WO1991015769A1

WO1991015769A1 - Bi-directional lateral chromatographic test methods

Info

Publication number: WO1991015769A1
Application number: PCT/US1991/002403
Authority: WO
Inventors: Frederick C. Horstman; Julia A. Whiteside
Original assignee: Disease Detection International
Priority date: 1990-04-09
Filing date: 1991-04-09
Publication date: 1991-10-17
Also published as: AU7672291A

Abstract

A chromatic test device (10, 30) having a unitary planar fibrous body (20, 36) incorporating a sample application zone (26, 38), a separation zone (42) and a reaction zone (28, 40) and methods of using the device are disclosed. The application zone (26, 38) of the device is in fluid communication with a first absorbent means (44) and the reaction zone (28, 40) is in fluid communication with a second absorbent means (48) in order to establish bilateral flow of the fluid component of a sample applied to the fibrous filter body.

Description

BI-DIRECTIONAL LATERAL CHROMATOGRAPHIC TEST METHODS

Background of the Invention

The invention relates to a diagnostic device for performing solid phase immunoassays to detect the presence of antigens or antibodies in biological or non-biological fluids. The teachings of the invention are incorporated in a bi-directional lateral chromatographic device for use in solid phase immunoassays, or for the non-immunological detection or quan ita ion of proteins , or substances in biological or non-biological fluids.

More particularly, the invention relates to devices and methods which utilize filter means for testing biological fluids to detect the presence of analytes, such as bacterial, viral, parasitic or fungal antigens, and immunoglobulins, hormones, serum proteins, drugs and the

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Typical of prior art devices presently in use are the teachings of U.S. Patent No. 4,623,461, which discloses a filter body located in a housing having an opening therein for the reception of a suspension sample to permit the upper face of the filter to trap colored or particulate matter contained within the specimen, and prevent such matter from reaching the bottom face of the reaction zone, which has been previously treated with a suitable reactant. The perimeter of the filter is engaged with a suitable reactant. The perimeter of the filter is engaged with a suitable absorbent body, and the absorbent body is intended to receive the outward diffusion of liquids applied to the filter.

One of the disadvantages of the '461 construction lies in the fact that the fluid flow from the point of application of the suspension to the absorbent body is uni¬ directional, and a substantial accumulation of solids at the point of application of the sample suspension is inevitable, which will seriously impinge upon the resultant chromatological or other type of test reading imparted by the device.

U.S. Letters Patent No. 3,825,410 discloses a disposable, combined storage and reaction cell for use in the performance of chemical and biological reactions which receives reactants dispensed therein and maintains the same in stored condition so that they remain stable. Reactants will not mutually react until such time as it is required to initiate the reaction.

The immobilization of the reactants is accomplished by such procedures as freeze drying, and. the reaction is initiated by the introduction of a sample to be analyzed, whereafter separation of bound and free ligand can be performed either within the unit itself or externally.

The reaction cell of the '410 patent may include a filter so that the entire process of separation can be completed within the reaction cell, and the filter can be removed from the reaction cell and submitted for radioactivity or other tracer counts.

U.S. Letters Patent No. 3,888,629 discloses a reaction cell for performing various types of assays which incorporates a matrix pad of absorbent material retaining the necessary reagents for the reaction and serving as a site in which the reaction totally occurs. A separable, lower chamber incorporates absorbent material abutting the matrix pad to promote filtration through the pad after the reaction has taken place.

Both patents are characterized by the mere utilization of the filter as a pass-through device, which is hindered by the deposition of solids out of the suspension sample. Objects and Advantages of the Invention

One of the objects of the invention is the provision of a diagnostic device which incorporates a planar filter body having sample application, separation and reaction zones, said filter body being configured in such a manner that the bulk of the solids in the suspension sample are retained in the sample application zone and the fluid is caused to migrate bilaterally through the interstices of the filter by the fluid communication of absorbent means with the application and reaction zones.

