Method and cell for detection.

The present invention relates to assays for ligands, e.g. antigens, in a liquid sample such as, for example, a body fluid. More particularly, the present invention relates to a method and apparatus for the detection of a ligand in a body fluid such as, for example, urine using a conjugate comprising colored particles and a novel flow-through test cell.

Many types of ligand-receptor assays have been used to detect the presence of various substances, often generally called ligands, in body fluids such as, for example urine. These assays involve antigen antibody reactions, synthetic conjugates comprising radioactive, enzymatic, fluorescent, or visually observable metal sol tags, and specially designed reactor chambers. In all these assays, there is a receptor, e.g. an antibody, which is specific for the selected ligand or antigen, and a means for detecting the presence, and often the amount, of the ligand-receptor reaction product. Most current tests are designed to make a quantitative determination, but in many circumstances all that is required is a positive/negative indication. Examples of such qualitative assays include blood typing and most types or urinalysis. For these tests, visually observable indicia such as the presence of agglutination or a color change are preferred.

Even the positive/negative assays must be very sensitive because of the often small concentration of the ligand of interest in the test fluid. False positives can also be troublesome, particularly with agglutination and other rapid detection methods such as, for example, dipstick and color change tests. Because of these problems, sandwich assays and other sensitive detection methods which use metal sols or other types of colored particles have been developed. These techniques have not solved all of the problems encountered in these rapid detection methods.

In EP-A-0258963 there is disclosed an immunoassay method for providing sensitive and rapid detection of ligands in body fluids utilizing colloidal gold labelled reagents and a matrix membrane having a immunologically active component therein. All liquid materials brought into contact with one side of the membrane flow through the membrane into an absorbent material in physical and fluid communication with the other side of the membrane. Reactions may be visually detected and in the case of hCG. a sensitivity of 50 mlU or less may be had within a total assay and manipulation time of one and a-half minutes.

In US-A-4366241 there is disclosed a method and apparatus for performing immunoassays employing a device comprising a relatively small test zone referred to as-an immunosorbing zone, and a relatively large liquid absorbing zone in liquid receiving relationship with said immunosorbing zone. The immunosorbing zone includes a member of an immunological pair ("MIP")-ligand and antiligand - bound to a support. A signal producing system is employed in conjunction with said device having as one component a signal label bound to a mip. The signal producing system provides for production of a detectible signal in the immunosorbing zone in relation to the amount of analyte in a sample.

It has now been found possible to provide a rapid, sensitive method for detecting ligands in body fluids. It has also been found possible to provide an assay which has high sensitivity and fewer false positives than conventional assays. It has further been found possible to provide a test cell for detection of low levels of ligands in body fluids. It has still further been found possible to provide an assay system which involves a minimal number of procedural steps, and yields reliable results even when used by untrained persons.

The invention features a method and test cell for detection of a preselected ligand in a liquid sample such as, for example, a body fluid.

According to the present invention there is provided a method of the detection of a ligand in a liquid sample, which comprises: <ul id="ul0001" list-style="none"><li>(A) transporting via permeable material along a flow path in a test cell a solution, including a liquid sample suspected to contain a ligand and including a conjugate, through a filtration means for filtering a liquid sample and then into contact with a test site visible through a window in a wall of the test cell, the test site having immobilized thereon a first protein having a binding site specific to a first epitope on the ligand, and the conjugate comprising colored particles coupled to a second protein selected from proteins having a binding site specific to a second epitope on the ligand and proteins which bind with the first protein in competition with the ligand; and</li><li>(B) continuing transport of the solution to progressively produce at the test site a complex comprising the ligand and conjugate for a time sufficient to visually determine through the window whether a color is developed at the test site.</li></ul>

The present invention also provides a test cell for detecting a ligand in a liquid sample, which comprises an elongate casing for housing a permeable material and defining a liquid sample inlet, a reservoir volume, a test volume interposed between the inlet and reservoir volume, and a window through the casing at the test volume, and a filtration means disposed between the inlet and the test volume, the permeable material being capable of transporting an aqueous solution disposed within the casing and defining a flow path extending from the sample inlet through the test volume and into communication with the reservoir volume, there being present a first protein having a binding site specific to a first epitope on the ligand, the first protein being immobilized at a test site visible through the window, and being disposed within the test volume in fluid communication with the flow path. Preferably there is sorbent material in the reservoir volume for drawing liquid sample along the flow path and into contact with the test site.

