The present invention relates to improved specific binding assay devices comprising a chromatographic medium including a reaction site at which a specific binding reagent is immobilized, a sample application well located adjacent to the chromatographic medium and offset upstream from the reaction site, and liquid absorption blotter offset downstream from the reaction site.
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0. 12. A test device for determining the presence or amount of an analyte substance in a sample by means of one or more specific binding reactions comprising:
a bibulous chromatographic medium-free of chromatographic transport-facilitating agent having capillarity and the capacity for lateral chromatographic transport of one or more reactive sample components and non-immobilized reagents, said medium including a reaction zone comprising an immobilized reagent which binds a member selected from the group consisting of said analyte substance and a labeled specific binding material, said reaction zone occupying less than the entire area of the medium such that there remains an upstream portion and a downstream portion;
a single undivided well defined by a housing element comprising an upper and lower portion; wherein said upper portion defines the spatial boundaries of said well and said lower portion comprises a part of said upstream portions, said well being in fluid contact with the upstream portion of said chromatographic medium and being of sufficient capacity to retain a volume of fluid sample for chromatographic movement of said volume of sample along said chromatographic medium from said well through said reaction zone and into a liquid absorption means;
said liquid absorption means consisting of a quantity of blotter material disposed in-fluid contact with said downstream zone of said chromatographic medium.
1. A test device for determining the presence or amount of an analyte substance in a sample by means of one or more specific binding reactions comprising:
a bibulous chromatographic medium which extends throughout said device and is free of chromatographic transport-facilitating agent, defining a flowpath having capillarity and the capacity for chromatographic solvent transport of one or more reactive sample components and non-immobilized reagents; said chromatographic medium comprising (1) a prefiltering zone; (2) a reaction site zone disposed downstream from said prefiltering zone at which is present containing an immobilized reagent capable of binding which specifically binds a member selected from the group consisting of said analyte substance and a labeled specific binding material, and at which the presence or amount of immobilized specific binding material, may be is capable of being detected; and (3) a downstream zone free of said immobilized reagent disposed downstream from said reaction site zone,
a sample application means selected from the group consisting of a single undivided well and an absorbent pad defined by a housing element comprising an upper and lower portion; wherein said upper portion defines the spatial boundaries of said well and said lower portion comprises a portion of said chromatographic medium which is upstream of said prefiltering zone, said well having sufficient capacity to hold a volume of sample for chromatographic movement of said volume of sample from said well to a liquid absorption means, said well being located adjacent to and disposed in fluid contact with said prefiltering zone of said chromatographic medium and offset upstream from said reaction site zone, and
said liquid absorption means consisting of a quantity of blotter material disposed in fluid contact with said downstream zone of said chromatographic medium and offset downstream from said reaction site zone.
7. A method for determining the presence or amount of an analyte substance in a sample which method utilizes a device comprising:
a bibulous chromatographic medium which extends throughout said device and is free of chromatographic transport-facilitating agent, defining a flowpath having capillarity and the capacity for chromatographic solvent transport of one or more non-immobilized reagents and reactive sample components; said chromatographic medium comprising (1) a prefiltering zone; (2) a reaction site zone disposed downward from said prefiltering zone at which is present containing an immobilized reagent capable of binding which specifically binds a member selected form the group consisting of said anlayte substance and labeled specific binding material, and at which the presence or amount of immobilized labeled specific binding material may be is capable of being detected and (3) a downstream zone free of said immobilized reagent disposed downstream from said reaction site zone,
a sample application means selected from the group consisting of a single, undivided well and an absorbent pad defined by a housing element having an upper and lower portion; wherein said upper portion defines the spatial boundaries of said well and said lower portion comprises a portion of said chromatographic medium which is upstream of said prefiltering zone, said well being of sufficient capacity to hold a volume of sample for chromatographic movement from said well to a liquid absorption means, said well being located adjacent to and disposed in fluid contact with said prefiltering zone of said chromatographic material medium and offset upstream from said reaction site zone, and
a said liquid absorption means consisting of a quantity of blotter material disposed in fluid contact with said downstream zone of said chromatographic chromatographic medium and offset downstream from said reaction site zone, said method comprising:
(a) applying a said volume of said sample sample application means to said well whereby said sample is free of chromatographic transport-facilitating agent and is transported absorbed onto said chromatographic medium at and is chromatographically transported through said prefiltering zone and along said chromatographic medium through said reaction site zone, and said downstream zone to said sample liquid absorption means,
(b) contacting said labeled specific binding material to said reaction site zone, and
(c) determining the presence or amount of said labeled specific binding material immobilized at said reaction sites zones as an indication of the presence or amount of the analyte substance in the sample.
