HK1058968A - Reagent delivery device and method of use - Google Patents

Description

Reagent delivery device and method of use

Background

The present invention relates to a method and apparatus for filtering a biologically derived sample and mixing the sample with a reagent for analyzing an analyte in the sample. Once the sample is filtered off and mixed with the reagents, it can be analyzed using conventional analytical techniques. The sample generally includes an analyte in the form of a bindable target in a biologically derived liquid sample that is expected to contain the target. Many different forms of analytical devices can be used to analyse the filtered reagent-mixed analyte. Such a device is disclosed, for example, in U.S. patent No.585,526, which is incorporated herein by reference. Many of these devices use a reaction membrane on which is immobilized a receptor that specifically binds to the target. Typically, the sample to be tested is added dropwise to the reaction membrane. If the target is present in the sample, it will bind to the immobilized receptor. Various methods are used to determine whether a target binds to a receptor, and thus whether the target is present in the sample. In one commonly used method, an immunologically active binding capable of specifically binding to a target attached to a detectable label is applied to the membrane. In the device of the above patent, the sample is added to the top of the reaction membrane and flows through the reaction membrane onto the absorbent material below the membrane, which pulls the sample through the membrane.

In testing blood sample solutions, one known device is available from EY Laboratories, inc^TMThe name "comprises a liquid container having a generally cylindrical reservoir body having an open end that mates with a movable closure member, the open end defining a liquid passageway. The closure takes the form of a tapered head. A filter is disposed in the conical head across the liquid passage. The blood is placed in a reservoir body which is sealed to the closure member. Liquid reagents may be added to the reservoir. The reservoir body is flexible so that by compressing it blood is forced through the filter and out the conical outlet in the form of droplets. The filtered blood sample is then introduced into an analysis device such as the membrane device described above using a filtration device of the type described above. Reagents for forming the assay, e.g. labels retained on the reaction surface, are added in separate steps after the sample has flowed through the membraneOnto the membrane. This separate step increases assay time and reduces the possibility of technician error.

Disclosure of Invention

According to the present invention there is provided apparatus for filtering a biologically derived sample and for mixing said sample with a reagent, said apparatus comprising a liquid container defining a reservoir chamber and including a container outlet, a filter and a liquid flow-through matrix disposed in a flow path between said container chamber and container outlet, water-soluble dry reagent retained on said matrix, and means for urging an aqueous liquid sample in said reservoir chamber through said filter and matrix and out of the container outlet.

According to another aspect of the present invention there is provided a method for filtering a biologically derived sample and mixing the sample with reagents, said sample including at least one analyte reagent for analysis of an analyte, said method comprising:

(a) applying pressure to an aqueous liquid sample in said receptacle chamber of said container to cause said sample to flow in said liquid form through a filter in said container and a liquid flow-through matrix carrying a dry water-soluble reagent thereby dissolving said reagent and producing a filtered aqueous liquid mixture of said reagent and sample, and

(b) allowing said filtered mixture to flow out of said container for subsequent analysis of said test substance.

Drawings

Fig. 1 and 2 are schematic cross-sectional views of a mixing and filtering device of the invention having separate reservoir bodies and closure members and showing one reaction surface.

Detailed description of the invention

The present invention provides a device for filtering a biologically derived sample and mixing the sample with a reagent that greatly simplifies sample analysis. Biologically derived samples can be analyzed using known analytical procedures. These samples include body fluids such as whole blood, serum, plasma, urine, saliva, and the like. In addition, the samples include extracts such as extracts from fecal samples, E.coli cultures, suspensions in a surface for food safety such as the surface of chickens, etc., liquid in sewage for various purposes, and various cells, bacteria and viruses, which are disrupted to release the analyte of interest.