The test cell useful in the practice of the invention has an elongate outer casing which houses an interior permeable material, e.g. glass fiber, capable of transporting an aqueous solution, for example, by capillary action, wicking, or simple wetting. The casing defines a sample inlet, and interior regions which, for ease of description, can be designated as a test volume and a reservoir volume. The reservoir volume is disposed in a section of the test cell spaced apart from the inlet, and preferably is filled with sorbent material. The reservoir acts to receive liquid transported along a flow path defined by the permeable material and extending from the inlet and through the test volume. In the test volume is a test site comprising a first protein having a binding site specific to a first epitope of the ligand immobilized in fluid communication with the flow path, e.g. bound to the permeable material or to latex particles entrapped in or bonded to the permeable material. A window such as, for example, a hole or transparent section of the casing permits observations of the test site through the casing wall.

In a preferred embodiment, the flow path is restricted or narrowed in the test area, thereby channeling and concentrating fluid flow into contact with the test site. The test cell includes a solution filtering means disposed in the flow path between the sample inlet and the test site. The filtration means can comprise a separate, conventional filter element disposed within the casing of the test cell in fluid communication with the permeable material defining the flow path, but preferably is defined simply by a portion of the permeable material itself. The provision of such a filtration means in the test cell has the effect of removing by entrapment from impure samples, such as, for example, urine samples, a portion of the particulates and non-specific interfering factors which sometimes cause false positive readings.

The method of the invention requires the use of a conjugate comprising a second protein bound to colored particles such as, for example, a metal sol or colloid, preferably gold. The conjugate can take two distinct forms, depending on whether the assay is designed to exploit the "sandwich" or "competitive" technique.

In the case of the sandwich technique, the second protein comprises a site which binds to a second epitope on the ligand. This type of conjugate reacts with the ligand to form a complex in the liquid sample. The complex is detected by visual observation of color development at the test site in the test cell. At the test site, the ligand bound with the conjugate reacts with the immobilized first binding protein to form a "sandwich" of the first protein, ligand, second protein, and colored particles. This sandwich complex is progressively produced at the test site as sample continuously passes by, filling the reservoir. As more and more conjugate is immobilized, the colored particles aggregate at the test site and become visible through the window, indicating the presence of ligand in the liquid sample.

In the case of the competitive technique, the second protein binds with the first protein in competition with the ligand. The second protein comprises, for example, an authentic sample of the ligand or a fraction thereof which has comparable affinity for the first protein. As the liquid sample is transported in contact with the test site, ligand, if any, and the conjugate compete for sites of attachment to the first protein. If no ligand is present, colored particles aggregate at the test site, and the presence of color indicates the absence of detectable levels of ligand in the sample. If ligand is present, the amount of conjugate which binds at the test site is reduced, and no color, or a paler color, develops.

In one embodiment of the invention, the test liquid is mixed with the conjugate outside the test cell. In another embodiment, the conjugate is disposed in freeze-dried or other preserved form on the permeable material between the inlet and the test site, and the sample liquid resolubilizes the conjugate as it passes along the flow path.

Color development at the test site may be compared with the color of one or more standards or internal controls to determine whether the development of color is a true indication of the presence or absence of the ligand, or an artifact caused by nonspecific sorption.

In one embodiment employing the sandwich technique, the standard consists of a negative control site, preferably disposed adjacent the test site, and visible through a second window proximate the first. The negative control site preferably is prepared identically to the test site, except immobilization of the first binding protein is omitted. Therefore, although the conjugate will reach the control site, it aggregates due only to non-specific binding. If the test site is not appreciably more intense in color than the control site, the assay is considered negative.

In another embodiment, the assay and test cell may include a positive control. Thus, when exploiting the sandwich technique, the cell may have an authentic sample of the ligand immobilized at a control site. If no color develops at this control site, the assay is considered inconclusive. When exploiting the competitive technique, the development of color at the positive control site means the assay results are inconclusive.