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Of of the quantity and/or binding affinity of the labelled specific binding material can be made in order to determine the quantity of analyte present in the sample.
Various other specific binding assays may be carried out on the devices of the present invention. As an example, the chromatographic medium may be impregnated at additional reaction sites with various immobilized specific binding agents. Such additional reaction sites may be used for the detection of additional analyte substances or may be used, as in a comparison-type assay to quantify the amount of analyte substance present in a sample. Additional reaction zones may be used to capture substances that interfere with accurate determination of the analyte.
The chromatographic medium may be impregnated with other reagents and materials including dye compounds, enzyme substrates, coenzymes and cofactors which react with enzyme labels to produce color signals as is well known in the art. According to one aspect of the present invention, where the labelled specific binding agents are enzyme-labelled the enzyme label being characterized in that its presence may be determined by reaction with members of a signal generating substrate/cofactor group, a first member of the signal generating substrate/cofactor group may be immobilized at a reaction site on the chromatographic medium. A second member of the substrate/cofactor group may be added at an appropriate time to activate the signal generating system if an enzyme label is present. By preimpregnating the reaction site with a member of the signal generating substrate/cofactor group, an additional step is avoided and problems relating to the instability or insolubility of substrates, cofactors and dye precursors when stored together are avoided. Moreover, certain members of the signal generating group of compounds may have low chromatographic mobility with the result that pre-impregnation at the reaction site is particularly preferred.
The devices may be housed singly, in pairs, or in multiple configurations. The housing should preferably be watertight to prevent leakage and may be manufactured from a variety of inert materials, with polymer materials being preferred for their ease of fabrication. The sample well should be of sufficient volume to contain any required amount of sample or reagents to be used with the invention. Where the liquid absorption means is enclosed within the housing of the device, it should be provided with sufficient volume that sample, wash, and reagent materials applied to the device during assay procedures may all be absorbed. According to a preferred embodiment of the present invention wherein the chromatographic media is a strip of nitrocellulose material 0.4 cm wide, sample and reagent volumes ranging from 10 μl to 150 μl are suitably accommodated.
The reaction site is located downstream from the sample application means and is visible to view from either side, either being unenclosed or covered with a transparent material. The reaction site is preferably unenclosed, thus facilitating observation of the presence or absence of signals from the site and also allowing the addition of reagents and/or wash solutions to the reaction site.
The liquid absorption means located downstream from the reaction site absorbs sample materials, reagents and wash solutions and thus allows chromatographic transport of sample and other materials from the first end of the chromatographic medium through the reaction site to the second end of the medium. Without such absorption chromatographic transport would cease and the efficiency advantage resulting from the lateral flow of sample through the reaction site would be lost.
The liquid absorption means can include the chromatographic medium itself wherein sample and reagent materials are transported and absorbed by an extended length of the medium. A particularly preferred aspect of the invention is that wherein the liquid absorption means is located adjacent to the chromatographic medium and is offset downstream from the reaction site. Locating the absorption means adjacent to the chromatographic medium provides additional absorption capacity. Offsetting the absorption means downstream from the reaction site ensures that sample materials, and all other materials introduced upstream of the reaction site flow primarily laterally and not vertically through the reaction site.
