RADIAL FLOW ASSAY

Abstract: A delivering member for filtering and delivering a sample to a test assay surface includes a prefilter having a sample receiving surface and a surface for delivering a sample to a test assay surface. The prefilter includes a diffusing material on its receiving surface for rapidly and evenly diffusing a sample across the prefilter receiving surface. In this way, a sample may be rapidly and evenly delivered to a test assay surface. A test assay solid phase has a surface including an area carrying an immobilized species and an area free of immobilized species. When a sample is applied to the surface, followed by application of a labeled species, the evenness of distribution of the labeled species is indicative of the evenness of application of the sample. A kit includes a delivering member and an assay surface.

Description: KADIAL FLOW ASSAY This application is being filed concurrently with a commonly-owned U.S. Patent Application entitled "Assay Employing Both Protein and Antibody", by Dorval et al., which is incorporated herein by reference for all purposes. FIELD OF THE INVENTION The present invention relates generally to assay reagents, methods, and apparatus, and more particularly to radial flow assay apparatus and methods providing rapid and sensitive determination of an analyte in a variety of test assays. BACKGROUND OF THE INVENTION Immunoassays can be used to detect the presence of unwanted agents in a sample of fluid, for example the presence of bacteria in a bodily fluid or a pollutant in water. They can be used also to test for the presence of antibodies in body fluids, as an indication of the presence of an infectious agent, a cancer cell or an autoimmune disease. A variety of test assay components have been developed for use in immunoassays, and some of these components exploit the capillary flow of fluid through a porous medium. Assays utilizing this phenomenon have occasionally been somewhat improperly termed "chromatographic assays", and some involve but a one-step procedure. For example, U.S. Patent Number 4,956,302, issued September 11, 1990 to Gordon et al. , describes an assay device including a chromatographic medium having 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 a liquid absorption blotter offset downstream from the reaction site. A fluid sample is applied to the application well and is transported through the chromatographic medium, through the reaction site, and to the blotter. The reaction site is then contacted with a labeled, specific binding material, is washed to remove unbound species, and the label is detected. U.K. Patent Application Number 8809867, published November 9, 1988 under Publication Number 2204398A, describes a nitrocellulose medium including a sample application area, non-immobilized, labeled binding reagent specific for a first analyte epitope located downstream therefrom, and immobilized, non-labeled binding reagent specific for a second analyte epitope located further downstream. The analyte is provided in a fluid which carries the analyte to the labeled reagent and which carries the analyte and labeled reagent together to the immobilized, non-labeled reagent. After the assay, observation of label at the zone of immobilization is indicative of the presence of analyte in the sample. In each of the above-described devices, the chromatographic medium provideε the function of filtering the fluid sample of unwanted particulates, and of slowing the flow of fluid to allow specific binding to occur and to efficiently carry non-immobilized reagents to sites where other reagents are immobilized. However, this slow flow contributes to non-specific binding as well, and one must wait for a period of time on the order of minutes for a test result. Filtration of large quantities of serum and blood has also been carried out using porous media such as nitrocellulose. However, a significant pressure differential must be applied across a nitrocellulose filter to achieve such filtration. Porous media have been used to form so-called "prefilters" in test assay apparatus, these prefilters being used to spread a sample from a localized application area over a larger test assay surface area. U.S. Patent No. 4,912,034, issued March 27, 1990 to Kalra et al. describes such a prefilter. These prefilters are typically constructed of glass fibers, and provide rapid wetting of a test assay surface, but provide poor filtration, as selection of a prefilter having a pore size providing adequate filtration generally slows the wetting process significantly. That is, to apply a sample using a prior art prefilter, good filtration and rapid wetting of an assay surface are generally mutually exclusive. Additionally, glass fibers have very large void (retention) volumes, hence they absorb significant quantities of sample. Therefore, if only a small sample is available, it must be diluted significantly if the assay surface is to be adequately wetted. Then, to boost sensitivity, an enzymatic tag is typically employed. Although many such assays represent extraordinary improvements over the prior methodology for determining the presence of an agent in a sample, the goal remains to develop immunoassays that are rapid and sensitive. It is a drawback of almost every immunoassay presently in use that the assay either is too slow (5-30 minutes) or, if faster, iε not sufficiently sensitive (or both). Where the detection of disease is concerned, it is unacceptable to compromise on sensitivity, and accordingly those assays that are among the most sensitive tend to be cumbersome and slow. SUMMARY OF THE INVENTION The present invention is directed to products and processes that are useful in a variety of test assays. The invention permits the detection of analytes in very small samples, with enhanced sensitivity. The invention also permits the rapid detection of analytes in a sample. It further eliminates many steps characteristic of prior art assays, including in particular wash steps. The foregoing is accomplished, generally, by rapidly dispersing the sample into a film, and then delivering this film uniformly to a test assay surface. Thus, according to one aspect of the invention, a delivering member for delivering a sample to a test assay surface is provided. The delivery member includes a prefilter having a sample receiving surface. The prefilter also includes a sample delivering surface for delivering the sample to the test assay surface. A diffusing material is provided on the prefilter sample receiving surface for rapidly and laterally dispersing the sample when applied to the delivering member. According to a preferred embodiment the delivering member is substantially circular, and a sample is applied to the center of the receiving surface. Then the sample iε rapidly dispersed radially from the location of sample application. The diffusing material can be, for example, a membrane or a surfactant. Thus, the delivering member may be prepared from a pair of membranes in face to face relation, one of the membranes acting as the diffusing material and the other being the prefilter. The diffusing membrane has a porosity and thickness selected to rapidly laterally diffuse a fluid sample over the prefilter receiving surface. The prefilter is a substantially planar membrane having a porosity and thickness selected to rapidly transfer the sample from the receiving surface to the delivering surface while filtering the sample of particulate material larger than a predetermined dimension. In one arrangement, the diffusing material is a membrane having a pore size of about 5-25 microns and the diffusing material is a membrane having a pore size of from about .1 micron to 1.0 microns and a thickness of from about 50 microns to about 300 microns. If the diffusing material is a surfactant, then the construction of the delivery member may be according to a wide variety of embodimentε, discussed in greater detail below. In any event, the delivering membrane is constructed and arranged preferably such that the sample is rapidly and laterally diffused over the prefilter receiving surface, and then delivered evenly and uniformly to the test asεay surface. According to another aεpect of the invention, a method for making a delivering member for delivering a sample to a test assay surface is provided. A porous, substantially planar prefilter having a sample receiving surface is provided. A diffusing material then is applied to the sample receiving surface. The diffusing material can be a diffusing membrane having a periphery and having a porous structure providing rapid lateral flow of a sample applied to the receiving surface. In this instance, the diffusing membrane is mounted on the prefilter in face to face relation, preferably by a connector about the periphery of the prefilter and diffusing membrane. According to another aspect of the invention, a method of delivering a sample to a test asεay εurface is provided. A delivering surface of a prefilter is held in even contact with a test assay surface. A fluid sample then is applied to a diffusing material on a receiving surface of the prefilter so as to cause rapid lateral flow of the fluid sample at the receiving surface. The fluid sample then permeates the prefilter in a direction perpendicular to the direction of the lateral flow. In other words, the sample first flows in a direction parallel to the plane defined by the receiving surface and the test assay surface, and then flows through the prefilter and onto the test asεay εurface in a direction perpendicular to the firεt direction. According to another aεpect of the invention, a teεt aεεay kit for the determination of an analyte iε provided. The teεt aεεay kit iε a package containing a teεt assay solid phase having a test asεay surface carrying an immobilized binding partner of an analyte. The package also contains a prefilter having a sample receiving surface and a sample delivering surface positionable in face to face relation with the test aεεay surface, and being removable therefrom, with a diffusing material on the sample receiving surface. The package also contains an urging member for holding the prefilter delivering surface and the test asεay surface in even contact. The urging member can be an absorbent material in contact with a test assay εolid phase. Most preferably the prefilter is a planar cellulosic membrane, the diffusing material is a subεtantially planar celluloεic membrane and the teεt aεεay εurface iε a εurface of a planar membrane, and the teεt assay surface has pores of a dimenεion larger than the analyte and the delivering member haε pores smaller than those of the asεay εurface. The package further may contain detection reagentε aε deεcribed herein, including labeled detection reagentε. The detailed conεtruction of the delivering member may be as described above, the kit may also contain instruction for applying a predetermined amount of sample to the delivering member. According to another aspect of the invention, a test asεay kit for the determination of an analytes is provided. The kit compriseε a package containing a teεt aεεay εurface for supporting a test asεay reaction and a porous cellulosic prefilter having a pore size of from about 0.1 to about 1.0 microns, and having a delivering surface positionable in face to face relation with the test assay surface. According to still another aspect of the invention, a method of making a test assay kit for the determination of an analyte is provided. The method involves immobilizing a binding partner of an analyte on a test asεay εurface; fabricating a delivering member by applying a diffuεing material to a sample receiving surface of a substantially planar prefilter; and providing an urging member positionable so as to hold the delivering surface in even contact with a test asεay surface. The test assay surface, delivering member and urging member then are packaged together to form the kit. The urging member can be positioned, prior to packaging the materials, in contact with one of the porous delivering member or the assay solid phase εo aε to hold the delivering surface in even contact with the assay surface, the method may alεo include formulating a detection reagent containing a labeled binding partner of the analyte. According to another aspect of the invention, a test assay method for the determination of an analyte is provided. A fluid sample suspected of containing the analyte is applied to a receiving surface of the porouε delivering member also having a delivering surface in even contact with a test assay surface. The fluid sample iε caused to first rapidly flow laterally. The fluid sample then is caused to permeate the delivering member and to be evenly transferred to the test asεay εurface. According to another aspect of the invention, a test assay method for the determination of an analyte is provided. A fluid sample