Broadly, the method of the invention involves the use of a test cell of the type described above to achieve an easily readable, sensitive, reproducible indication of the presence of a ligand, e.g., human chorionic gonadotropin (hCG), in a test sample such as, for example, a human urine sample. The method involves the step of transporting the sample and a conjugate comprising a protein bound to a metal sol or other colored particle along a flow path and in contact with a test site comprising immobilized binding protein specific to an epitope of the ligand, and preferably also in contact with a control site. Preferably, the colored particle comprises a gold sol; the flow path in the region of the test site is reduced in cross-section relative to other parts of the flow path; the sample is passed through a filtration means after it enters the test cell but before it contacts the test site; and the test site comprises latex particles entrapped or otherwise fixed in the flow path having the immobilized protein on their surface. In the practice of the process, either the conjugate is premixed with the sample, or the conjugate is disposed in preserved form, e.g., lyophilized, in the flow path between the inlet and the test site. In either case, placement of the test cell in the sample, or application of the sample to the inlet, initiates flow, and the result is read by observing color development at the test site, or by comparing the color of the test site and control site.

The use of the colored particle detection system in combination with the filtration means, the concentrating effect of flow of the sample, and the ease of comparison between the colors of the test and control sites, together enable construction of a family of extremely sensitive assay systems which minimize false positives and can be used effectively by untrained persons.

The present invention provides a test cell for conducting a sandwich or competitive immunoassay, and a process which utilizes the test cell and a conjugate comprising colored particles. As disclosed below, various features of the process and test cell of the invention cooperate to enable untrained personnel reliably to assay a liquid sample for the presence of extremely small quantities of a particular ligand while avoiding false positives and simplifying test procedures. The invention is ideal for use in over-the-counter assay test kits which will enable a consumer to self diagnose, for example, pregnancy, venereal disease, and other disease, infection, or clinical abnormality which results in the presence of an antigenic marker substance in a body fluid, including determination of the presence of metabolites of drugs or toxins. The assay process and the cell are engineered specifically to detect the presence of a preselected individual ligand present in a body or other fluids.

Broadly, the test cell and process of the invention can be used to detect any ligand which has heretofore been assayed using known immunoassay procedures, or known to be detectable by such procedures, using polyclonal or monoclonal antibodies or other proteins comprising binding sites for ligands. Various specific assay protocols, reagents, and analytes useful in the practice of the invention are known per se, see, e.g., US-A- 4 313 734 (columns 4-18), and US-A- 4 366 241 (columns 5-40).

The combination of features believed to be responsible for the excellent sensitivity and reproducibility of assays constructed in accordance with the invention is the use of the novel test cell which serves to concentrate ligand from a test sample at a test site in the cell, and the use of a metal sol or other colored particle as a marker system which permits direct visual observation of color development. False positives are reduced while maintaining excellent sensitivity by including in the test cell a negative control or control site whose color is compared with the test site, and by including a filtration means which limits the introduction to the test site of contaminants from the sample.

The assay is conducted by simply placing the inlet of the test cell in contact with a liquid test sample. One then merely waits for the test sample to pass through the cell and into reactive contact with the test site (and optionally one or more control sites) visible through a window or windows in the cell's exterior casing. In one embodiment, the conjugate is mixed with the sample and incubated briefly before the test cell is inserted. In another embodiment, the conjugate is disposed in preserved form in the flow path within the cell. If the ligand is present in the sample, it passes through the inlet and the interior of the cell along the flow path past the test and control sites, where, in the sandwich embodiment, it reacts with immobilized binding protein, e.g., antibody, at the test site, and perhaps also non-specifically at the control site. A "sandwich" forms at the test site comprising immobilized binding protein-ligand binding protein-colored particle. The presence of the sandwich complex and thus the ligand is indicated by the development of color caused by aggregation of the metal sol particles at the test site. A deeper color at the test site than at the negative control site is a positive indication of the presence of the ligand.

By providing a reservoir of sorbent material disposed beyond the test and control sites, a relatively large volume of the test liquid and any ligand it contains can be drawn through the test area to aid sensitivity. Optionally, the region of the flow path in the test cell defining the test and control sites is restricted in cross-sectional area relative to other regions of the flow path. This feature produces a "bottle-neck" effect wherein all ligand in the entire volume of sorbed sample must pass through the restricted flow area immediately about the test site where it will be immobilized by reaction with binding protein.

