; a plunger having a first end, said plunger being effective to fit within said hollow bore, and a second end extending out of said open end when said first end is located within said hollow bore; and a concentrating material located between said first end and said outlet when said first en is located within said hollow bore; drawing at least a portion of said liquid sample into the applicator; mixing and incubating said portion with said concentrating material for a period of time sufficient to concentrate said portion by absorbing fluid into said concentrating material; and expelling an amount of said portion through said outlet. 20. The method of claim 19 wherein the applicator is a syringe. 21. The method according to claim 20 wherein the first end of the plunger defines a piston. 22. A method for reacting an analyte present in a liquid sample, comprising the steps of: providing an applicator comprising an applicator body defining a hollow bore with a open end and an outlet; a plunger having a first end, said plunger being effective to fit within said hollow bore, and a second end extending out of said open end when said first end is located within said hollow bore; and a concentrating material located between said first end and said outlet when said first emn is located within said hollow bore; drawing at least a portion of said liquid sample into the applicator; mixing and incubating said portion with said reagent for a period of time sufficient to react said analyte in said portion with said reagent; and expelling an amount of said portion through said outlet. 23. The method of claim 22 wherein the applicator is a syringe. 24. The method according to claim 22 wherein the first end of the plunger defines a piston. -12- WO 2006/007711 PCT/CA2005/001129 25. A method according to claim 22 further comprising the step of: placing the resultant analyte-reagent complex in contact with an immobilized specific binding partner. - 13 -