suεpected of containing an analyte iε applied to a diffuεing material on a receiving surface of a porous delivering member. The porous delivering member also includes a delivering surface in even contact with a test asεay surface carrying an immobilized binding partner of an analyte. The fluid sample is thus caused to rapidly and laterally flow in a direction subεtantially parallel to the receiving εurface. The fluid εample then iε allowed to permeate the delivering member and to be tranεferred evenly to the test assay surface. The delivering member then is removed from the test assay surface. The teεt aεεay surface is contacted with a detection reagent containing one of the labeled binding partner of the analyte or a labeled binding partner of the immobilized binding partner. Then the label is detected. Preferably, the test asεay εurface haε pores of the dimension larger than the analyte and the delivering member has pores of a dimension larger than those of the test asεay εurface. According to another aspect of the invention, a test assay method for the determination of an analyte is provided. A sample is applied to a test asεay εurface having regionε capable of non-specific binding. The sample includeε blocking specieε in an amount to only partially block the regionε. The regionε then are contacted with a detection reagent including a species carrying a label. Then, the uniformity of the distribution of non-specific binding of the labeled species is meaεured, the uniformity being determinative of the evenneεε of the application of the εample to the teεt aεεay εurface. Preferably, the regionε are free of any immobilized biological reagentε. According to another aεpect of the invention, a teεt assay solid phase having a test assay surface is provided. The test asεay εolid phaεe may be uεed in accordance with the method of the invention deεcribed immediately above. The teεt assay surface includeε an area having a chemical functionality that allowε non-specific binding of labeled reagent to a degree indicative of the extent of sample first applied to the area. Thus, when a sample is first applied to the area to a particular extent, followed by application of the labeled reagent, labeled reagent is preεent to a particular degree. When εample iε first applied to the area to a second extent different from the first extent, followed by application of labeled reagent, labeled reagent non-specifically binds to the area to a second degree distinguishable from the first degree. The test assay solid surface may include a second area carrying an immobilized binding partner of an analyte. According to another aεpect of the invention, a method of delivering a fluid εample to a teεt aεεay surface is provided. The method includes the stepε of holding a delivering εurface of a prefilter in even contact with a teεt aεεay surface, forming a film of the fluid sample on a receiving surface of the prefilter, and allowing the film to permeate the prefilter and to evenly wet the test asεay εurface. According to εtill another aεpect of the invention, an improvement to immunoaεεayε including kits and devices employing prefilters is provided. The improvement involves in one aspect the use of a prefilter having a pore size of from about .1 to 1.0 microns. The improvement in another aspect involves a prefilter comprising a pair of membranes in face to face relation. The improvement in another aspect involves a prefilter having a sample receiving surface, and constructed and arranged to rapidly laterally diffuse a fluid sample over the receiving surface and to then allow the sample to permeate the prefilter and to be delivered uniformly and evenly to a test assay surface. The improvement in still another aspect involves a prefilter having a sample receiving surface and a surface for delivering the sample to the test assay surface, and a diffusing material on the prefilter sample receiving surface. These and other aspects of the invention will be described in greater detail below in connection with the drawings. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates a delivering member and a test a-.say solid phase including a test assay surface according to one embodiment of the present invention; Figs. 2a-2f schematically illustrate a test assay procedure for the determination of both IgG and one of IgA, IgM, or a combination in a single sample according to one of embodiment of the invention, using an assay kit according to one embodiment of the invention; Figs. 3(a-c) illustrate the appearance of a test assay surface according to one embodiment of the present invention after a test assay as illustrated in Fig. 2 has been performed in which the result is error, negative, and positive, respectively; Fig. 4 is a top view of a test assay kit according to one embodiment of the present invention; and Fig. 5 is a croεε-section through lines 5-5 of Fig. 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Many pairs of biological species exhibit mutual affinity, or mutual binding capacity, typically defined by specific or non-specific binding or interaction. Such interaction RECTIFIED SHEET (RULE 91) ISA/EP generally occurs between pairs of molecular species in which one member posseεεes one or more particular exposed areas which bind to one or more particular exposed areaε of the other member of the pair. The exposed areas allowing such binding are typically defined by εpecific εpatial and/or polar organization. The term "binding partnerε" may be uεed, and iε uεed herein, to define pairε of εpecies exhibiting such mutual affinity. For example, such binding partners include antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, biotin/avidin, binding protein/εubstrate, carrier protein/subεtrate, lectin/carbohydrate, receptor/hormone, receptor/effector, complementary εtrandε of nucleic acid, repreεεor/inducer, and the like. In many teεt assays, it is desirable to capture, or determine, an analyte defined by one member of a pair of biological species exhibiting mutual affinity. Many immunoasεays have been developed which are designed to capture such an analyte, for example to capture an antibody produced by an animal in response to an antigen. According to one such immunoasεay, the antigen iε immobilized on a test asεay surface, a serum sample is contacted by the test asεay surface so that antibodies specific for the immobilized antigen are captured at the surface, and a detection reagent including a labeled binding partner of the analyte antibody is then contacted with the test asεay εurface εo that the preεence of the analyte iε indicated by label immobilized at the εurface. The preεent invention provides test assays that are rapid, sensitive, and useful with small sample sizes. It provides in particular a delivering member for filtering, and rapidly and evenly delivering an aεsay sample to a test assay surface. Additionally, a test assay εolid phaεe iε provided, the aεεay solid phase exposing a test asεay εurface εelected so as to provide advantageous measurement of the evennesε of distribution of a sample over the εurface. Aε uεed herein, the term "εample" iε meant to define a fluid mixture or solution suspected of carrying an analyte to be determined by a test asεay. When components of the present invention are used in a biological assay, the sample will generally be delivered as an aqueous solution or suspenεion. The delivering member and teεt assay surface may, together, be included in a test asεay kit in accordance with another embodiment of the invention, and theεe two componentε are illuεtrated in Fig. l. Referring to Fig. 1, a porouε delivering member in accordance with the invention iε illuεtrated at 20, and iε compriεed of prefilter 28 which haε a sample receiving surface 24 and a εample delivering εurface 22. A diffusing material 26 is provided on receiving surface 24 of prefilter 28. Teεt aεεay εolid phaεe 11 includeε test assay surface 10, and delivering εurface 22 of prefilter 28 iε conformable to test assay surface 10. Prefilter 28 and test asεay εolid phaεe 11 are substantially planar according to the embodiment illustrated, but need not be. It is important only that delivering member 20 is constructed so as to evenly wet test asεay εurface 10 with a fluid sample, and this may be achieved if delivering surface 22 and test assay surface 10 are of conforming shape, and/or if delivering εurface 22 and teεt aεεay εurface 10 are conformable to each other. Prefilter 28 and, optionally, aεsay solid phase 11 are constructed of a matrix having a porous structure that is inert with reεpect to εample componentε, detection reagentε or other species with which these elements come into contact during a test assay, or is capable of being made inert. As used herein, the term "inert" referε to a chemical or biological εtate in which the material doeε not interact with any component in a teεt εample in the manner in which any binding partner pairs in the assay interact, or act to hinder any desired assay reactions. When any test assay components non-εpecifically bind to material defining prefilter 28 and assay solid phase 11, it is a feature of the invention that the degree of εuch non-εpecific binding iε εmall in compariεon to the degree of εpecific binding which iε exploited by aεεays performed in accordance with the invention to determine analytes in a fluid sample. As discussed more fully below, it is desirable that a certain degree of labeled reagent non-specifically bindε to teεt aεεay solid phase in accordance with one aspect of the invention. Additionally, the material used to construct prefilter 28, and preferably assay solid phase 11, exhibits capillarity when exposed to the asεay fluid medium, and the capacity for fluid transport of non-immobilized reagents and εample componentε. The term "capillarity" defines a relationship between a porouε material and an aεεay εample fluid medium in which the contact angle between the fluid medium of the sample and the material is acute, or, a relationship in which the fluid sample is drawn through the material irrespective of the force of gravity on the fluid sample. Exemplary materials suitable for use as prefilter 28 and, optionally, test asεay εolid phase 11 include woven and non-woven fibrous materialε uεed for paper chromatography, microporous or microgranular thin layer chromatography subεtrateε, porouε polymeric materials including polyalkenes εuch aε polyethylene and polypropylene; polyvinyl chloride; polyamideε εuch aε nylons; polyaryls εuch aε polystyrene; polyarylsolfoneε; polyvinyl and polyvinylidene halideε; polyvinyl and polyvinylidene alcohol; polycarbonate; polysaccharides including celluloεicε εuch aε celluloεe acetate butyrate and nitrocelluloεe; polyeεterε εuch as polyethylene terephthalate; polyimides and polyurethanes such as polyether polyurethanes and combinations thereof, optionally pre-treated (in the case of solid phase 11) so as to provide binding capability as described in U.S. Patent No. 4,757,014, isεued July 12, 1988, and incorporated herein by reference; celluloεe hydrateε, charge-modified microporous membranes such as those described in U. S. Patent No. 4,604,208, issued August 5, 1986, and incorporated herein by reference; fluorinated polymeric species such as polytetrafluoroethylene and polyvinylidene fluoride; porous acrylic polymers including polyacrylates such as polymethyl methacrylate; porous so-called biomoεaic polymers, that is, polymers formed from a polymerized emulsion of at least one polymerizable monomer polymerized in the presence of a biologically active material, such as are described in EP application No. 90312532.6, published under publication No. 0430517A2 on June 5, 1991, incorporated herein by reference; silica; alumina; diatomaceous earth; magnesium sulfate or other inorganic finely divided material conveniently substantially uniformly disbursed in a porous polymer matrix; cloth, both naturally occurring and synthetic; agarose; dextran; gelatin, and the like. Exemplary materials suitable for use as prefilter 28 may also be found in U.S. Patent No. 4,366,241, iεsued December 28, 1982 to Tom et al. , and incorporated herein by reference. A variety of microporous membranes suitable for use as prefilter 28 and, optionally, asεay solid phase 11 are available commercially from Amicon, Geleman, Millipore, Schleicher and Schuell, and Pall Corporation. According to one embodiment of the invention, diffusing material 26 on receiving surface 24 of prefilter 28 is a surfactant. Aε uεed herein, the term "εurfactant" iε defined aε any εpecies which may be attached to or coated onto receiving surface 24, and which results in a depresεion in the εurface tenεion, or interfacial tenεion, of a fluid sample applied to receiving εurface 24. Thuε, the term encompaεεeε wetting agentε and emulsifiers, including detergents. A surfactant may be selected to chemically bind to