From the foregoing, it will be apparent that the success of the test procedure is dependent on ligand present in the sample reacting with the conjugate, or on reproducible competition between the ligand and the conjugate for sites of attachment at the test site. In accordance with the invention, as noted above, the assays can be conducted by premixing the conjugate with the liquid sample prior to introduction into the elongate test cell. Alternatively, the conjugate may be disposed in preserved form, e.g., freeze-dried, in the flow path within the test cell upstream of the test and control sites. In this case, the cell is placed directly in the liquid sample solution without premixing. Ligand, if any, passing up through the cell and entrained within the liquid moves into contact with the conjugate forming an immune complex or initiating competition <u style="single">in situ</u> as flow continues. This latter technique has the advantage that it eliminates a manipulative step in the assay procedure, and accordingly a possible source of error.

The present invention will now be more particularly described with reference to and as illustrated in, but in no manner limited to, the accompanying drawings, in which:- <ul id="ul0002" list-style="none"><li>Figure 1 is a cut-away, schematic, top view of an embodiment of a test cell useful in explaining the test cell and process of the present invention;</li><li>Figure 2 is a cross-sectional side view of the test cell of Figure 1;</li><li>Figure 3 is a perspective view of a currently preferred test cell constructed in accordance with the present invention;</li><li>Figure 4A is a cross-sectional, top view of the test cell of Figure 3;</li><li>Figure 4B is a cross-sectional, side view of the test cell of Figure 3 taken at line 4B-4B of Figure 4A;</li><li>Figure 5 is a cross-sectional view of the cell of Figure 3 taken at line 5-5 of Figure 4B; and</li><li>Figure 6 is a perspective view of another embodiment of a test cell constructed in accordance with the invention.</li></ul>

In the drawings, like reference characters in the respective drawn Figures indicate corresponding parts.

Referring to the drawings, Figures 1 and 2 illustrate schematically an embodiment of a test cell 5 constructed in accordance with the invention useful in explaining its principles of construction. It comprises an outer, moulded casing 10 which defines a hollow, elongate enclosure filled with a permeable, sorbent material 12. Casing 10 also defines a test liquid inlet 14 and a pair of circular openings 16, 18 comprising windows through which sorbent material 12 is visible.

Sorbent material 12 and the interior of casing 10 together define a flow path passing generally from left to right in Figures 1 and 2. When the test cell is placed with inlet 14 disposed within or otherwise in contact with a liquid sample, the liquid is transported by capillary action, wicking, or simple wetting along the flow path through upstream flow section 20, test volume 22, and into reservoir volume 24, generally as depicted by the arrows. The flow section 20 of the flow path disposed inwardly of the inlet 14 serves as a filter which can remove from impure test samples particulate matter and interfering factors. The provision of such a filtration means 20 downstream of the inlet 14 is believed to contribute to the success of the system and its ability to avoid false positives.

Disposed within sorbent material 12 is a band 26 of dehydrated conjugate, e.g., antibody-metal sol. As the liquid sample moves past band 26, the conjugate is entrained in the liquid, reconstituted, and reacts or competes with ligand, if present, dissolved in the liquid sample. Of course, conjugate band 26 may be eliminated, and the conjugate added to the test liquid prior to introduction of the cell 5 as previously noted.

Within the volume of sorbent material 12 disposed directly beneath circular openings 16 and 18 in casing 10 is disposed, respectively, control site 16′ and test site 18′. In the drawing, the control and test site are illustrated as being disposed serially along the flow path. Alternatively, the control and test site or sites may be disposed side by side or in other spacial relationships.

Test site 18′ comprises a preselected quantity of antibody against an epitope of the ligand to be detected immobilized in place within the flow path. Its detailed chemical structure can vary widely. Control site 16′ is preferably identical in size and chemical makeup to test site 18′, excepting that the immobilized antibody present at the test site 18′ is omitted at the control site 16′. Thus, any nonspecific aggregation of, e.g., ligand-conjugate or free conjugate, which occurs at test site 18′ also will occur at control site 16′. A deeper color at test site 18′ as compared with control site 16′ is a positive indication of ligand in the sample in the sandwich assay.

