The present invention relates to methods and apparati for testing a sample of biological fluid for the presence or absence of a selected substance and more particularly to testing for the presence or absence of antibodies and their corresponding antigens.
A variety of testing devices are known that use what has been termed “lateral flow” technology to separate cell, solid materials, certain large molecules and the like from selected substances of interest in a biological sample. Such prior devices typically comprise a single linear or planar, thin elongated generally horizontally disposed strip or column of chromatography material that wicks aqueous materials along the length of the continuous strip of wicking/chromatography material. An aqueous sample of biological fluid to be tested for the presence or absence of a selected antigen material is applied to a discrete spot on the horizontally disposed column/strip of chromatography material. The applied sample is then wicked laterally along the length of the column in a lateral direction toward a sink. A test site is located between the sample application site and the sink such that when a sample of aqueous fluid is applied to the sample application site, the sample must first pass by and make contact with the test site. A known antibody to a selected antigen of interest is bound to the matrix of the chromatography material at the test site so that any antigen to the antibody in the sample is captured and bound at the test site. A visually identifiable material that selectively binds to the antigen of interest in the sample is applied to the chromatography strip at the same time as the biological fluid is applied to the same site as the sample is applied. The visual label is alternatively pre-deposited on the strip at the sample application site or at a site slightly downstream of the sample application site/position. Additional drops of aqueous buffer are applied to the sample application site to cause the sample material and the visual label material to wick simultaneously together in the same body of fluid toward the test site. The visual label material typically binds specifically to the antigen of interest that is present in the sample upon intermixing of the visual label material and the sample. The bound antigen/label are wicked downstream together to the test site. The visual label material is reaction-specific to the one preselected sample antigen of interest and thus other chemical entities that may be present in the biological sample do not bind to or deplete the visual label material.
The present invention comprises an apparatus and method for testing for the presence or absence of a selected biological substance. The selected biological substance can be, for example, a preselected antibody, antigen, protein, enzyme or any molecule, cell or biological moiety (hereinafter individually and collectively “analyte”) that can be wicked along and through a series of separate, but fluidly communicating, wicking components that are placed in overlapping wicking engagement with each other. The analyte is captured on the chromatography medium by a capture substance that is bound to the chromatography medium at a test site. The test site is located at a discrete predetermined location along the path of wicking of the analyte. In one embodiment, a complementary antigen that is reactive with the select antibody is used as the capture material at the test site. And, vice versa, a select antigen can be tested for by using a complementary antibody that is reactive with the select antigen as the capture substance at the test site.
The invention further provides a two step chromatography, elution or aqueous chase process. First, the biological sample is delivered to a sample application site on a chromatographic pathway and wicked/chased toward the position of a capture test site. Next, in a second step subsequent to the time that wicking of the sample material has been started/initiated, a visual label material is wicked along the chromatography path toward the test site such that the visual label material does not contact or engage with the sample material at any time before reaching the location of the capture test site. The visual label material either reacts with the sample in its unbound configurational form or in the configurational form that the sample material assumes after it reacts with the capture material at the test site. The sample material is typically wicked all the way to the position of the test site before the visual label material is applied to the chromatography path.
In another aspect of the invention, a test device is provided that has two chromatographic or wicking pathways, one pathway for wicking of the sample material to the location of the test site and another pathway being at least partially physically separate from the first pathway for wicking the visual label material to the test site.
In accordance with the invention there is provided a fluid flow test device comprising:
The device can include a third strip of aqueous wicking material that is in fluid flow communication with the second strip at a second location upstream of the test site, the visual label material being delivered to the second strip from the third strip for aqueous wicking delivery to the second strip at the predetermined point in time after the first delivery time. The target material is typically a selected antibody and the capture material a selected antigen that complexes the selected antibody. The visual label material is either applied to the third strip of material at the time of use of the test device or the visual label material is pre-applied to third strip of material and embedded therein before use of the test device.
Further in accordance with the invention there is provided a method of testing for the presence or absence of a target material in an aqueous biological sample, comprising:
The second predetermined point in time is preferably long enough after the first selected delivery time to ensure that the biological sample has been wicked downstream to the test site.
The target material is a selected antibody that binds to the visual label material, the visual label material comprising an antigen that non-selectively binds to the selected antibody and to other antibodies that are contained in the biological sample. The visual label material can alternatively comprise an antibody to the target material that binds to the target material. The capture material typically comprises an antigen that selectively binds to the antibody.
Preferably, the visual label material is applied to a third strip of aqueous wicking material that is in fluid communication with the second strip at a second location that is upstream of the first location. A separation distance is preferably selected between the first and second locations such that the biological sample is ensured of being wicked downstream to the test site before the visual label material is wicked downstream to the test site. The visual label material is typically delivered to a second location on the second strip that is upstream of the first location.