receiving surface 24, or to coat surface 24 in an adεorptive manner. Secure chemical attachment of the surfactant to receiving εurface 24 may optionally be effected by heating the prefilter for an appropriate period of time, or other known chemical or phyεical methods. Although it may be advantageous to select a surfactant to firmly bind to receiving surface 24 so aε not to be dissolved in or otherwise swept away be a sample passed through prefilter 28, this is not εtrictly necessary. If a one-use, or dispoεable, prefilter iε εelected, then the degree to which the εurfactant is removed during application of the sample is not critical so long as the surfactant serves to rapidly diffuse the sample laterally acroεs receiving surface 24, and does not interfere with the reactivity or detection in the assay. A surfactant may permeate into prefilter 28 beyond receiving surface 24 somewhat, although it is preferable that the surfactant reside substantially at receiving surface 24. A wide variety of εurfactants may be selected for employment as diffusing material 26 of delivering member 20 of the preεent invention, εelected from a variety of claεses, including but not limited to alkyl sulfates and quaternarieε, including heterogeneouε polyoxyethyleneε, glucamides, digitonin, bile acids, sulfo betaineε, betaineε, alkyldimethylamine oxides, alkyl glucosides, alkyl maltosides, lecithins and lyεolecithins, homogeneous polyoxyethylenes, and alkyl thioglucosides. Specifically, polyethylene glycol dodecyl ether, octanoyl-N-methylglucamide, nonanoyl-N-methylglucamide, decanoyl-N-methylglucamide, nonyl glucoεide, nonaethylene glycol octylphenyl ether, octyl glucoside, octyl thioglucoside, polyethylene polypropylene glycol, monosodium taurocholic acid, sodium taurodesoxycholate, nonaethylene glycol mono-dodecyl ether, nonaethylene glycol octylphenol ether, nonaethylene glycol octylcyclohexyl ether, heptaethylene glycol octylphenyl ether, heptaethylene glycol octylcyclohexyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, n-octylsulfobetaine, n-decylεulfobetaine, n-dodecylsulfobetaine, n-tetradecylsulfobetaine, n-hexadecylsulfobetaine, N,N,bis(3-D-gluconamidopropyl) cholamide, polyoxyethylene lauryl ether, octaethylene glycol monododecyl ether, nonaethylene glycol monododecyl ether, cetrimmonium bromide, 3-[ (3-cholamidopropyl) dimethylammonio]- 1-propaneεulfonate, 3-[(3-cholamidopropyl) dimethylammonio]-2- hydroxypropane-1-sulfonate, cholic acid (monosodium salt), decyl glucoside, decyl maltoside, N,N-bis(3-D- gluconamidopropyDdeoxycholamide, deoxycholic acid (εodium salt), digitin, dodecyl maltoside, N-Dodecyl-N,N- dimethylglycine, dioctyl sodium sulfosuccinate, lauryldimethylamine oxide, decaoxyethylene monolauryl ether, octaethylene glycol iεotridecyl ether, polyoxyethylene iεotridecyl ether, glycocholic acid (sodium salt), glycodeoxycholic acid (sodium salt), heptyl glucoside, hexyl glucoside, heptyl thioglucoεide, N,N-Dimethyl-N-dodecylamine oxide, εodium lauryl εulfate, and equivalent εurfactantε may be selected. A variety of suitable εurfactants are εold under the trademarks TRITON, TWEEN, ZWITTERGENT, MEGA, PLURONIC, GENAMINIOX, GENAPOL, available from Calbiochem Corporation, La Jolla, California. When a surfactant iε εelected as diffusing material 26, it is generally advantageous to position delivering member 20 with receiving surface facing upward during its use in a test asεay procedure, that iε, εubstantially parallel to the ground. In this way, the εample is asεured of diffusing equally in all directions a radial manner. Alternatively, the delivering member may be held in non-parallel relation with the ground, and the sample may be added to a location of the receiving εurface other than at the center, εo that diffuεion across the receiving εurface iε aided by gravity. When delivering member 20 in accordance with the present invention is conεtructed of a εurfactant as diffusing material 26 at receiving εurface 24 of prefilter 28, it iε to be understood that the diffusing material/prefilter combination may be arranged in one of a number of ways. According to one, the diffusing material is defined by a surfactant that coats only receiving surface 24 of prefilter 28, while the remainder of prefilter 28 is free of any surfactant. According to another, a first surfactant, or blocking agent, may coat εome or all portionε of the porouε network of prefilter 28, while a εecond εurfactant coatε receiving εurface 24 and defineε diffuεing material 26. According to thiε arrangement, the preεence of the εecond surfactant at receiving surface 24 depresεeε the εurface tenεion of a fluid sample applied to the prefilter relative to the remainder of the prefilter. For example, the second surfactant may be a better wetting agent than the first surfactant or may be applied in a greater amount. According to another arrangement, a surfactant coats any or all portionε of prefilter 28, and the εame εurfactant iε provided in excess at receiving surface 24, such that the surface tension of a fluid sample is depreεεed to a greater extent at receiving εurface 24 than at other portions of prefilter 28. According to yet another arrangement, a surfactant coats receiving surface 24 and the same or another surfactant may coat other portions of prefilter 28, but the surfactant coated receiving εurface 24 iε εeparated from any εurfactant coating any other region of prefilter 28 by at least a portion of prefilter 28 remaining free of surfactant. For example, receiving εurface 24 may be coated with a firεt εurfactant, and delivering surface 22 may be coated with the same or another surfactant, while portions of prefilter 28 between receiving surface 24 and delivering surface 22 remain free of any surfactant. Thus, when diffusing material 26 is defined by a surfactant, one or more surfactantε may be applied to one or more portions of prefilter 28, so long as delivering member 20 is constructed and arranged such that the surface tension of a fluid sample iε depreεεed at receiving surface 24 to a greater extent than it iε depreεεed at portionε of prefilter 28 immediately adjacent to receiving εurface 24, whereby a εample will be rapidly laterally diεtributed prior to reaching the teεt aεεay εurface. According to another embodiment of the invention, diffuεing material 26 on receiving εurface 24 of prefilter 28 iε a porouε, inert matrix possesses capillarity with respect to an applied εample, preferably a porouε membrane εelected to rapidly laterally diffuεe a fluid εample over receiving εurface 24 via capillary action, and according to thiε embodiment reference will be made to diffuεing material 26 aε diffuεing membrane 26. Diffuεing membrane 26 may be fabricated from one of or a combination of the materials described above with respect to fabrication of prefilter 28. Additionally, a frit such aε a glass or nylon frit material may be used. Diffusing membrane 26 and prefilter 28 may or may not be fabricated from the εame material in a εingle delivering member 20. Diffuεing membrane 26 may be juxtaposed with receiving surface 24 of prefilter 28 by a variety of methods, for example via chemical or physical binding, such aε with adhesive, preferably applied in very small, isolated regions between the membranes, or such as via stitching or microrivets or the like. Preferably, delivering member 20 includes a connector, such as a label or tab (not εhown in Fig. l), connected to the periphery of prefilter 28 and to the periphery of diffusing membrane 26, holding the diffusing membrane in face to face relation with receiving surface 24 of the prefilter. As used herein, the phraεe "face to face relation" is meant to define close and juxtaposed relation, but not necessarily even contact. As uεed herein, the phraεe "even contact" is meant to define a relationship in which a surface of one component is urged into contact with a surface of another component with application of a constant force, such as with a resilient member, so that the two surfaces evenly contact each other across the entire operative area between them defined by each surface. When membranes of the present invention are held in face to face relation, they may be brought into even contact with application of an external force, other than the force of gravity, onto one or both membranes. As will be apparent, diffusing membrane 26 and receiving surface 24 can be held in even contact during tranεfer of a fluid εample to aεεay εurface 10 by delivering member 20, but need not be at other timeε. To achieve rapid lateral diffuεion of a fluid εample over prefilter receiving surface 24, using diffusing membrane 26, a variety of factorε can be considered in the selection of diffusing membrane 26 and prefilter 28. Some of these factors include porosity, capillarity, void volume, thicknesε, and wetability. As used herein, the term "porosity" is meant to define characteristics of the porous structure of a membrane including pore size, directional pore orientation, the degree of pore blockage or openness, and the like. As one example of a suitable combination of a membrane selected as diffuεing membrane 26 and a membrane εelected aε prefilter 28, both membraneε may be conεtructed of the εame material, with diffuεing membrane 26 being selected to have a pore size larger than that of prefilter 28. According to this arrangement, and with reference to Fig. 1, application of a sample at the center of diffusing member 26 will result in rapid permeation of the fluid sample laterally through membrane 26. Such rapid lateral diffusion is due in part to the relatively large pore size and resultant relatively rapid flow within membrane 26, in combination with the relatively slower flow through prefilter 28 due to its relatively smaller pore size. The sample is forced to flow laterally through diffusing membrane 26 aε it contactε receiving surface 24 of prefilter 28. According to another arrangement, diffusing member 26 contains pores oriented substantially parallel to receiving surface 24, that is, the distribution of pore orientation favorε lateral capillary flow within the membrane. In thiε way, rapid lateral diffuεion of a sample by diffuεing membrane 26 occurε. According to another arrangement, the capillarity of diffusing membrane 26 is greater than the capillarity of prefilter 28, due to a greater depresεion in fluid εample εurface tension within diffusing membrane 26. In any case, when delivering member 20 is constructed of diffusing membrane 26 and prefilter 28, membrane 26 and prefilter 28 are selected so that, when a sample is applied to diffusing membrane 26, a fluid sample film is rapidly formed on receiving εurface 24 of prefilter 28, whereupon the film is allowed to permeate prefilter 28 and to wet test aεεay εurface 10 evenly and equally. Aε uεed herein, the term "film" is meant to define a fluid front that is substantially parallel to receiving surface 24 and teεt aεεay εurface 10. This is to be distinguiεhed from a fluid front which reεultε from application of a fluid εample to a prior art prefilter having no diffuεing material at a receiving εurface thereof. Fluid applied to εuch prior art prefilterε radiateε into the prefilter equally in all directionε, that iε, the fluid will reach a delivering εurface of the prefilter and contact the teεt aεεay εurface prior to itε complete lateral permeation to the edgeε of the prefilter, and fluid therefore will wet gradually a teεt aεεay εurface radially from a central location. When εuch a prior art prefilter iε uεed in aεsociation with a test aεsay surface and detection reagent in accordance with the present invention, uneven distribution of label on teεt aεεay εurface 10 reεultε. This phenomenon is deεcribed more fully below. Prefilter 28 and diffuεing material 26 of the preεent invention are selected such that delivering member 20 uniformly wets teεt aεεay surface 10 substantially instantaneously acrosε itε entire εurface, due to the formation of a fluid sample film at receiving surface 28 and permeation of that film through the prefilter and to test assay surface 10. This ensures that analytes in the sample are applied in uniform amounts acrosε the teεt aεsay surface. To achieve rapid lateral flow of a fluid sample acrosε receiving εurface 24 of prefilter 28, eεpecially when diffuεing membrane 26 and prefilter 28 are conεtructed of the εame material, the dimensions of pores in diffusing membrane 26 and the thickness of this membrane can be selected within prescribed ranges relative to the pore dimensions of prefilter 28. Similarly, to achieve adequate filtration while rapidly delivering a sample from receiving surface 24, through prefilter 26 in a direction perpendicular to receiving εurface 24 and evenly to teεt aεεay surface 10, prefilter 28 can be selected to have a pore size within a preεcribed range. Additionally, delivering member 20 iε conεtructed so as to provide rapid and even