The test substances include a wide variety of proteins, such as those listed in U.S. Pat. No.5,006,464, which are typically assayed by immunoassay systems, and others which are not classified as immunoassay systems, because these assays use the reaction of known binding pairs. In addition, the system can be applied to assay system analysis that is not classified as an immunoassay, such as the detection of unknown deoxyribonucleic acid (DNA) sequences. The assay device of U.S. patent No.5,006,464 is particularly effective for the purposes of the present invention, unlike a flow-through assay device.

Referring to fig. 1, fig. 1 shows one embodiment of a filtration and mixing apparatus according to the present invention. The apparatus comprises a container 10, the container 10 having a tank body 12, said tank body 12 having a tank body opening 12a at its lowermost end and an internal reservoir 12 b. The reservoir body 12 includes a generally cylindrical side wall 12c, preferably made of a flexible material such as plastic, which side wall 12c is squeezable to urge the liquid in the reservoir to the filter and a substrate and out of the container as will be described below. Suitable plastics are polyvinyl chloride (PVC) or polyhexene or polyesters.

The lower wall of the tank body 12 cooperates with and forms a seal with the movable closure member 14. As shown in fig. 1, the reservoir body 12 is separate from the closure 14. The closure 14 includes an upwardly extending cylindrical sidewall 14a, the cylindrical sidewall 14a being sized to form a press-fit seal with the adjacent sidewall of the reservoir body 12, the sidewall 14a sealing against liquid leakage. Additional sealing means may be used such as a screw fit. The closure 14 also includes an opening 14b at its upper end, both the opening 14b and the opening of the tank body 12 forming an internal liquid passage through the container 10. Closure 14 is generally cylindrical at an upper end and generally conical at a lower end and defines an outlet opening 14c, which outlet opening 14c is sized so that when flexible wall 12 is squeezed to force liquid in reservoir 12b through a filter and base as described below, liquid flows dropwise from container 10 and out opening 14 c.

Instead of using a flexible wall 12, another method may be used to push the aqueous liquid in the reservoir chamber through the filter. For example, a piston may be used at the top of the container.

The filter 16, suitably in the form of a disc, is positioned across the flow path between the reservoir body 12 and the opening 14c so that liquid in the reservoir 12b flows through the filter to retain debris in the liquid that may interfere with the assay. For example, for blood analysis, particulate matter may be filtered out by a filter. Suitable filters include filter membranes made of nitrocellulose, polycarbonate, nylon, polyester or other porous polymer or other material lined with paper. Suitable filter porosities for blood are about 0.2-1.0 microns. Other porosities may also be used, depending on the sample and the desired application. The thickness of the filter is typically about 0.5mm to 1.5mm or less, depending on the manufacturer's specifications. Suitable filters for filtering blood in a WBT device may be used in the context of the present invention (e.g., filters manufactured by Schleicher & Schuell, Millipore OrMDI). As shown in fig. 1, the closure 14 includes a shoulder 14d, the shoulder 14d extending inwardly of the closure to form a support for the outer periphery of the filter 16.

When the container 10 is used with whole blood or serum, plasma, urine, saliva, or other secretions having particulate matter, the selective filter filters out debris, dirt, debris from red or white blood cells, sediment in the sample, and the like.

In the embodiment of fig. 1, the base 18 is shown on the outlet side of the closure 14 from the filter 16. Water-soluble dry reagents for use in subsequent analysis of the sample for the analyte are immobilized on the substrate. The dry reagent is preferably dissolved by a liquid forced through the matrix by pulling pressure against the flexible wall of the reservoir body 12.

The matrix 18 may be made of any material that can be placed in a container and the dry reagents may be immobilized on the matrix 18 for subsequent dissolution in the passage of the aqueous liquid through the matrix. The matrix 18 has sufficient porosity so that liquid can flow through without excessive pressure drop. Suitable matrix materials include filter glass, flow-through screens, porous membranes such as nitrocellulose or coated nitrocellulose, or a packed particle bed with a device to obtain a particle bed such as a porous frit.