In another aspect of the invention there is provided, a method of testing for the presence or absence of a target material in an aqueous biological sample, comprising:
Further in accordance with the invention there is provided, a fluid flow test device comprising:
The visual label material can either be applied to the third strip of material at the time of use of the test device or the visual label material is pre-applied to third strip of material and embedded therein before use of the test device.
Further in accordance with the invention there is provided a fluid flow test device comprising:
The above and further advantages of the invention may be better understood by referring to the following description in conjunction with the accompanying drawings in which:
As best shown in
A strip of water impervious double sided tape 160 is disposed between the sample application pad 140 and the plastic barrier 150 to ensure that the pad 140 is firmly held in position relative to the other underlying components of the assembly 80 as shown in
As shown in a specific embodiment in
The sample application spot 142 is aligned directly under a sample application aperture 40 provided in the housing component 20. In practice, the sample 190 is first applied to spot 142 and allowed for a relatively brief amount of time, e.g. 1-10 seconds, to wick in a downstream direction, i.e. wicking occurs from right to left as shown in
A predetermined amount of time after the sample 190 has been applied to spot 142, and preferably after the sample 190 has been allowed to wick to the location of test site T, an aqueous buffer 200 that does not contain analyte material is applied to spot 102 on upstream pad 100. Spot 102 is positionally aligned with an aperture 30 provided in housing component 20 for ready and precise application of the buffer 200 to the pad 100. When applied, the buffer 200 wicks continuously downstream first through pad 100, then through mating surfaces 104 to and through glass pad 180 then through mating surfaces 182 to and through visual label containing pad 170, then through mating surfaces 134 to and through membrane 130 past the position of both test site T and control site C and then through mating surfaces 136 to sink pad 120. Pad 120 is relatively larger in volume than the other pads such that it attracts aqueous fluids 190 and 200 that are applied at the upstream end of the stepped chromatography paths leading to pad 120, the pad 120 thus acting as a sink to cause fluids 190 and 200 to tend to travel in the downstream direction, i.e. right to left as shown in
Typically a substantially greater volume of buffer 200 is applied to spot 102 than the volume of sample material 190 that is applied to spot 142, e.g. 2-10 times the volume. Pad 170 contains a selected amount of a predetermined visual label material that is capable of binding to the selected analyte material, i.e. capable of binding either directly to the analyte in its free unbound form or capable of binding to the analyte in its bound form after being captured by the capture material resident at the test site T. A large volume of buffer 200 is applied to spot 102 (relative to the volume of sample 190) so that there is a sufficient, if not excess, amount of buffer available to assure that the visual label material is fully dissolved or otherwise entrained in the buffer and wicked completely downstream along the chromatography path from pad 170 to test site T and control site C.
In the sequence of steps described, the sample 190 is first wicked downstream through pad 130. Only after the sample 190 has been fully wicked to at least the downstream location of test site T is the buffer 200 and the visual label material resident in pad 170 then wicked along at least the same chromatography path through pad 130 to the location of test sites T and C. The precise amount of time in sequencing the application of sample 190 and buffer 200 and the precise volume of sample 190 and buffer 200 that is applied can be varied depending on the precise length and number of separate pads that comprise the chromatography paths between the sample application spot 142 and the test site T and between the location of the visual label pad 170 and the test site T. In any case, the sample material 190 is wicked at/during such a period of time and along such a path that the visual label material does not mix or combine with the sample at any time within the matrix of the chromatography path prior the time that the sample 190 has wicked to the location of test site T.
The materials of which the pads 100, 120, 140, 170, 180, and membrane 130 are comprised are capable of readily wicking aqueous fluid. The mating surfaces 104, 182, 134, 132, 136 are arranged such that opposing surfaces of the respectively mating pads are engaged sufficiently with each other to enable aqueous fluid to readily wick from one pad to another with which it is mated. The overlapping of separate generally planar, but bendable pads, 100, 180, 170, 140, 130, 120 creates a wicking or chromatography path between the pads that is both continuous and interrupted or stepped from horizontal to vertical at mating surfaces 104, 182, 134, 132 and 136. Extraneous materials contained within the buffer 200 or the sample 190 that can interfere with the reaction between the analyte and the visual label material or the capture material at the test site T can be selectively filtered by both the matrices of the chromatography pads themselves and by the mating surfaces that make up an interrupted or stepped chromatography path. The sample application pad 140 initially filters cells out of the aqueous flow that wicks through to the test site T. The interrupted or stepped chromatography path as specifically shown and described herein can be varied in many ways, e.g. by varying the number and length of mating surfaces and chromatography pads such that the overall configuration and length of the chromatography path and the number of mating surfaces is tailored to achieve any desired degree of filtering or separation of sample from the visual label. As shown best in
The visual label material resident in pad 170 and the capture material resident at test site T are typically preselected to be reactive with and bind to the predetermined analyte being tested for. Alternatively, the visual label material may be selected to be reactive with the reaction product of the analyte and the capture material. Thus when the visual label material is wicked to the location of the test site T, the visual label material will be captured and accumulate at the test site (being visible to the eye indicating the presence of analyte in the sample) only when the analyte has been captured at the test site T. To ensure that the visual label material is in fact responsible for a positive visual presence at the test site T, a control capture substance that reacts with and captures the visual label material is bound to the membrane 130 at a control site C located downstream of the test site T. The visual label material is provided in a molar amount on pad 170 in excess of the maximum molar amount of capture material present at test site T such that upon wicking of the visual label material to the point of the test site T an excess of amount of visual label material is guaranteed to flow past test site T to the location of the control site C. If, after application of sufficient buffer 200 to cause the visual label material to wick to the point of control site C, a positive visual identification of the visual label material can be made at control site C, then the user of the device is assured that a positive identification of visual label material present at the test site is not a false positive from an extraneous substance binding to the test site T and that the device is working correctly.