The absorption means may consist of an extended length of chromatographic medium but preferably consists of a quantity of blotter material. Cellulosic blotter materials derived from wood pulp or cotton are suitable. The blotter material need not have dimensions similar to those of the chromatographic medium and is generally wider and thicker than the chromatographic medium. The blotter need only be adjacent to the chromatographic medium such that fluid from the chromatographic medium can flow into the blotter. A particularly preferred material is James River Type 52 point blotter material (James River Paper Co., Inc., Richmond, Va.). The absorption means preferably must be capable of absorbing the entire volume of sample material and is preferably capable of absorbing the additional reagents and doing so rather rapidly.
Chromatographic media useful with the present invention include those chromatographic substrate materials having capillarity and the capacity for chromatographic solvent transport of non-immobilized reagents and sample components. The chromatographic media used with the invention are preferably in the form of strips. While a wide variety of chromatographic materials such as woven and non-woven fibrous materials used for paper chromatography are suitable for use with the invention, the use of microporous or microgranular thin layer chromatography substrates is particularly preferred as the use of such materials improves the speed and resolution of the assays according to the invention. The materials should preferably be inert and generally not react physically or chemically with any of the sample components, reagents buffers or reaction products. Co-owned and copending U.S. patent application Ser. No. 912,878 filed Sept. 29, 1986 U.S. Pat. No. 4,960,691 and U.S. patent application Ser. No. 072,459 filed July 13, 1987 Pat. No. 5,120,643, the disclosure of which are hereby incorporated by reference, disclose a wide variety of suitable chromatographic substrate materials. Particularly preferred, however, is the use of a microporous nitrocellulose material with a pore size of 5 μm designated Type SMWP (Millipore Corp., Bedford, Massachusetts).
Because the chromatographic media of the device is preferably chemically inert, it may have to be activated at any reaction site where it is desired to immobilize a specific binding reagent against solvent transport. Various methods will be required to render the reagent immobilized according to the particular chemical nature of the reagent. Generally, when the media is nitrocellulose or a mixed nitrocellulose ester, no special chemical linkage is required for the immobilization of reagents. Various techniques may be used for other materials and reagents which include functionalization with materials such as carbonyldiimidazole, glutaraldehyde or succinic acid, or treatment with materials such as cyanogen bromide. Other suitable reactions include treatment with Schiff bases and borohydride for reduction of aldehyde, carbonyl and amino groups. DNA, RNA and certain antigens may be immobilized against solvent transport by baking onto the chromatographic material. Baking may be carried out at temperatures ranging from about 60° C. to about 120° C. for times varying from about five minutes to about 12 hours, but preferably at about 80° C. for about two hours.
Specific binding reagents useful with the present invention would be readily identifiable to one of skill in the art and include those materials which are members of a specific binding pair consisting of a ligand and a receptor. The ligand and receptor are related in that the receptor specifically binds to the ligands, being capable of distinguishing the ligand from other materials having similar characteristics. The methods and devices according to the present invention are particularly useful in the practice of immunological assay techniques where the specific binding reagents are antigens and antibodies including antibody fragments and synthetic antibodies. Specific binding materials such as avidin, biotin, strepatavidin and antibiotin may also be labelled and utilized in specific binding chromatographic assays according to the invention. The methods, kits and devices may also prove useful in the practice of DNA and RNA hybridization assays and other specific binding assays such as those involving receptors for hormones or other biologically active agents.
Blocking agents useful in preparation of devices for the specific binding of the present invention are those agents capable of blocking excess binding sites on the chromatographic media which might hinder chromatographic solvent transport of sample materials or reagents of the invention. In the construction of devices of the present invention, the chromatographic medium is impregnated with the reagent to be immobilized at the location desired. Once the reagent has been immobilized at the desired site, the strip is then preferably processed so as to block excess binding sites for other reagents or sample materials. Particularly suitable is the use of blocking solutions comprising proteins such as casein, gelatin, albumin or total serum. Such proteins are selected to not interfere with or cross-react with reagent materials of the assays. Blocking of the sites may preferably be conducted by dipping the chromatographic substrate materials in a solution of 0.2% casein in physiological saline and air drying the strip materials. Other methods include dipping in solutions of 0.1% gelatin or 0.1% bovine serum albumin followed by air drying of the substrate materials.