The invention is not limited by the precise nature of the test site 18′ and corresponding control site 16′, and in fact, control site 16′ may be entirely eliminated if a reduction in sensitivity can be tolerated. Generally, antibody or other binding protein may be immobilized at test site 18′ using adsorption, absorption, or ionic or covalent coupling, in accordance with methods known <u style="single">per</u><u style="single">se</u>. A currently preferred formulation for test site 18′ is to immobilize monoclonal antibody against an epitope of the ligand on latex beads, and then to entrap or otherwise link the beads in sorbent material 12 at region 18′. Control site 16′ is fabricated identically, except that the latex beads contain non specific immunoglobulin, e.g., immunoglobulin from bleedings from an animal that has not been immunized.

Disposed beyond test volume 22 is a reservoir volume 24 comprising a relatively large mass of sorbent or supersorbent material. The purpose of reservoir volume 24 is to ensure that a reasonably large amount of test liquid is drawn through test volume 22. Increasing the volume of reservoir 24 can have the effect of increasing the sensitivity of the assay procedure, as it results in an increase in the amount of ligand passing through the test area 22. Suitable sorbents include commercial materials of the type available, for example, from The Dow Chemical Company of Midland, Michigan, and the Chemical division of American Colloid, Arlington Heights, I11. These materials can absorb many times their weight in water and are commonly used in disposable diapers. They comprise lightly crosslinked polyacrylate salts, typically alkali metal salts.

Polyclonal antisera and indeed monoclonal antibodies or fractions thereof having specific binding properties and high affinity for virtually any antigenic substance are known and commercially available or can be produced from stable cell lines using well known cell fusion and screening techniques. The literature is replete with protein immobilization protocols. See, for example, <u style="single">Laboratory Techniques in Biochemistry and Molecular Biology</u>, Tijssen, Vol. 15, Practice and Theory of Enzyme immunoassays, chapter 13, The Immobilization of Immunoreactants on Solid Phases, pp. 297-328, and the references cited therein.

Metal sols and other types of colored particles useful as marker substances in immunoassay procedures are also known <u style="single">per</u><u style="single">se</u>. See, for example, US-A- 4 313 734. For details and engineering principles involved in the synthesis of colored particle conjugates see Horisberger, Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and Scanning Electron Microscopy, Biol. Cellulaire, <u style="single">36</u>, 253-258 (1979); Leuvering et al, Sol Particle Immunoassay, J. Immunoassay 1 (1), 77-91 (1980), and Frens, Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions, Nature, Physical Science, <u style="single">241</u>, pp. 20-21 (1973).

The cell can take various forms. It will usually comprise an elongate casing comprising interfitting parts made of polyvinyl chloride, polypropylene, or other thermoplastic resin. Its interior flow path will contain a relatively inert material or a combination of materials suitable for transporting the liquid. In some circumstances it may be preferable to use a material of higher sorptivity as the reservoir, promoting the flow of liquid, and a different material for remaining portions of the flow path.

From the foregoing it should be apparent that the advantages in reproducibility, sensitivity, and avoidance of false positives of assay systems constructed in accordance with the invention are traceable to a combination of features of the invention. In use, the test cell of the invention and the metal sol particles used as a marker together cooperate to result in an increase in color intensity progressively as ligand complexed with conjugate is trapped at the test site by the immobilized binding protein. This approach can be utilized to design assays and test cells for essentially any antigenic material.

The present invention will now be further particularly described with reference to the following Examples. In the Examples, the test devices are described with reference to Figures 3,4A,4B,5 and 6 of the accompanying drawings which have been briefly referenced to hereinabove.

Example 1

The currently preferred test device embodying the invention is shown in Figures 3, 4A, 4B, and 5. A modification of the device depicted in Figure 3 is shown in Figure 6, and includes a second control site 19 in addition to control site 16′ and test site 18′, as well as a stand 21 useful for maintaining the test cell in an incline position.