In one specific embodiment of a test device according to the invention, the sample analyte comprises an antibody to a selected condition or antigen present in the biological subject, e.g. an immunoglobulin (IgG, IgM, IgA, IgE, IgD) and variants thereof; and the capture material comprises one or more antigens to the antibody. And, the visual label material comprises an antibody or antigen to the selected analyte-antibody
For example the analyte could comprise a specific anti-condition X IgG, such as anti-Candida immunoglobulin G (“IgG”). In such an example, the capture material that is bound to the matrix of the membrane at test site T comprises one or more antigens (typically proteins) to the anti-Candida IgG. When the anti-Candida IgG containing sample 190 is wicked from the sample application site 142 to the test site T, at least one antigen at the test site has an epitope which is recognized by the antibody and to which the antibody binds. Once the anti-Candida antibody is bound to the antigen at the test site T, the antibody unfolds exposing another site on the antibody that is reactive with a visual label protein that recognizes the exposed reactive site on the bound antibody. In such a case, the visual label material can be selected to comprise a gold conjugate of another different protein reactive with the anti-Candida IgG, the visual label material typically also being reactive with any IgG that may be present in the sample and not just with anti-Candida IgG. In such an example, the visual label material must be separated from contact with the sample 190 within the chromatography path prior to the sample's being wicked to the test site T in order to avoid the visual label material being depleted by IgG other than the specific anti-Candida IgG (or other specific anti-X IgG) whose presence or absence is being tested for. Separation of the generally reactive visual label material from the sample 190 is achieved by sequenced timing of application of the sample 190 and the buffer 200 as described above and by physical separation of the application pad 140 from the visual label impregnated pad 170 as described above.
In the specific embodiment described regarding anti-Candida IgG, the capture material resident at the test site T can comprise a mixture of one or more antigens derived from a cell line of one or more specific Candida species (e.g. C. albicans, C. tropicalis, C. parapsilosis, C. Iusitaneae, C. glabrata and C. krusei) and is reactive with antibodies to one or more or all of such species. The antigens are sprayed on and bound to the membrane at the test site T. As can be readily imagined the test and control line capture materials can comprise antigens derived from and peculiar to any preselected biologic subject's condition and the substance/analyte to be tested for in the sample 190 can be the antibody to the antigen that is peculiar to any such preselected condition. A visual label material that is either reaction specific to the analyte or generally reactive with the class of molecules of the analyte can be used.
Alternatively, the material selected for binding to the test site T can comprise one or more antibodies to a preselected biologic subject's condition (instead of antigen) and the analyte can comprise one or more antigens (instead of antibody) peculiar to the condition. In such an embodiment, the visual label material is selected to be reactive with the antigen, e.g. another different antibody to the antigen.
This application claims the benefit of priority under 35 U.S.C. Section 119 to U.S. provisional application Ser. No. 60/665,824 filed Mar. 28, 2005 and 60/676,248 filed Apr. 29, 2005 the disclosures of both of which are incorporated herein by reference in their entirety as if fully set forth herein. This application is a continuation-in-part of and claims the benefit of priority under 35 U.S.C. Section 120 to U.S. application Ser. No. 10/682,807 filed Oct. 10, 2003 and Ser. No. 09/841,188 filed Apr. 25, 2001 the disclosures of both of which are incorporated herein by reference in their entirety as if fully set forth herein.
Number | Date | Country | |
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60665824 | Mar 2005 | US | |
60676248 | Apr 2005 | US |
Number | Date | Country | |
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Parent | 10682807 | Oct 2003 | US |
Child | 11186289 | Jul 2005 | US |
Parent | 09841188 | Apr 2001 | US |
Child | 11186289 | Jul 2005 | US |