delivery of a small, undiluted fluid sample to test assay surface 10. These objects may be achieved, for example, by selecting a prefilter 28 having a pore size of from about 0.1 micron to about 1 micron, preferably from about 0.2 micron to about 0.7 micron, more preferably from about 0.33 micron to about 0.53 micron, and a diffusing membrane 26 having a pore size of from about 5 to about 40 microns, preferably from about 8 to about 30 microns, and more preferably from about 10 to about 20 microns. According to a particularly preferred selection of material for fabrication of prefilter 28 and diffusing membrane 26, both are fabricated from a cellulosic membrane material, preferably nitrocellulose, the prefilter 28 having a thicknesε of from about 50 micronε to about 300 micronε, more preferably from about 110 microns to about 185 microns, and the diffusing material 26 having a thickness from about 105 to about 165 microns. If a prefilter of a diameter different from that illustrated and deεcribed herein is selected, it may be desirable to construct delivering member 20 such that the amount of diffusing material 26 applied to receiving surface 24 of prefilter 28 is adjusted accordingly. For example, if receiving surface 26 comprises a surfactant, it may be deεirable to increaεe the amount of εurfactant applied to receiving εurface 24 as the diameter of prefilter 28 is increased. Or, delivering member 20 may be conεtructed with εurfactant arranged nonuniformily on receiving εurface 24 of prefilter 28, with more εurfactant being applied at the center of the receiving surface and the amount of εurfactant decreaεing radially acroεε the prefilter. When a membrane is selected to serve as diffusing material 26, delivering member 20 may be conεtructed εuch that a membrane serving aε diffuεing material 26 decreaεes in thickneεε radially, so as to have a greater thicknesε at the center of prefilter 28 and a leεser thicknesε at the periphery thereof. Or, when the diameter of prefilter 28 iε increaεed to cover a larger teεt surface area, it may be desirable to increase the thickneεε of diffuεing membrane 26. Additionally, diffuεing material 26 may include a combination of a membrane and a εurfactant applied to any portion of the membrane, or applied to the receiving εurface 24 of prefilter 28. That iε, delivering member 20 may include prefilter 28, a εurfactant applied to receiving surface 24 thereof, and a diffusing membrane 26 applied to receiving surface .24 following surfactant application. This may be especially advantageous if a delivering member having a particularly large diameter is constructed. The thicknesε of prefilter 28 may remain substantially the same over a wide range of diameters, if diffusing material 26 is selected εo as to rapidly and evenly diεtribute a sample across receiving surface 24 of prefilter 28 of large diameter. However, the thicknesε of prefilter 28 may be adjuεted εo aε to provide greater or leεεer εample retention, greater or leεser filtration, or for a variety of reaεonε. When delivering member 20 iε poεitioned εo aε to tranεfer a sample to test assay surface 10, delivering member 20 is oriented substantially parallel to test aεεay surface 10, and delivering surface 22 is held in even contact with aεεay surface 10. Referring to Fig. 1 (in which, for purpoεes of illustration, delivering surface 22 is not held in even contact with assay surface 10) this is illustrated by application of a sample near the center of diffusing material 26, whereupon the sample is rapidly and evenly diffuεed laterally, or radially from the center, by diffuεing material 26 in the direction of arrows 30, so aε to rapidly and evenly spread the sample acrosε the entire receiving εurface 24 of prefilter 28. Diffusing material 26 and prefilter 28 are selected such that lateral diffusion is fast enough, relative to any permeation of sample through prefilter 28, that a sample fluid film is formed at receiving surface 24. Prefilter 28 rapidly and evenly transferε the sample, as a film, from receiving surface 24, in a direction εubεtantially perpendicular to the plane of receiving εurface 24, to teεt aεεay εurface 10, while filtering the sample of small particles. As used herein, the term "film" is meant to define a thin layer of sample fluid, having a fluid front that is maintained subεtantially parallel to teεt aεεay εurface 10, εo that surface 10 is contacted by the εample at all regions thereof subεtantially inεtantaneouεly and with a uniform portion of the εample being applied at any particular region of the surface 10. Selection of material for use as delivering member 20 can be made in conjunction with selection of fluid as the test asεay medium. When the teεt assay fluid medium is water, cellulosicε, and in particular nitrocelluloεe, iε preferred for both prefilter 28 and diffusing member 26. When delivering member 20 is to be used to tranεfer a biological εample, such as a εerum or blood εample, to an aεεay εurface, delivering member 20 (including prefilter 28 and, according to one embodiment, diffusing membrane 26) may advantageously be blocked to prevent binding of sample constituents thereto. Blocking agents useful for achieving thiε are thoεe agentε capable of blocking all binding εiteε that could hinder solvent tranεport of sample componentε or reagentε of the invention. Any blocking agent can be εelected εo aε to be inert with reεpect to any εample componentε. That iε, any agent that would εpecifically interact with any εample component can be avoided. A εuitable blocking agent may comprise a naturally occurring or synthetic, proteinaceous or non-proteinaceouε material. A non-limiting exemplary liεt of material suitable for such a blocking agent includes casein, gelatin, albumins εuch aε bovine serum albumin, bovine gamma globulin, apoferritin, ovalbumin, avidin, peptides, polyamino acids and synthetic polyamino compounds, total serum, non-proteinaceous polyamino acids and synthetic polyamino compounds. Additionally, prefilter 28 and diffusing membrane 26 may be coated with a surfactant, as described above, to achieve blocking. The size of any sample applied to sample receiving surface 24 of delivering member 20 can be selected in conjunction with the size of delivering member 20 and the retention (void) volume thereof εuch that the sample volume does not exceed that amount that would adversely effect the desired flow characteristicε of delivering member 20. When porous material is selected as solid phase 11, it is desirable to select a sample volume of a εize εmaller than that which would completely fill the retention volume of delivering member 20 and that of εolid phaεe 11. Preferably, a εample εize iε εelected to permeate delivering member 20, and to wet teεt aεεay εurface 10 of teεt aεsay solid phaεe 11 only to the extent neceεεary to perform the teεt aεεay. For example, if all immobilized teεt assay reagents at teεt assay surface 10 reside substantially at the surface, rather than within test aεεay εolid phaεe 11, it iε desirable that the fluid εample does not permeate into test assay solid phase 11 to a significant extent. On the other hand, if test assay reagents are immobilized within asεay εolid phaεe 11, that iε, below surface 10, it would be desirable for a fluid εample to permeate into teεt aεεay solid phase 11 to the extent necesεary to contact the immobilized reagentε. Additionally, it iε deεirable for a teεt assay fluid to wet test aεεay εurface 10 to the extent that a deεired degree of non-εpecific binding of a later-applied labeled reagent occurε. This is described more fully below. Additionally, it is desirable to select a sample volume of a εize facilitating permeation of delivering member 20 via such as thoεe deεcribed in U.S. Patent No. 4,366,241, issued December 28, 1982 to Tom et al. , referenced above. Test aεεay solid phase 11, and in particular surface 10 thereof, can be selected such that a test asεay reagent may be conveniently immobilized thereon. Surface 10 may include one, or a plurality of specific areas at which one or more test assay reagents are immobilized. Typically, an immobilized asεay reagent is selected aε one member of a binding partner pair. Immobilization of a member of a binding partner pair onto teεt aεεay surface 10 may be achieved via covalent or absorptive attachment. According to one embodiment, test asεay surface 10 includes exposed functional groups including, but not limited to, carboxyl groups, εulphonic acid groups, amino groups, thio groups, hydroxyl groups, pyridyl groups, phosphoryl groups, derivativeε of any of theεe, or other functional groupε known to thoεe of ordinary εkill in the art that would facilitate immobilization. Theεe functional groupε can alεo be preεent in an activated form, activated by the introduction of a εubstituent such as, for example, halogen, phenyl, carboxyl or sulphonyl or the like. Test asεay εolid phase 11 may be formed with an analyte binding partner immobilized therein or thereon as a result of the formation procesε, aε deεcribed in U. S. Patent No. 4,098,645, iεεued July 4, 1978, and incorporated herein by reference. Additionally, plaεma immobilization of proteinε on polymeric matrices are described in U.S. Patent No. 5,028,657, issued July 2, 1991 and incorporated herein by reference, and such immobilization is within the scope of the present invention. DNA, RNA and certain antigens may be immobilized against solvent transport by baking onto the test aεεay εurface 10. Generally, when teεt aεεay εurface 10 compriεeε nitrocelluloεe or a mixed nitrocelluloεe eεter, no special chemical linkage is required for the immobilization of assay reagents thereon. That is, immobilization may be abεorptive. As illustrated in Fig. 1, surface 10 includes first area 12 having a first aεεay reagent 14 immobilized thereon (Protein A is illustrated) and a second area 16 carrying a second immobilized assay reagent 18 (HIV is illustrated). Any number of areaε carrying immobilized aεsay reagents may be provided on test asεay surface 10, and these reagents may εerve aε analyte binding partners, control binding partners, or a combination. Test aεεay εurface 10 of teεt aεεay εolid phaεe 11 may be blocked according to the invention, but need not be. As noted, according to one embodiment test assay solid phase 11 iε porouε, and may be fabricated from any of the materialε deεcribed above which are εuitable for use in fabrication of prefilter 28. According to this embodiment, and especially when teεt aεεay εolid phaεe 11 and prefilter 28 are fabricated from the εame material, εolid phaεe 11 haε a pore εize as large as or larger than that of prefilter 28. Also, solid phase 11 can have a pore size εelected to facilitate rapid flow of sample, and detection reagent (described more fully below), so as to minimize non-specific binding at test asεay εurface 10. Specifically, εolid phaεe 11 can have a pore εize of from 1 to 100 microns, preferably from about 5 to 40 microns, more preferably from about 8 to about 30 microns, and most preferably from about 10 to about 20 microns. The εize and εhape of delivering member 20 and teεt aεεay εolid phaεe 11 may take a variety of formε and valueε. For example, delivering member 20 and test assay solid phase 11 need not be the same size or shape. Only a portion of the exposed surface of solid phase 11 as illustrated may define test assay surface 10, or the entire exposed surface may define the test assay surface. Additionally, delivering surface 22 of member 20 may be defined by only a portion, or all, of an exposed surface of member 20. It is important only that any portion of delivering member 20 defining delivering εurface 22 iε brought into even contact with the entire exposed surface of solid phase 11 that defines test asεay surface 10. According to the embodiment illuεtrated, delivering member 20 and test assay solid phase 11 are both substantially circular, and each has a diameter of approximately 1.5 centimeter. The thickness of test assay solid phase 11 is not critical to the present invention, but if a porous solid phase 11 is selected, it is advantageous to select one with a retention volume equal to or greater than the difference between the volume of sample applied to delivering member 20 and its retention volume. In this way, solid phase 11 adds to the rapid an even transfer of a fluid sample from receiving surface 24 onto asεay surface 10. Referring now to Figs. 2a-2f, an exemplary teεt assay is illustrated, utilizing a test aεεay kit in accordance with the present invention. In all of the figures, each component common to several of the figures is given a single numerical designation. The kit includes (Fig. 2a) delivering member 20, and utilizes a detection reagent deεcribed more fully in co-pending Application entitled "Aεεay Employing Both Protein and Antibody", by Dorval et al. , referenced above. Specifically, a