One or more dry reagents are immobilized on a matrix material. In one embodiment, the reagent is a detectable label such as freeze-dried colloidal gold, a fluorescent label, an enzyme, or the like, conjugated to a chemical moiety capable of binding to a biomolecule. Suitable freeze-dried gold conjugates are available from EY Laboratories, Inc. Sold as listed in its catalog. The label may be conjugated to any material capable of binding directly or indirectly to the analyte in the sample using known techniques, such as in a competitive or sandwich binding assay. Specifically, the bioconjugate portion of the label is typically bound to a capture reagent 22 that is immobilized on a reaction surface 24. Suitable chemicals include, in part, various proteins, carbohydrates, and polynucleotides, including, specifically, antibodies, antigens, deoxyribonucleic acid (DNA) ribonucleic acid (RNA), xenobiotins, biotin, biotinylated specific proteins, biotinylated carbohydrates, biotinylated polynucleotides, avidin, antignathons, and the like. Other dry reagents that are not conjugated to the label may also be retained on the matrix 18. For example, such other labels include enzymes, fluorescent dyes, bioluminescent or chemiluminescent compounds, or encapsulated dyes such as microspheres containing dye or activated carbon, or microspheres linked to enzyme or dye, or colloidal gold. A stabilizer such as sucrose, maltose, melibiose, polyethylene glycol or bovine serum albumin or a buffered saline solution, or combinations thereof, may be added to the reagent on the substrate 18.

In one immobilization method, a reagent, such as a known concentration of Fluorescein Isothiocyanate (FITC) labeled antibody, is deposited on and adsorbed to a substrate, such as a substrate in the form of glass fibers. The reagent is then allowed to air dry on the substrate and the substrate disk is cut open for installation into an enclosure 14 for use in the apparatus. The same procedure is used for other reagents such as bioconjugates labeled with colloidal gold, enzymes, biotin, avidin, etc.

Another procedure for fixing the reagent to the substrate 18 is by freezing and drying. Here, the substrate (e.g., glass fiber) is immersed in a reagent solution, and the substrate with the reagent solution is then flash frozen, such as by immersion in ethanol and dry ice solution or liquid nitrogen. The disk-shaped frozen film to be placed in the apparatus is then placed in a freeze-dryer to be frozen and dried using well-known techniques.

Referring to fig. 1, an optional bag 20 is disposed in the reservoir body 12, the bag 20 including an aqueous carrier liquid 20 a. The bag 20 is made of a material that is easily ruptured, such as polyvinyl chloride, so that the bag can be ruptured when the wall 12c is squeezed. Such rupturable pouches are commercially available. Typically, carrier liquid 20a is an aqueous buffer solution that is mixed with the sample and flows through the filter and matrix and out of outlet 14 c. Liquid buffers are a well known common type for later use in the analysis of samples for analytes. The buffer solution is also used to reconstitute the desiccant on the substrate 18 for later analysis. Suitable aqueous buffer solutions include conventional ingredients such as phosphates, borates or bicarbonates. In addition, surfactants or detergents may be added to reduce viscosity, provide lubrication and form micelles. The liquid is preferably at a pH of 7.0-8 or above or below this value, depending on the stability of the reagents and the optimal conditions for the binding pair.

In another embodiment shown in fig. 1, an optional filter 22 may be inserted above filter 16 (between reservoir 12b and filter 16) to provide additional filtering capacity. In this case, the filter 22 may be of the same type as described above for the substrate or filter. This may be required for samples comprising large amounts of material to be filtered, such as urine.

In yet another embodiment shown in fig. 1, the base 18 may be disposed in the sump chamber side of the filter, which in turn is disposed on the outlet side of the base 18. One advantage of this configuration is that the dissolved reagent and sample are thoroughly mixed. As shown, filters 16 and 22 are disposed above substrate 18, and additional filters are disposed below substrate 18, a layer such as filter 24 disposed between substrate 18 and outlet 14c may be used to further mix the reagents and sample solution prior to capture with capture reagent 24 on membrane 22. Depending on the application, all of the filters 16, 22 and 24 may be used or one or more of them may be used.