As shown in Figures 3, 4A, 4B, and 5, the preferred test cell of the invention differs from the exemplary device discussed above and shown in Figures 1 and 2 in certain of its more specific internal features. Specifically, the casing comprises a pair of interfitting polymeric parts including a U-shaped top part 10 which, when the device is assembled, interfits with lower part 10′. Top and bottom parts 10 and 10′ may be connected through a hinge region 11. The bottom section 10′ defines a pair of channels 28 above which is disposed a strip of glass fiber paper 13 (available commercially from Eaton Dikeman, Grade 111, or Whatman, Grade GFA). Test liquid applied through inlet 14 soaks along the paper strip 13 which defines the flow path and a filtering means region 20, as well as a positive control site 16′ and test site 18′ visible through windows 16 and 18 consisting of openings through upper mating member 10. The paper strip 13 overlaps into reservoir volume 24, which is defined by a cavity between the interfitting top and bottom mating members 10 and 10′. The cavity in this case is filled with sorbent blotting paper 12 comprising the sorbent reservoir. A suitable paper is sold as Grade 12A absorbent paper, a cellulose product available from Schleicher and Schuell. In one preferred embodiment, the dimensions of the glass fiber paper 13 were approximately 0.64 by 7.62 cm (one quarter inch by three inches), and those of the absorbent material 12 approximately 5.08 by 0.40 cm (two inches by five thirty seconds of an inch) on each side. A number of these devices were produced and further treated to adapt them to detect pregnancy by assay of urine.

Test site 18′ in each device was fabricated as a spot within fiber paper 13 using the following technique. Latex beads available commercially and comprising polystyrene particles 0.0003mm (0.3 micron) in diameter were passively coated with purified rabbit anti-human chorionic gonadotropin. The polyclonal antibody was purified using conventional techniques from bleedings of a rabbit previously immunized with human chorionic gonadotropin in a manner know <u style="single">per</u><u style="single">se</u>. Equal parts of a latex (0.6% by weight) having a continuous phase of glycine buffer, pH = 8.3, and a 1 mg/ml antibody solution in the same buffer were mixed and incubated at 37°C. 15 microliters of this solution, comprising approximately 0.6% solids, were added, one drop at a time, to the glass fiber paper 13 to produce spot 18′ after the devices had been assembled. The spots were then allowed to dry at 37°C. The control site 16′ was produced identically to the test site disclosed immediately above, excepting that rabbit polyclonal non-immune gamma globulin was used in place of the anti-hCG gamma globulin.

Metal sol particles were prepared in accordance with the method of Frens, Controlled Nucleation for the Regulation of the Particle Size in Mono Dispersed Gold Solutions (1973), <u style="single">supra</u>. Briefly, the gold sol was prepared by reducing a 4% solution of gold chloride with 1% sodium citrate to produce gold particles of approximately 18nm in diameter. The particles were made immunochemically reactive by admixture with a monoclonal antibody specific for human chorionic gonadotropin with no detectable cross-reactivity with human leutinizing hormone. The antibody was purchased from Charles River Labs, and is produced using standard techniques including purification from ascites using HPLC ion exchange chromatography. It was added to the gold sol as a 10 »g/ml solution in borate buffer, pH-6. The bound antibody fraction is separated from the free fraction by either density centrifugation or gel filtration chromatography. Additional details of the currently preferred procedure for making the antibody sol conjugate are disclosed by Leuvering et al, J. Immunoassay (1980) <u style="single">supra</u>. Individual batches of the latex and the conjugate are titrated to optimize activity so that a suitable amount of latex is applied to the test site and a suitable amount of conjugate is used in conducting the test.

Test Protocol

To a 10 X 50 mm test tube of lyophilized gold sol antibody conjugate is added 0.5 ml urine sample containing a known quantities of hCG. The samples comprised hCG standards purchased from Sigma Chemical Company diluted in processed, hCG negative urine. The contents of the tube are mixed by shaking in a horizontal motion until the lyophilized antibody is dissolved. The device depicted in Figures 3 to 5 is then inserted into the tube, and the results are read after the entire fluid volume has been absorbed.

The results of this qualitative procedure are as follows: <tables id="tabl0001" num="0001"><img file="EP0349215B1_D0001.tif" /></tables>

The pink color clearly visible at 50 mIU of human chorionic gonadotropin means that the test can detect pregnancy one day after a missed menstrual period. In initial stages of testing, approximately 50 negative samples from various sources have been run with no false positives or even border-line cases. It is anticipated that the commercial device will have less than 1% false positives.

Non-limiting examples of materials which may be assayed in accordance with the invention in addition to the human chorionic gonadotropin noted above include human leutinizing hormone, progesterone, estrogen, and streptococcus.