highly-εenεitive HIV detection analyεiε according to the present invention is- illustrated. With reference to Fig. 2, the assay includes Protein A as a first immobilized assay reagent 14 and HIV as a second immobilized asεay reagent 18 on first area 12 and second area 16, respectively, of test assay surface 10. In Fig. 2, delivering member 20 is represented schematically, and diffusing material 26 is not shown. Therefore, receiving surface 24 of prefilter 28 (not shown) is represented as a receiving surface of delivering member 20 as a whole. Delivering member 20 is placed upon test aεεay solid phase 11 such that delivering surface 22 evenly contacts test assay surface 10. Sample is added to sample receiving surface 24 of delivering member 20 (Figs. 2b, 2e). preferably substantially in the center of surface 24, whereupon the RECTIFIED SHEET (RULE 91) εample is rapidly radially diffused by diffusing material 26 (not specifically illustrated) so as to form a film, and the film is evenly transferred to test asεay εurface 10, εo as to contact all portions of surface 10 substantially simultaneously. Delivering member 20 includes a prefilter (not specifically illustrated) which filters the sample of particulate material 32 of a dimension larger than the smallest pore dimension of the prefilter. After a sample has been delivered to asεay εurface 11 via delivering member 20, any binding partnerε of firεt or εecond immobilized assay reagents 14 and 18 will bind thereto. As illustrated in Fig. 2b, a sample containing all of IgG, IgA and IgM specific for HIV are captured by HIV immobilized at second immobilized aεεay reagent area 16. IgG haε alεo been captured at firεt immobilized assay reagent area 12 by immobilized Protein A, and this serves as a control as IgG is present in large amounts in all serum samples. If no disease is present, that is, if no antibodies to HIV are present in the sample, species in the sample will not bind to the immobilized HIV at area 16 (Fig. 2e) . Inevitably, some sample components will remain at test assay surface 10, unbound to any immobilized asεay reagents, and this is indicated at 34. Subsequently, delivering member 20 is removed and an excess of a detection reagent is deposited upon test asεay surface 10 (Figs. 2e, 2f). According to the asεay illustrated, the detection reagent includes Protein A 36 coupled to a hydrophobic label, specifically indigo, which binds to IgG bound to Protein A at area 12 of surface 10, and to IgG bound to HIV at area 16 of surface 10. The reagent also includes anti-IgA-IgG 38 which binds to IgA bound to HIV at area 16 of surface 10, and anti-IgM-IgG 40 which binds to IgM bound to HIV at area 16 of surface 10. Indigo is hydrophobically coupled to each of anti-IgA-IgG 38 and anti-IgM-IgG 40, serving both as a label and a blocking agent blocking the binding site on each from interaction with Protein A 36. RECTIFIED SHEET(RULE 91) fSA/EP Aε noted above, the pore dimension of test asεay solid phase 11 is most preferably from about 10 to about 20 microns. The relatively large pore size of test assay solid phase 11 and the resultant rapid rate of flow of the excess detection reagent through the solid phase 11 results in a reduction in non-specific binding as extraneous species 34 is swept through solid phase 11 from test asεay εurface 10, and eliminates the need for a wash step between the step of applying sample and the step of applying detection reagent. Additionally, the rapidity of application of sample to asεay εurface 10, followed by rapid introduction of detection reagent, allows insufficient time for significant non-specific binding to be established. After the addition of the detection reagent, the presence of label, specifically indigo detected visually without the aid of a microscope, at both first immobilized assay reagent area 12 and second immobilized asεay reagent area 16 is indicative of a sample containing antibodies to HIV (Fig. 2c). The presence of label at first immobilized asεay reagent area 12 only (i.e. label at area 16) indicates the abεence of antibodieε to HIV (Fig. 2f). An aεpect of the assay is the ability to detect the evenness of the distribution of the .sample across the test assay surface. Asεay reagents are immobilized at areas 12 and 16, and optionally additional areas (not shown), and other areas of surface 10 remain free of reagent immobilization. Surface 10 is selected so that the areas remaining free of immobilized asεay reagent expoεe a chemical functionality that allowε non-εpecific binding of labeled reagent to one degree when εurface 10 is first addressed by a sample to a first extent, and to a different degree when surface 10 is first addressed by a sample to a second extent. In other words, even after sample iε applied, a low level of non-specific binding of labeled reagent to surface 10 occurε. If the sample is applied to surface 10 unevenly, then those regions with less sample applied will bind more RECTIFIED SHEET(RULE 91) label non-εpecifically. Thus, an even tint acrosε the surface 10 indicateε an even diεtribution of εample, whereas uneven tint indicates uneven diεtribution. According to the invention, if no εample iε applied to any particular region, then non-εpecific binding of the label will be very εtrong and the surface 10 will have dark blotcheε on it. Preferably, however, the sample acts to only partially block non-εpecific binding of label to provide not only an abεolute, but alεo a relative meaεure of εample diεtribution. Therefore, the evenneεs of distribution of excesε indigo 42 acroεε teεt aεεay surface 10 is an indication of the evenness of the preceding distribution of the serum sample on surface 10 by delivering member 20, as the serum sample serves to block surface 10 during the asεay. Thus, this may serve an indication of the reliability of the particular test. Referring now to Figs. 3a-c, the appearance of test aεεay εurface 10 after the aεεay illuεtrated in Fig. 2 haε been performed and indicating error (a), a negative teεt (b) , and a poεitive teεt (c), is illustrated. Fig. 3a indicates error as dye has been unevenly non-εpecifically bound to test asεay εurface 10, indicating that serum has been unevenly applied to surface 10. Another indicator of a test error (not illustrated) is a completely dark test asεay surface 10, indicating dye completely coating surface 10. This may be the case if serum does not adequately coat surface 10 prior to the addition of the detection reagent. Referring now to Fig. 3b, the preεence of dye at area 12 and the preεence of lightly shaded dye background acroεs surface 10, and the absence of dye at area 16 signifieε abεence of infection. IgG haε bound to Protein A immobilized at area 12 which haε in turn been coupled to dye-coupled Protein A in the detection reagent. However, no antibody to HIV haε been immobilized at area 16, indicated by the lack of labeled detection reagent immobilized at area 16. Referring to Fig. 3c, infection is indicated as labeled components in the detection reagent are bound at both areas 12 and 16, signifying the presence of antibodies to HIV in the serum sample bound to immobilized HIV at area 16. In all cases label bound at area 12 serves as a control according to the assay illustrated. Referring to now Figs. 4 and 5, an exemplary assay kit arrangement in accordance with the present invention is illustrated generally at 44 and includes base 46 having cavity 48 formed therein. Cavity 48 has a dome 60 protruding upward from its bottom surface. Cavity 48 houses a resilient, absorbent member 50, such as a cotton ball, which is arranged to protrude above the top of base 46 when naturally expanded. Resilient, absorbent member 50 supports test assay solid phase 11 at the top of cavity 48, that is, even with the top of base 46, when a force is directed downwardly against the expansive force of member 50, as described below. Sample delivering member 20 is mounted within and is integral with rectangular tab 52 which extends across a portion of base 46, and across cavity 48 so as to place delivering member 20 directly above test assay solid phase 11. When membranes define both prefilter 28 and diffusing member 26 (not illustrated in Figs. 4 and 5), tab 52 acts as a connector connected to the periphery of prefilter 28 and the periphery of diffusing membrane 26, and holding the receiving surface of prefilter 28 in face to face relation with diffusing membrane 26. Tab 52 and delivering member 20 are held in place by a cover 54 which may be attached to baεe 46 by way of rivetε or εnapε 56, by εonication welding, or the like. Cover 54 includes an opening 58 which exposes most of-delivering member 20, and the periphery of which resiεts an upward force provided by absorbent member 50 onto delivering member 20 via test assay εolid phaεe 11. In thiε way, delivering εurface 22 of delivering member 20 is held in even contact with test assay surface 10 of test assay solid phase 11. If a membrane defines diffusing member 26, diffusing membrane 26 and RECTIFIEDSHEET(RULE91) ISA/EP receiving εurface 24 of prefilter 28 are held in even contact in thiε manner as well. A downward force on the periphery of diffusing material 26 of delivering member 20 is supplied by the periphery of opening 58 of cover 54, and an upward force on test asεay εolid phaεe 11 iε εupplied by reεilient abεorbent member 50, acting aε an urging member. Tab 52 extendε out from under cover 54 εo that delivering member 20 can be quickly and easily removed from under cover 54 and from test asεay εolid phase 11. When a sample is applied to delivering member 20 at receiving surface 24 (prefilter 28 is not shown), it is filtered, and is rapidly and evenly delivered by member 20 to test asεay εurface 10. This may be observed during the assay by the radial wetting of the visible receiving surface 24 (or the top surface of diffusing membrane 26). When εurface 24 is completely wet, a few εecondε are allowed for the fluid sample film to completely permeate prefilter 28 and to wet test assay surface 10. Then, tab 52 may be removed from under cover 54. Absorbent, resilient member 50 then urges test assay solid phase 11 upward against the periphery of opening 58 of cover 54. Cover 54 iε conεtructed εo as to provide a fluid well about opening 58. A detection reagent may then be introduced into the fluid well, which detection reagent rapidly flows through test asεay solid phase 11 and is absorbed by absorbent, reεilient member 50. Teεt assay surface 10 will then give a visual indication as to a positive or negative test result, or an error, as illustrated in Figs. 3(a-c) . The entire process takes lesε than 1 minute, typically about 30 seconds. If tab 52 completely covers the cavity 48 formed in baεe 46, a hole may be formed in the cavity so as to allow air displaced by fluid absorbed by absorbent member 50 to escape. If tab 52 does not completely cover cavity 48, no hole iε needed aε air will eεcape around tab 52. Aε illuεtrated, teεt aεεay εolid phase and delivering member form a slight dome due to the upward force of reεilient, abεorbent member 50. This arrangement is underεtood to be consistent with the description of components of the test asεay kit of the preεent invention, aε deεcribed above, in which it iε deεirable that delivering member, and in particular delivering surface 24 of prefilter 28, and test asεay surface 10, are arranged substantially parallel to each other. Additionally, the above-described radial diffusion of test assay fluid by diffusing material 26 in a manner subεtantially parallel to delivering surface 24 is to be understood to include diffusion acroεε the slightly dome-shaped delivering member 20 as illustrated in Fig. 5. The following examples are intended to illustrate the benefitε of the preεent invention, but do not exemplify the full εcope of the invention. For example, although dyes and pigments are excluεively exemplified as labels, a variety of labels may be employed. Although nitrocellulose as delivering member and test asεay εurface material iε excluεively exemplified, a variety of porouε materialε would be εuitable in fabrication of theεe componentε. Aε a substantially circular test assay surface and delivering member are exemplified, a variety of εhapeε and εizes of these features may exiεt. While immobilization of antigen to detect antibodieε εpecific for the immobilized antigen in a εerum sample, with immobilization of protein to capture IgG as a control iε excluεively exemplified, any of a variety of teεt aεεayε may be carried out. The above-deεcribed and other modificationε and their equivalentε are underεtood to be within the scope of the present invention. EXAMPLE 1 PROCEDURE FOR PRODUCING FROZEN STOCKS OF HIVI In this and the following examples, all chemicals were obtained from Sigma Chemical Corporation (St. Louis, MO) unlesε otherwiεe noted. Preparation of required solutions: An