In another embodiment, not shown, the system does not include a rupturable pouch 20. In this case, the sample may be a liquid such as blood, with no additional carrier liquid added to the reservoir chamber 12 b. Alternatively, the sample may be loaded into chamber 12b and a carrier liquid, such as the same aqueous buffer solution, mixed with the sample before closure 14 is sealed to reservoir body 12.

In another embodiment not shown, the sample may be deposited on a surface at the outlet end of the reservoir chamber 12b, such as by swabbing. In this case, the deposited sample is not in liquid form. Therefore, it is necessary to add a carrier liquid such as a buffer solution to the reservoir chamber 12 b. As mentioned above, the carrier liquid may be in the bag 20 or just fill the reservoir. In the illustrated embodiment, the sample is deposited on the filter 16 prior to loading into the container. By squeezing the reservoir body 12, the analyte in the carrier liquid deposition sample is suspended or dissolved for flow through the filter 16 and matrix 18 and out the outlet 14 c.

Referring again to FIG. 1, the sample and reagents flow out of the outlet 14c and against a reaction surface 30 of a conventional apparatus 32. One such reaction surface comprises an exposed membrane that forms the reaction surface with an absorbent material beneath the membrane, the absorbent material beneath the membrane being contained in a plastic housing. The capture reagent 34, which is immobilized on the reaction surface 30, interacts with the sample analyte as in an immunoassay. One suitable capture reagent is an antibody or antigen or other binding reagent (e.g., a reagent known in capture assays). The liquid is pulled through the membrane by the absorbent material. One suitable device of this type is disclosed in U.S. patent No.5,885,526, which is incorporated by reference herein.

The analytical method may be carried out in a single step by flowing the dissolved reagent out of the outlet 14c and onto the reaction surface. Alternatively, additional reagents (e.g., wash solution) not from container 10 may be added to the surface if desired. Conventionally, the sample analyte is bound directly or indirectly to the reaction surface, such as by an immunological reaction pair, by a DNA hybridization pair, an lectin, carbohydrate pair or other molecule in the biological system or molecule used to bind dual standard RNA.

Referring to fig. 2, fig. 2 shows an embodiment of the present invention that includes an alternative to a rupturable pouch. Like parts in fig. 1 and 2 are indicated by like reference numerals. As shown in fig. 2, the bag includes an outlet drip 20b, the opening of the drip 20b facing the opening of the tank body 12a, with a movable closure such as a press-fit flexible cap 20 c. In use, cap 20c is removed and closure 14 is sealed with reservoir body 12. The liquid in the bag is squeezed into the reservoir body 12 at the time of testing. The following examples are provided to illustrate the invention.Example 1

The reagent fixing film 18 of FIG. 1 is produced by the following procedure. A glass fiber disc (e.g., 3/8 "diameter) was dipped into the reagent (protein a colloidal gold labeled conjugate). The A-tag was made using the procedure of U.S. Pat. No.5,541,059, which is incorporated herein by reference. Reagent solution was 20nM OD⁵²⁰(wavelength 520nM) colloidal gold labeled protein A (or a specific immunoglobulin) in 20nM phosphate buffer containing 1% Bovine Serum Albumin (BSA) at pH 7.4. 50 μ l of this solution was added to the top of the membrane and absorbed. The discs were placed in a freeze-dryer and freeze-dried.Example 2

In an example, the WBT device is used when the container 10 is made of plastic with a flexible wall having a reservoir body. The filter is a nitrocellulose type lined paper. The reagent-holding base disc made in fig. 1 is placed in the closure 14 between the filter 16 and the outlet 14 c.