According to this example, sandwich-type immunoassay devices for the detection of Hepatitis B surface antigen (HBsAg) were constructed and used. Microporous nitrocellulose material with a thickness of approximately 0.15 mm and a pore size of 5μm (Millipore SMWP) was laminated to Mylar and adhesive (Monokote, Top Flite Models, Inc., Chicago, Ill.) at 60° to 65° C. in a film dryer apparatus. The membrane and backing was cut to strips 0.4 cm wide and 2.5 cm long. Anti-HBsAg antibodies (Abbott Laboratories, North Chicago, Ill.) (0.1-0.2 μl, 1.5 mg/ml) were applied to the reaction sites and incubated under ambient conditions for 15 minutes. Non-specific binding sites on the chromatographic medium were then blocked and cosubstrate bound by incubation for ten minutes at ambient temperature with a solution comprising 0.1% fish gelatin, 1% sucrose and 0.14 mg/ml nitro blue tetrazolium in 10 mM Tris and 150 mM NaCl (pH 7.6). The strips were then dried at 40° C. for about one hour and stored at ambient in the presence of a desiccant. Devices generally similar to the device of
To the sample application well of each test device was added a 30 μl sample of decalcified plasma which had been spiked with varying concentrations of HBsAg. After the sample had soaked into the first end of the nitrocellulose material, and had been transported through the reaction site to the second end of the nitrocellulose material, been absorbed by the blotter material a 15 μl aliquot of biotin labelled Anti-HBsAg antibodies (1 μg/ml) was added to each sample well. After this solution was transported through the device, a 15 μl aliquot of alkaline phosphatase labelled antibiotin (2 μg/ml) (Abbott Laboratories, North Chicago, Ill.) was added to each sample well and reaction sites were twice washed by addition to each sample well of 15 μl of wash solution comprising 1% Triton X 100 Triton X 100® brand of octylphenoxypolyethoxy ethanol nonionic detergent Tris buffered saline solution. A 15 μl aliquot of developing solution comprising 0.5 mg/ml bromochloroindolylphosphate, 0.1 mg/ml MgCl2 and 1% 2-amino-2-methyl propanol (pH 9.8) was then added to each sample well and a color reaction was allowed to develop for five minutes before the reaction was inhibited by addition of 15 μl of a 10 mM ethylene diamine tetraacetic acid (EDTA) solution. Sample solutions containing no HbsAg produced no signal, the reaction site was white. Sample solutions containing as little as 0.1 ng/ml HbsAg produced readily visible blue-gray spots at the reaction site. The total assay time was 25 minutes.
According to this example, sandwich-type immunoassay devices for the detection of HbsAg were constructed and used wherein the labelled specific binding material is labelled with colloidal gold particles according to the method of co-owned and copending U.S. Ser. No. 072,459 U.S. Pat. No. 5,120,643. The device and materials used were identical to that described according to Example 1, with the exception that nitro blue tetrazolium was omitted from the blocking solution.
To the sample well of each device was added a 50 μl sample of recalcified plasma which had been spiked with varying amounts of HBsAg. After the sample had soaked into the first end of the nitrocellulose material, a 50 μl aliquot of anti-HBsAg antibodies labelled with 40 nm colloidal gold particles adsorbed at 5 μg/ml (Abbott Laboratories) was added to each sample well. The total time for the assay was 15 minutes. Sample solutions containing no HBsAg produced no signal over background. Sample solutions as little as 1.0 ng/ml of HBsAg produced visible purple spots at the reaction site. No wash step was required since the antibody gold particles were sufficiently dilute.
Numerous modifications and variations in practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing descriptions of preferred embodiments thereof. It is well within the skill in the art to practice the present invention according to a wide variety of methods and formats. Consequently, only such limitations should be placed on the invention as appear in the following claims.
Gordon, Julian, Pugh, Charles Stuart Gredell
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