LB solution was prepared by disεolving 5 grams of yeast extract (DIFCO/VWR) , 10 gramε of tryptone (DIFCO/VWR) , and 10 gramε of NaCl in 1 liter of MilliQ water, adjuεting the pH to 7.2, and εterilizing by autoclaving. MilliQ water was obtained by filtration of water through a MilliQ water system (Millipore Corp., Bedford, MA). The solution was allowed to cool and Ampicillin was added to bring the final concentration to 0.1 mg/ml. SOB Broth was prepared by disεolving 5 gramε of yeaεt extract and 20 grams of tryptone in 1 liter of MilliQ Water containing 10ml of a IM NaCl stock, and the pH was adjusted to 7.5. The solution was autoclaved and allowed to cool. The final solution was adjusted to 20mM MgCl 2 /MgSO., by the addition of 10ml of a IM MgCl, stock and 10ml of a IM MgSO. stock. FSB solution was prepared as described in Maniatis (Molecular Cloning), §1.78. The following componentε were added to MilliQ water: 10 ml of IM potaεεium acetate (pH 7.5), 8.91g of MnCl 2 4H 2 0, 1.47g of CaCl^H^, 7.46g of KCl, 0.80g of hexaminecobalt chloride, and 100ml of glycerol (Amreεco) . The εolution was adjusted to 1 liter with MilliQ water and the pH adjusted to 6.4 with 0. IN HC1 (J. T. Baker, Phillipsburg, NJ) . Preparation of E. Coli HIV1 stocks: LB Broth (10ml) was innoculated with 0.2 ml of a glycerol (Amreεco) stock and then grown overnight in an incubator εhaker at 28°C and 300 RPM. The overnight culture waε added to 200ml SOB containing 20mM MgCl 2 /MgS0 4 in a 1 Liter Pyrex Fernbach Flask and the mixture was grown until the optical density was 0.6 to 0.7. The cells were then transferred to sterile, diεpoεable, ice-cold 50 ml polypropylene tubeε (Falcon) and the cultureε cooled to 0°C by εtoring the tubeε on ice for 10 minuteε. The remaining εtepε were carried out aseptically. The cells were recovered by centrifugation at 4000 rpm for 10 minuteε at 4°C, using a TZ-28 rotor (Sorvall) in a RC-5B centrifuge (Sorvall). After the supernatant waε completely removed and diεcarded, the pellet was resuεpended by gentle vortexing, in approximately 80 ml (20ml per 50 ml tube) of ice-cold FSB. The cellε were allowed to εtand on ice for 10 minuteε and were recovered by centrifugation at 4000 rpm for 10 minuteε at 4°C. After the εupernatant waε completely removed and diεcarded, the pellet waε reεuεpended by gentle vortexing in approximately 20 ml (5ml per 50 ml tube) of ice-cold FSB. 0.7 ml (175ul per 50ml tube) of DMSO was gently mixed into the solution and the suspension was returned to an ice bath. An additional 175ul (per 50ml tube) of DMSO was added to the εuεpenεions, mixed gently and then returned to an ice bath. The suεpenεionε were aliquoted (0.2mlε) into chilled, εterile microfuge tubes and then frozen by immersion of the tightly closed tubes in a methanol dry ice bath. The tubes were stored at -80°C until needed. Production of HIV1 from a frozen DMSO stock: At least two dayε after freezing the cellε, the high yield induction of HIV1 from a εtarter culture, which was grown from a frozen DMSO stock, may be determined. In order to perform thiε analysis the stock was removed from an -80°C freezer and thawed on ice. 0.2ml of cells were added to 1ml of SOB medium, and then incubated for 1 hour at 28°C. The εtarter culture waε prepared by adding this 1.2 ml culture to 200 ml LB medium (O.lmg/ml Ampicillin (Amresco)) and incubating at 28°C overnight. The HIVl was then purified as described in Example 2. EXAMPLE 2 PROCEDURE FOR PRODUCING AND PURIFYING HIV 1 Preparation of required εolutions: LB Broth was prepared by dissolving 17 grams of yeast extract (DIFCO/VWR), 34 grams of tryptone (DIFCO/VWR), and 34 grams of NaCl in 3.4 liters of MilliQ water (Millipore Corp.), adjusting the pH to 7.2, and sterilizing by autoclaving. The solution was allowed to cool and Ampicillin (Amreεco) was added to a final concentration of 0.1 mg/ml. One Liter of 50mM Tris-HCl, 2mM EDTA, pH8.0 was prepared as follows: 6 grams of TRIZMA BASE and 744 milligrams of EDTA were dissolved in MilliQ water (Millipore Corp.), the pH was adjusted to 8.0 with HC1 (J. T. Baker), and the final volume of the solution was adjusted to 1 liter with MilliQ water. In order to prepare one liter of Extract Buffer 480.5 grams of UREA (8M) (Amreεco), 4.08 gramε of BICINE (25 mM) , 1.54 grams of DTT (10 mM) , and 1.86 grams of EDTA (5 mM) were disεolved in MilliQ water containing 1 ml of TWEEN 20 (0.1%, polyoxyethylene εorbitan monolaurate, Calbioche , La Jolla, CA) . The pH of the εolution was brought to 8.5, and the volume was adjusted to one liter with MilliQ Water. One liter of 25% Ammonium Sulfate was prepared by disεolving 250 gramε of ammonium εulfate in one liter of MilliQ water. Growth of and induction of 3 liters of E. coli containing HIVl: The culture from Example 1 was innoculated into a 1 Liter Pyrex Fernbach Flask containing 200ml LB Broth, and grown overnight in an incubator εhaker at 25°C and 300 RPM. 10ml of this culture was saved and stored at 4°C for an experimental control (UNINDUCED CONTROL) . The next day 50 ml of the culture was used to innolculate 3 2.8 Liter Pyrex Fernbach Flaskε each containing 1 liter LB Broth. The cultures were grown in the incubator shaker at 30°C and 300 RPM until the OD was 0.6-1.0 at 600nm (2 to 3 hourε) . The cultureε were removed from the 30°C incubator εhaker and a 160-170 ml aliquot waε transferred into a sterile 0.5 Liter Pyrex Fernbach Flask. The temperature of the culture aliquot was quickly raised by transferring the 0.5 liter flaεk into a 60°-70°C water bath. While it waε immersed in the water bath the flask was shaken and the temperature of the culture inside was monitored. As soon as the culture reached 42°C (after 2 to 3 minutes), the contentε of the flask was transferred into a preheated 2.8 Liter Pyrex Fernbach Flaεk (6 empty, sterile 2.8 Liter Pyrex Fernbach Flasks were preheated in a shaker at 42°C and 200 RPM). This procedure waε repeated until each of the 6 flaεkε contained 0.5 literε of induced culture. The flaεkε were covered and the εhaker εpeed waε raiεed to 400 RPM. A 1 ml aliquot waε removed from each of the 6 flaεkε and εaved aε an experimental control for induction (INDUCED CONTROL). The cultureε were incubated at 42°C and 400 RPM overnight. The next day the cellε were harveεted by centrifugation in a rotor (Sorvall) in a Sorvall RC-5B centrifuge at 8000 RPM for 20 minutes. If the cells were not required for immediate purification, they were stored at -80°C. Extraction and purification of incluεion bodieε: The cell pellet waε thawed and resuspended in a total of 300ml of 50mM Tris-HCl pH 8.0, 2mM EDTA (lOOml/lL culture ratio) . In order to enable efficient resuspension, the solution waε εonicated (Branεon εonifier Model 450) and lyεozyme waε added to a final concentration of lOOmg/ml (3ml of a lOmg/ml stock freshly prepared in 50mM Tris-HCl pH 8.0, 2mM EDTA was uεed). 1/100 of a volume of 10% Triton X-100 (3ml) waε added, and the solution was incubated at 30°C for 15 minuteε. The solution was then cenrifuged in a Sorvall RC-5B at 8000 RPM in a GS-3 rotor for 1 hour. If the supernatant waε not clear the centrifugation waε continued for another hour or the solution waε spun at 15000 RPM in a TZ-28 rotor. The supernatant waε collected and εtored at 4°C until an aliquot (100 ulε) had been analyzed by Polyacrylimide Gel Electrophoreεiε (PAGE) (LYSOZYME SUP). The pellet waε resuspended with sonication in a total of 300ml of 50mM Tris-HCl pH 8.0, 2mM EDTA (lOOml/lL culture ratio) and lOOul waε removed (LYSOZYME PREC) for PAGE analysis. Centrifugation was performed as described above (in a Sorvall RC-5B at 8000 RPM in a GS-3 rotor (Sorvall) for 1 hour) and lOOul of the supernatant waε removed for PAGE analyεiε (WASH SUP) . The pellet waε reεuεpended with sonication in a total of 60ml of Extract Buffer (20ml/lL culture ratio) . A 20 ul aliquot was saved for an experimental control (EXTRACT 1). The cell suspension was then centrifuged in a Sorvall RC-5B at 15000 RPM in a TZ-28 rotor for 30 minutes. The supernatant was transferred to a fresh tube for further purification (SOLUTION l). The pellet waε reεuεpended in 30ml of Extract Buffer and centrifuged aε deεcribed in the previouε εtep (15000 RPM in a TZ-28 rotor for 30 minutes). Before centrifugation a 20 ml aliquot was removed for an experimental control (EXTRACT 2). The supernatant was transferred to a fresh tube for further purification (SOLUTION 2). 20 ml aliquots of SOLUTION 1 and SOLUTION 2 were removed for an experimental control. SOLUTION l and 2 were combined and the inclusion bodies were precipitated by adding 5 volumes of 25% Ammonium Sulfate (450ml). The solution was centrifuged in a Sorvall RC-5B at 8000 RPM using a GS-3 rotor (Sorvall) for 1 hour. If the supernatant was not clear the centrifugation was continued for another hour or the solution was centrifuged at 15000 RPM in a TZ-28 rotor. A 20 ul aliquot of the supernatant was removed for uεe aε an experimental control (SOLUTION 3). The pellet waε resolubilized by the addition of 60ml of Extract Buffer with sonication. A 20ul aliquot of the resuεpended pellet waε removed for an experimental control (EXTRACT 3). The solution was precipitated with 300ml (5 volumes) of 25% Ammonium Sulfate and centrifuged as described above (at 8000 RPM in a GS-3 rotor (Sorvall) for 1 hour). A 20 ul aliquot of the supernatant was removed for use as an experimental control (SOLUTION 4). The pellet waε reεuεpended with sonication in 100ml (1/30 of the original culture volume) of Extract Buffer (INCLUSION BODIES). Purification of HIVl by electroelution: 13 ml Glycerol (Amreεco) and 13 ml of 10% SDS (GIBCO BRL, Helgerman, CT) were added to the εuεpenεion of incluεion bodies (100ml) obtained in the previous step and aliquots of 16-17ml were removed for electrophoresis. The remainder of the samples were stored at 4°C (or at -80°C for long-term storage) . Preparative SDS Polyacrylamide Gels (37mm ID) were prepared as described in the BIO-RAD Model 491 Prep Cell Instruction Manual (pages 13-15). 80 ml (10cm) of a 12% Separating Gel solution and 28 ml (3.5cm) of Stacking Gel solution were used to pour the gels. The Model 491 Prep Cell (BIO_RAD) was asεe bled and operated according to the Inεtruction Manual (pageε 15-20) . Prior to loading, 50ul of εample buffer containing Bromophenol Blue and 160ul of b-Mercaptoethanol were added to each 16-17ml aliquot of εample. The εample waε heated to 90°C for 10 minuteε (thiε waε done by tranεferring εmall aliquots of the sample into eppendorf tubes and placing them into a heat block) . A total of 16-17ml of εample waε loaded onto each preparative gel. After loading, the Model 491 Prep Cellε waε operated under a conεtant current of 40mA, for 22h, with an elution buffer flow rate of 30ml/h (0.5ml/min), and fractionε were collected for 20 min. lOul aliquots of sample were collected after the elution of the Bromophenol Blue. These aliquotε were run on a 12% SDS (GIBCO/BRL) polyacrylamide gel in order to determine which of them contained the 27.3 kd HIVl protein. The fractionε containing HIVl were then pooled and further purified by precipitation and reεuspenεion aε described below, The experimental controls were procesεed as deεcribed for each control and 10 ul aliquots of each sample were run on 12% SDS (GIBCO/BRL) polyacrylimide gels under reducing conditions. INDUCED AND UNINDUCED CONTROLS: Each sample was tranεferred to an eppendorf tube and εpun to separate out inεoluble material. The εupernatant waε diεcarded and the pellet was resuεpended in 0.1ml MilliQ water. LYSOZYME SUP, LYSOZYME PREC, SOLUTION 1,2,3 & 4 and EXTRACTS 1, 2 & 3: Theεe controls were diluted with 80ul of water and run in lOul aliquots on PAGE gels. Precipitation and resuεpenεion of HIVl for test asεay surface blotting: The pooled HIVl fractions from electroelution were added to a new centrifuge tube (50 ml, Sarstedt, Newton, NC; 250 ml, Corning, Corning, NY, or equivalent) which is capable of holding three times the starting volume. Two volumes of 100% ethanol were added to the fraction and mixed well. After incubation for at least 30 minuteε at room temperature, the εuεpension waε centrifuged in a Sorvall RC-5, SS34 rotor (Sorvall) at 4000 rpm for 30 minuteε, with no brake. The supernatant was decanted and saved, and the tube was inverted to blot off all fluid. The pellet waε white, waxy and εhiny with a very εmooth texture (i.e. not granular). The pellet was resuspended in the smalleεt amount of 0.1 N NaOH poεsible. To do this, the NaOH must be added in 200 ul increments until there are no viεible εolidε remaining. The εolution waε then centrifuged in a microfuge for 5 minutes to remove any remaining solids. The protein concentration of this solution was determined and then 5 ug/lane of sample were run on a 15% SDS-PAGE reduced and non-reduced. 