Reservoir 12b is partially filled with blood. A phosphate buffered saline solution is placed into rupturable pouch 20. When it is desired to perform the assay, the wall 12c is squeezed until the bag 20 ruptures so that the buffer solution mixes with the blood and is filtered by the filter 16 and through the matrix 18 so as to dissolve the colloidal gold-labelled reagent. The aqueous mixture of sample and reagent is dripped through the outlet 14c for deposition onto one of the reaction surfaces 30 (nitrocellulose in the backing paper) for reaction with the capture reagent 34 (as described in us patent 5,885,526).Example 3

In this case, a solid sample is tested for microorganisms. The sample was scraped from the skin of the chicken with a swab. Optionally, the sample is then concentrated by depositing the sample on a filter 16, which filter 16 has a porosity sufficient to pass the microorganisms. The solution in the bag was discharged to carry the microorganisms through the filter and the matrix containing the freeze-dried colloidal gold labeled anti-E.coli 0157.H7 specific antibody. The red color in the subsequent analysis indicates that the test is positive.

Claims (22)

1. An apparatus for filtering a biologically derived sample and for mixing said sample with a reagent, said apparatus comprising a fluid container defining a reservoir chamber and including a container outlet, a filter and a fluid flow-through matrix disposed in a flow path between said container chamber and container outlet, a water-soluble dry reagent retained on said matrix, and means for urging an aqueous liquid sample in said reservoir chamber through said filter and matrix and out of said container outlet.

2. The apparatus of claim 1 wherein said urging means comprises a flexible wall portion of said container adjacent said reservoir chamber.

3. The apparatus of claim 1 wherein said container includes a reservoir body defining an opening, said container further including a movable closure defining said container outlet at one end and an opening at its other end, said opening cooperating with said reservoir opening to define a fluid passageway therebetween, said filter and base being disposed in said closure.

4. The device of claim 1, further comprising a fluid-containing bag disposed in said reservoir chamber.

5. The method of claim 4 wherein said bag includes an outlet opening and a removable cap.

6. The device of claim 1, wherein said substrate is filter glass, a porous membrane, a packed particulate bed or a screen.

7. The device of claim 1, wherein said reagent comprises a detectable label.

8. The device of claim 6, wherein said detectable label is conjugated to a moiety of a chemical that is capable of binding to a biomolecule.

9. The apparatus of claim 1 wherein said filter is on the reservoir chamber side of said substrate.

10. The device of claim 9, further comprising a porous material disposed between said matrix and the outlet of the container for mixing said dissolved reagent and sample.

11. The apparatus of claim 1 wherein said substrate is on the reservoir chamber side of said filter.

12. A method for filtering a biologically derived sample and mixing the sample with reagents, said sample including at least one analyte reagent for analysis of an analyte, said method comprising:

(a) applying pressure to an aqueous liquid sample in said receptacle chamber of said container to cause said sample to flow in said liquid form through a filter in said container and a liquid flow-through matrix carrying a dry water-soluble reagent thereby dissolving said reagent and producing a filtered aqueous liquid mixture of said reagent and sample, and

(b) allowing said filtered mixture to flow out of said container for subsequent analysis of said test substance.

13. The method of claim 12, wherein said sample comprises whole blood, serum, plasma, urine, or saliva.

14. The method of claim 12 wherein said substrate is a filter glass, a permeable membrane, a bed of packed particles, or a screen.

15. The method of claim 12 wherein said container has a flexible wall portion adjacent said reservoir chamber and said pressure is applied by squeezing said wall portion.

16. The method of claim 12 wherein said sample is passed through said filter in an aqueous carrier liquid.

17. The method of claim 16 wherein said carrier liquid is added to said container in a rupturable pouch, said rupturable pouch being ruptured under said applied pressure.

18. The method of claim 17 wherein said sample is deposited on a surface in said container and at least partially dissolved in said carrier liquid prior to flowing through said filter.

19. The method of claim 12 wherein said sample is passed through said filter before being passed through said matrix.

20. The method of claim 12 wherein said sample is passed through said matrix before passing through said filter.

21. The method of claim 12, wherein said reagent comprises a detectable label.

22. The method of claim 12, wherein said label is conjugated to a moiety of a chemical that is bound to a biomolecule.