0.5 ul containing 2 ug of HIVl was then be blotted as deεcribed in Example 4. EXAMPLE 3 PREPARATION OF AMYLOSE COLUMN AND ELUTE BUFFER AND PROCEDURE FOR PRODUCING AND PURIFYING HIV 2 Preparation of required solutions: Amylose column buffer was prepared by diεεolving 1.2 gramε of trizma baεe, 11.7 gramε of NaCl, and 372 milligramε of EDTA in 900 ml of MilliQ water. Once the chemicalε were completely diεεolved 700 ul of b-mercapatoethanol was added and pH was adjusted to 7.4 (within 0.1) with ION HC1. Sterilization was accomplished by filtration of the solution through a 0.45 mm filter unit (Costar, Cambridge, MA). The sterile solution was stored at 4°C. This εolution εhould not be uεed if stored for longer than 30 days. Amylose elute buffer waε prepared by dissolving 1.2g of Trizma base, 11.7g of NaCl, 372 mg of EDTA, and 3.42 g of Maltose in 900 ml of MilliQ water. Once the chemicals were completely dissolved 700 ul of b-mercapatoethanol was added and the pH of the solution was adjuεted to 7.4 (within 0.1) with ION HC1 (J. T. Baker). The solution was sterile filtered through a 0.45 mm filter unit and stored at 4°C. This εolution εhould not be uεed if εtored for longer than 30 dayε. LB Broth was prepared by dissolving 5 gramε of yeast extract (DIFCO/VWR), 10 grams of tryptone (DIFCO/VWR), and 10 grams of NaCl in 1 liter of MilliQ water, adjusting the pH to 7.2, and sterilizing by autoclaving. The solution was allowed to cool and Ampicillin (Amresco) waε added to a final concentration of 0.1 mg/ml. One liter of LB broth was aliquoted into each of 6, 2.8 Liter Pyrex Fernbach Flasks and 400 ml of LB Broth was aliquoted into a 1 Liter Pyrex Fernbach Flask. 100 ml of 100 mM isopropylthio-b-D-galactoεide (IPTG) 500 ml of 20% Ethanol (V/V) 8 Literε of IX Phoεphate Buffered Saline (PBS) pH 7.2 Growth of 6 liters of E. coli/MBP construct: A 1 Liter Pyrex Fernbach Flask containing 400ml LB Broth (100 mg/ml Ampicillin, Amresco) was innoculated with the transfected culture and grown overnight in the incubator shaker at_37°C and 250 RPM. 10ml of this culture was saved at 4°C for an experimental control (UNINDUCED) Each of 6, 2.8 Liter Pyrex Fernbach Flasks containing 1 liter of LB Broth were innoculated with 50 ml of culture grown in the previous εtep. Cultureε were grown in the incubator shaker at 37°C and 250 RPM until the O.D. at 600nm was between 0.6 and 1.0 (2 to 2.5 hourε). 3 ml of culture were removed for SDS-PAGE analyεiε of induction (UNINDUCED 2). The cultures were induced by adding 10ml of 100 mM IPTG to each flask. The flaskε were covered and incubated for 3 hours at 37°C and 250 RPM. The final O.D. was measured and an aliquot of 3 ml of culture was removed for SDS-PAGE analysiε of induction (INDUCED). The cells were harvested by centrifugation in the Sorvall RC-5B at 8000 RPM for 20 minutes. If cells were not required for immediate purification, they were stored at -80°C. Induction waε analyzed by electrophoreεiε of the control εampleε iεolated in the above steps. 1 ml of the UNINDUCED, UNINDUCED 2, and INDUCED sampleε were εpun in a microcentrifuge and the pellets were resuεpended in 100 ul of MilliQ water. 10 ul aliquotε of theεe cell εuspensions are run on a 15% SDS-PAGE gel under reducing conditions. Extraction of inclusion bodies: The cell pellet was thawed and resuspend in Amylose Column Buffer at a ratio of 10 ml per every gram of pellet. The sampleε were εonicated until total εuεpenεion waε achieved. The εuspension waε kept chilled by εtorage in an ice bath during εonication. Once totally suspended, the sampleε were incubated at room temperature on a tumbler or rocking platform for 4 hourε (or overnight). However, one ml of the εuεpenεion waε εaved for SDS-PAGE analyεiε of the extraction (SOLUTION 1). The samples were transferred to high speed centrifuge tubes and centrifuged in a Sorvall RC-5B (TZ-28 rotor) at 18000 RPM for 20 minutes. The supernatant was decanted into a 250 ml conical tube and diluted with an equal volume of Amylose Column Buffer. The mixture was then εterile filtered uεing a 500 ml 0.45 mm filter unit. The filtered solution contains the cell-free extract (CFE) . Aliquotε of SOLUTION 1 and CFE were run on a gel to determine the efficiency of the above procedureε. 0.5ml of SOLUTION 1 was aliquoted into a new eppendorf tube and spun to pellet in the microcentrifuge. The supernatant was discarded and the pellet was resuεpended in 0.5ml of MilliQ water. A 5 ul aliquot of the pellet suεpenεion of SOLUTION 1 along with 5 ul of CFE waε run on a 15% SDS-PAGE gel under reducing conditionε. Purification of MBP construct on amylose column: A 2.5 cm x 30 cm HPLC column (Amicon, Beverly, MA) was assembled with a Peristaltic Pump (Pharmacia LKB Pump P-l, Piscataway, NJ), UV Detector (Pharmacia LKB Uvicord SII), and Chart Recorder (Pharmacia LKB Reel). Once the equipment waε warmed up (30-60 minuteε before uεe), 60-80 ml of Amyloεe reεin waε poured over the column and the column waε packed by paεεing through 4-5 column volumeε of MilliQ water. The reεin waε equilibrated with 2 column volumes of Amylose Column Buffer. The chart recorder was started and once a stable baseline was achieved, the CFE was loaded using Pump Setting 4 (x 10). 100ml aliquots of the flow through (FT) waε collected in 250 ml conical tubes (Corning/VWR, Corning, NY) (Fractions which are collected after the saturation of the column waε reached were run over another column) Once the whole CFE had paεεed the reεin, the column was washed with Amylose Column Buffer until the baseline was reached. Once baseline was achieved, the Amylose Elute Buffer was run through the column. The construct began to elute in the firεt 3-5 fractions causing a sharp peak to appear on the chromatogram. The fractions were collected until baseline was reattained. The purity of the peak fractions was determined by running 2-5 ul aliquots of each fraction, varying the amount depending on the εtrength of the peak (very weak peak requiring more εample), on a 15% SDS-PAGE gel under reducing conditionε. The 2 fractionε flanking each peak as well as the FT fractions were also run. The FT fractions was run in order to determine if the saturation of the reεin was reached. If saturation was reached, the whole flow through or part of it was run over a second column. If the protein was >90% pure aε estimated by SDS-PAGE, the peak fractions were pooled and dialyzed in dialysiε membrane of MWCO 25,000 (Spectrum, Loε Angeleε, CA) , againεt four liter volumeε of IX PBS pH 7.2 at 4°C. At leaεt four hourε waε allowed for each cycle. If significant impurities were preεent, SDS-PAGE and Weεtern Blot Analyεiε were performed on the dialyzed protein. The Amyloεe Reεin waε reuεed up to five timeε before loεing itε binding capacity. Storage of the column waε done in 20% Ethanol (Aaper Alcohol, Shelbyville, KY) by running 2-3 column volumeε through the reεin and storing at room temperature. EXAMPLE 4 PREPARING HIV 1 AND HIV2 IMMOBILIZED ON A TEST ASSAY SURFACE A porous, nitrocellulose membrane (Millipore 704, 10-20 um, SA3M214H2, nitrocellulose), to which HIVl and HIV2 antigenε were to be attached, was cut into 20 mm squares and placed on a piece of blue interleaf on a benchtop. The front of each square at the bottom center was marked to inεure that the blotted side was up and the orientation was clear. The HIVl antigen waε diluted to 8 mg/ml with 0.1 N NaOH and the HIV2 antigen waε diluted with 0.2 um filtered diεtilled water (FDW) to 4 mg/ml. An equal volume of HIV2 εolution waε added to the HIVl εolution and mixed well without foaming (HIV 1/2 combo-antigen) . Protein A (Repligen, RPA-100) waε diεεolved at 1 mg/ml in FDW for a control. Theεe εolutionε were prepared freεh and used the same day. 0.5 ul of the HIV 1/2 protein solution was blotted onto the test asεay surface at the bottom center (near the identification mark) and 0.5 ul of the Protein A was blotted at the top center. The blotε were εpaced in the center of the teεt aεεay εurface, without any overlap, and allowed to dry for at leaεt 30 minuteε at room temperature. The blotted membraneε were stored at 4 °C in a zip-lock bag. EXAMPLE 5 PROCEDURE FOR BLOCKING PREFILTER A porous, nitrocellulose prefilter membrane (Millipore, HATF 08250, 0.45 um, nitrocellulose) was cut into 20 mm squareε, and εubmerged in PBS containing 5% fetal bovine εerum (FBS, JRH 12-10378P) for 20 minuteε. The reεultant individual filterε were then removed from blocking buffer, dipped in PBS to waεh off the exceεε FBS, and transferred to PBS-2% polyethylene glycol (PEG; 3000-3700 MW) . Once the filters were removed from the PBS-PEG they were placed on dry filter paper and allowed to dry at leaεt 30 minuteε at room temperature. The membraneε were stored at 4 °C in a zip-lock bag. EXAMPLE 6 PROCEDURE FOR ASSEMBLING DELIVERING MEMBER A porous diffusing material was formed of a membrane (Millipore 704, 10-20 um, SA3M214H2, nitrocellulose) which was cut into 20 mm εquares. Each square was placed on the εticky εide of the top half of a two-piece adheεive label, or tab, so as to completely cover a 1.5 cm hole in the label. The top half was marked with typing to distinguiεh it from the bottom half of the label. The blocked teεt aεεay εurface membraneε from Example 5 were removed from the plastic bag (Millipore, HATF 08250, 0.45 um, nitrocellulose; in some cases Millipore HAHY0000 was used). Each was tamped onto a 1.5 cm hole on the sticky side of the bottom half of the label, which was not marked with any typing. Both membranes were carefully tamped onto the sticky sideε of the labelε to insure even contact. The labels were carefully stuck together (adhesive sides facing each other) so that the label holeε were aligned and then any excess membrane was trimmed off. The label halveε thus define a connector, holding the diffusing membrane and prefilter membrane in face to face relation. This asεembled delivering member waε εtored at 4°C in a zip-lock bag. EXAMPLE 7 PROCEDURE FOR ASSEMBLING TEST ASSAY KIT A plastic base having a cavity approximately 0.7 cm deep, 2.1 cm wide and 3.5 cm long was obtained from Porex Technologies. At the bottom of the cavity, a dome approximately 0.3 cm high, with a diameter of approximately 1.5 cm was formed in an upward direction. An urging member in the form of an absorbent material, specifically a cotton ball, was pressed into the cavity of the base so that the cotton waε about half an inch above the top of the baεe. On top of the cotton, waε placed one blotted-membrane from Example 4 and on top of that waε placed one prefilter (prefilter from Example 6). A plastic cover, also from Porex Technologies, including an opening approximately 1.0 cm in diameter, and forming a well immediately about the opening, was then placed over the membranes so that they were centered within the opening. The top was presεed down and riveted into place. The membrane appeared with a εlight "dome" which, when preεsed, was very firm, indicating that the device waε packed correctly. That iε, the test assay surface of the test asεay solid phaεe (blotted membrane of Example 4) waε held in even contact with the delivering surface of the delivering member. Similarly, the diffusing membrane and prefilter membrane of the delivering member, held in face to face relation with the label connector of Example 6, were held in even contact according to this arrangement. EXAMPLE 8 PROCEDURE FOR PRODUCING PROTEIN A LABELED WITH INDIGO Indigo (Aldrich, 22,929-6) waε placed into a mortar and ground with a peεtle until the powder waε very fine and uniform (about 15 minutes). 1.5 gm of ground Indigo was suspended in 300 ml of milliQ water in a 500 ml Erlenmeyer flask and the solution was heated at 65°C for 10 minutes. The solution was εonicated for 90 minuteε (Branεon 450, duty cycle 70, output 4) and then allowed to come to room temperature (about 90 minuteε). The pH of the εolution was adjusted to pH 5.5 uεing 0.1 N HCL. 1 mg/ml εolution of Protein A (Repligen, RPA-100) was prepared in D-H 2 0 and 1.75 ml was added to 35 ml of the Indigo suεpenεion in a 50 ml tube (Nalgene polypropylene-capped, 8 tubeε required) . This gave a 50 ug/ml solution. After incubating for 10 minutes at room temperature the solution was centrifuged at 18000 rpm for 50 minutes in an SS34 rotor (Sorvall, RC-5). The supernatant waε removed and the pellet waε resuspended in 30 ml of 10 mM Na 2 HP0 4 , pH 7.4 containing 0.5 M NaCl and 1% BSA (PBSBSA). This solution was centrifuged at 18000 rpm for 25 minuteε in an SS34 rotor (Sorvall, RC-5) and the pellet was resuεpended in 30 ml of PBSBSA containing 10% glycerol (PBSBSAG) . The solution was again centrifuged at 18000 rpm for 25 minutes in an SS34 rotor (Sorvall, RC-5) and the pellet was resuεpended in 30 ml of PBSBSAG. The εolution was filtered through an 8 um filter (Whatman) . This took about 20 minutes and resulted in very little losε of material. Then the εolution waε filtered through a 2 um filter (Whatman) . Thiε took about 1 hour and resulted in approximately 30% losε of material. 4 ul of PAI waε added to 750 ul of D-H 2 0 and the OD 61Q waε adjuεted to 0.0075 with PBSBSAG. 1.5 ml of the diluted PAI waε aliquoted into vialε (Sarstedt, 72730-005) and stored at 4°C. EXAMPLE 9 PROCEDURE FOR PRODUCING ANTI-IqM-IqG AND ANTI-IqA-IgG LABELED WITH INDIGO 2g of Indigo (Aldrich, 22,929-6) was placed into a mortar and ground with a pestle until the powder waε very fine and uniform (about 15 minuteε). 1.5 g of the ground Indigo waε suspended in 300 ml of MilliQ water in a 500 ml Erlenmeyer flask, covered with tin foil. The solution was heated at 65°C for 10 minutes and then allowed to come to room temperature (about 90 minutes). The pH of the solution was adjusted to pH 5.5 using 0.1 N HCL. A 450 ug/ml solution of Antibody was prepared as followε: 12 ml Bethyl Goat anti-Human IgA ttA920-6 (Affinity Purified) © 3.4 mg/ml was added to 16 ml Bethyl Goat anti-Human IgM tt M21-6 (Affinity Purified) @ 2 mg/ml with a total volume of 28 ml and a total of 78 mg. 18 ml of the Indigo suspenεion waε placed into eight 40 ml tubeε (Sorvall, 03530) and 3.5 ml of Ab combo waε added to each tube, reεulting in a 500 ug/ml εolution. The εolution waε then incubated for 30 minuteε at room temperature before centrifugation (Sorvall, RC-5 with SS34 rotor) at 18000 rpm for 50 minuteε. The εupernatant waε removed and the pellet was resuspended in 18 ml of 10 mM Na 2 HPO. (Sigma), pH 7.4 containing 0.5 M NaCl (Sigma) and 1% BSA (Miles, Pentax, Fract V, 81003-40 [PBSBSA]). Thiε εolution was centrifuged at 18000 rpm for 25 minutes in an SS34 rotor (Sorvall, RC-5) with the brake off. The pellet was resuεpended in 18 ml of PBSBSA containing 10% glycerol (Amreεco, 0854) (PBSBSAG). Thiε solution was again centrifuged at 18000 rpm for 25 minutes in an SS34 rotor (Sorvall, RC-5) with the brake off, and the pellet was reεuspended in 18 ml of PBSBSAG. The solution was filtered through an 8 um filter (S&S Immobilization membrane, #AE99), followed by a 5 um filter (MSI, magna nylon transfer membrane), then a 2 um filter (Millipore, Immobilion AV-2, SA3J898E8). The OD of the εolution waε checked by meaεuring at 610 nm. 8 ul of Ab-I waε added to 1.5 ml PBSBSAG and the OD was adjusted to 0.03 with PBSBSAG. For example, if the 8 ul gave an OD of 0.12 the PAI waε diluted 1:4 with PBSBSAG. NaN 3 waε added to a final concentration of ug/ml and the εolution waε εtored at 4°C. EXAMPLE 10 PROCEDURE FOR PRODUCING DETECTION REAGENT 15.6 mlε of IMA (Solution obtained in Example 9) waε mixed with 109.4 mis of IPA (Solution obtained in Example 8). 375 mis of PBSBSAG containing 40 ug/ml of NaN 3 was added to this solution. The total volume was 500 mis and the solution had an OD gl0 =0.015. 500 mis of PBSBSAG containing 40 ug/ml of NaN 3 waε added and the final εolution had an OD 610 =0.0075. EXAMPLE 11 PROCEDURE FOR PRODUCING PROTEIN A, ANTI-IqA, ANTI-IqM-PIGMENT DETECTOR 100 mg of pigment, either Red 17=part tt235-7515; Violet 23=part tt246-1670 or Green 7=part tt264-3120; from Sun Chemical, 4526 Chickering Avenue, Cincinnati, OH 45232, waε meaεured out and placed into a 50 ml conical tube (Sarεtedt). One of the following waε added to the tube containing the pigment: 1) 20 ml of a 1 mg/ml-diεtilled water εolution of Protein A (RPA-100, Repligen), 2) 6 ml of Goat anti-Human IgA (A80-102A, Lot A92G, 3.4 mg/ml, Bethyl Labs, Montgomery, TX) , or 3) 8 ml of Goat anti-Human IgM (A80-100A, Lot M121G, 2.0 mg/ml, Bethyl Labs). The material in each vial was suspended by inversion but it was done with care εo as not to create too much foam (about 30 minutes). The suspension which was clumpy and not well εuspended contained fluid which was clear and did not take on the color of the pigment. Each tube was brought to a final volume of 45 ml by adding PBSBSAG (10 mM Na2HP04, 0.5 M NaC) , 1% BSA:#81003-40, Miles, Pentax, 10% Glycerol:tt0854-l, Amresco). The material in each vial was suspended by inversion and by crushing the clumpε with a εtainleεε-εteel εpatula, while taking care not to create too much foam (approx 15 minuteε). The suspension which started to take on the color of the pigment but which was εtill clumpy and not well suspended, was incubated at room temperature for 45 minuteε, and then centrifuged at 18000 rpm for 25 minuteε, with the brake off (Sorvall, RC-5 centrifuge, SS34 rotor uεing 50 ml tubeε, Sorvall, tt03530). The εupernatant waε removed and the waε pellet resuεpend in 100 ml PBSBSAG. The εuspension was first filtered through an 8 um filter (S&S, AE99) and then through a 2 um filter (Milliport. Immobilion AV-2, SA31898E8) . NaN3 waε then added to the εolution to a final concentration of 20 ug/ml. EXAMPLE 12 TEST ASSAY 120 ul of a εerum εample waε added directly onto the center of the delivering member of the teεt aεεay kit of the preεent invention. After from 10 to 15 εecondε, the delivering member waε removed and diεcarded. 1.5 ml (0.015 OD unitε) of the detection reagent of Example 10 was added into the well and allowed to flow through the membrane (about 10 seconds). The resultε were recorded. EXAMPLE 13 COMPARATIVE POSITIVE, NEGATIVE, AND ERROR RESULTS USING AN ASSAY KIT 120 ul of each of a poεitive and negative HIV serum sample was added to the center of a delivering member of a test asεay kit prepared in accordance with the preεent invention. After from 10 to 15 seconds, in each case, the prefilter was removed and discarded. In each case, 1.5 ml of the detection reagent prepared in accordance with Example 10 was added into the assay kit well and allowed to flow through the test assay membrane, which was completed in about 10 seconds. Fig. 2c illustrateε the appearance of the teεt aεεay surface to which a positive serum sample had been applied, following the application of the detection reagent. Fig. 2b illustrates the appearance of the test assay surface to which a negative serum sample had been applied, after application of the detection reagent. As a comparative (error) example, the prefilter was removed from a test assay kit prepared in accordance with Example 10 and the test aεεay εurface waε wetted unevenly with a negative εerum εample. That iε, a portion of the test asεay εurface was contacted with serum to a greater extent than another portion. Subsequently, 1.5 ml of the detection reagent prepared in accordance with Example 10 was added allowed to flow through the teεt aεεay membrane. The reεultε are illuεtrated in Fig. 2a. The unevenness of the diεtribution of the label on the test asεay εurface can be measured with the naked eye, indicating the unevennesε of the εerum application to the teεt aεεay εurface. The preceding examples are set forth to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention. Additional embodiments and advantages within the scope of the claimed invention will be apparent to those of ordinary skill in the art. What is claimed is:

Claims: 1. A delivering member for delivering a sample to a test asεay εurface, compriεing: a prefilter having a εample receiving εurface and a surface for delivering the sample to the test asεay εurface; and a diffuεing material on the prefilter εample receiving surface. 2. The delivering member as recited in claim 1, wherein the diffusing material compriseε a εurfactant. 3. The delivering member aε recited in claim 1, wherein the diffuεing material compriεeε a porouε, inert, hydrophobic matrix. 4. The delivering member as recited in claim 3, wherein the diffusing material is chemically bound to the prefilter. 5. The delivering member aε recited in claim 3, wherein the diffuεing material iε phyεically bound to the prefilter. 6. The delivering member aε recited in claim 3, wherein the diffusing material comprises a diffusing membrane having a porosity and thicknesε εelected to rapidly laterally diffuεe a fluid εample over the prefilter receiving εurface, and the prefilter compriεes a subεtantially planar membrane having a porosity and thicknesε selected to rapidly transfer the sample from the receiving surface to the delivering surface while filtering the sample of particulate material larger than a predetermined dimension. 7. The delivering member as recited in claim 3, wherein the diffusing material compriseε a diffuεing membrane having a periphery, and the delivering member further comprises a connector connected to the prefilter periphery and the diffusing membrane periphery holding the diffusing membrane in face to face relation with the prefilter εample receiving surface. 8. The delivering member as recited in claim 7, wherein the diffusing membrane has pores larger than those of the prefilter. 9. The delivering member as recited in claim 3, wherein the prefilter comprises a subεtantially planar membrane having a pore εize of from about 0.1 micronε to about 1.0 micronε. 10. The delivering member aε recited in claim 3, wherein the prefilter haε a thickneεε of from about 50 micronε to about 300 micronε. 11. The delivering member aε recited in claim 1, wherein the prefilter comprises a subεtantially planar celluloεic membrane. 12. The delivering member as recited in claim 1, wherein the sample delivering surface is conformable to a test asεay εurface. 13. A teεt aεεay kit for the determination of an analyte compriεing a package containing: a teεt aεεay εurface for supporting a test assay reaction; and a porouε celluloεic prefilter having a pore εize of from about 0.1 to about 1.0 micronε, and having a delivering εurface poεitionable in face to face relation with the teεt asεay εurface. 14. A teεt assay εolid phaεe having a test asεay εurface including an area having a chemical functionality allowing non-specific binding of labeled reagent to a first degree when a sample iε firεt applied to the area to a first extent, and non-specific binding of labeled reagent to a second degree, distinguiεhable from the firεt degree, when a εample iε firεt applied to the area to a εecond extent different from the firεt extent. 15. The test assay solid phase as recited in claim 14, the teεt aεεay εurface including a εecond area carrying an immobilized binding partner of an analyte. 16. A teεt aεsay method for the determination of an analyte comprising: applying to a test asεay surface, having regions capable of non-specific binding, a sample including blocking species in an amount to only partially block the regions; contacting the regions with a detection reagent including a specieε carrying a label; and determining of the evenness of the application of the sample to the test asεay surface by measuring a uniform or non-uniform distribution of the label over the regions. 17. The test aεεay method aε recited in claim 16, the regionε capable of non-εpecific binding being free of any immobilized biological reagentε. 18. A method of delivering a fluid εample to a teεt aεεay εurface compriεing: holding a delivering εurface of a prefilter in even contact with a teεt assay surface; forming a film of the fluid sample on a receiving surface of the prefilter; allowing the film to permeate the prefilter and to evenly wet the test assay surface. 19. A delivering member for delivering a sample to a test asεay εurface comprising: a prefilter having a sample receiving surface and a surface for delivering a sample to a test aεεay εurface, the prefilter being conεtructed and arranged to rapidly laterally diffuεe a fluid εample over the receiving εurface and to then allow the sample to permeate the prefilter and to be delivered by the delivering surface to the test asεay surface. 20. In a test assay kit employing a prefilter for delivering a sample to a test assay surface, the improvement comprising: a prefilter having a pore size of from about 0.1 to about 1.0 microns. 21. In a test assay kit as recited in claim 20, the improvement comprising a prefilter having a pore size of from about 0.2 to about 0.7 microns. 22. In a test assay kit aε recited in claim 21, the improvement compriεing a prefilter having a pore size of from about 0.33 to about 0.53 microns. 23. In a teεt aεεay kit aε recited in claim 22, the improvement comprising a prefilter having a pore size of about 0.45 microns. 24. In a test asεay kit employing a prefilter for delivering a εample to a teεt aεεay surface, the improvement comprising: a prefilter comprising a pair of membranes in face to face relation. 25. In a test assay kit employing a prefilter for delivering a sample to a test aεsay εurface, the improvement compriεing: a prefilter having a εample receiving surface, and constructed and arranged to rapidly laterally diffuse a fluid εample over the receiving surface and to then allow the sample to permeate the prefilter and to be delivered to the test asεay εurface.

Inventors: DENHAM LILIBETH K, DORVAL BRENT L, KLIBANOV ALEXANDER M, DENHAM, LILIBETH, K., DORVAL, BRENT, L., KLIBANOV, ALEXANDER, M.
Applicants: INTRACEL CORP, INTRACEL CORPORATION
Application year: 1995
Main classification: G01N33
IPC codes: G01N33