Consequently, the unilateral directional flow which causes accumulation of solids in the reaction zone in the previously-discussed prior art devices is eliminated in the test device of our construction because the rapid bilateral flow achieved by the construction of the device causes immediate deposition of solids out of the fluid component of the suspension. Another object of the invention is the provision of a diagnostic test device of the aforementioned character, wherein the planar filter incorporates a sample application zone which is relatively large, and which is connected to the reaction zone by a separation zone, the length of the separation zone being proportioned to the character of the suspension sample applied to the application zone; and the separation and application zones cooperate to retain the bulk of the solids or particulate matter in the application and separation zones so that the immuno-reagent or chemical test reagent deposited in the reaction zone, when subjected to the test procedures and analytes, will have a minimum of, or no, particulates embodied therein which could cause the emission of high background signals, thus creating a negative effect on the test readout. Another important object of the invention is the provision of a composite housing filter conjugate which is characterized by ease of assembly and application. The test device of the invention is designed particularly for use in the field for the testing of various human and animal diseases, or for various chemical tests involving the utilization of blood samples from humans and animals; and it is capable of giving test results equal to laboratory results within a matter of minutes, depending upon the sample solution which is applied to the specific device.

Another object of the invention is the provision of a method of performing a test by the utilization of the device of the invention which incorporates a plurality of simple steps which can be carried forth by non-laboratory personnel in the field, and which can provide such personnel with an almost immediate readout of the presence or absence of the sought-after infection or drug, or the like.

Still another object of the invention is the provision of a simple two-step method of determining the presence or absence of an analyte in a sample fluid. In the practice of the two step-method, necessary reagents may be embedded within a filter matrix prior to use. However, when using the two-step method with an enzymatic tracer requiring a chromatic-eliciting substrate for detection, the chromatic eliciting substrate reagent should be added after application of the sample. The two-step method requires only the application of the sample itself, followed by application of a washing solution, which may be a solution of a chromatic eliciting substrate reagent.

Brief Description of the Drawings Other objects and advantages of the invention will be apparent from the following specification and the accompanying drawings, which are for the purpose of illustration only, and in which:

Figure 1 is a top plan view of a typical device of the invention;

Figure 2 is a device similar to Figure 1, with the exception that it incorporates a plurality of application and reaction zones;

Figure 3 is a top plan view showing the housing of the device of Figure 2 with the cover removed therefrom to illustrate the location of and configuration of the filter and the relation thereof with the absorbent means and the particular design of the housing to encapsulate the filter and absorbent means:

Figure 4 is a view similar to Figure 3 illustrating removal of the filter to disclose the relationship of the absorbent means with the housing and the particular configuration thereof;

Figure 5 is a vertical sectional view taken on the broken line 5-5 of Figure 2 and illustrates the fluid relationship of the filter with the absorbent means and the filter and absorbent components with the specific design of the housing and cover therefor; and

Figure 6 is an exploded view illustrating the various components of the test device. Description of the Preferred Embodiments of the Invention

The chromatic assay device of the present invention may be used to perform solid phase immunoassays for the detection of antigens or antibodies, hereinafter referred to as analytes, in biological and non-biological fluids. The device may be non-immunologically used to identify and/or quantitate proteins or substances in biological and non-biological fluids. The device may be utilized to perform assays such as competitive or non-competitive enzyme-linked immunoassays, enzyme-multiplied immunoassays, enzyme-inhibition assays, heterogeneous or homogeneous fluorescent immunoassays, chemiluminescent and bioluminescent assays, these assays utilizing various labelled probes, and the like.

Obviously, the particular analyte test to be used will depend upon the chosen sample and the desired result to be achieved.

Referring to the drawings, and particularly to Figure 1 thereof, we show a test device 10 constructed in accordance with the teachings of our invention which is incorporated in a housing 12 said housing consisting of a lower or bottom component 14 and a cover or closure 16. The bottom component 14 of the housing may be fabricated by injection molding from suitable synthetic plastic materials such as polyethylene and, as will appear further hereinbelow, is specifically designed to receive a flat co- planar filter 20. The closure 16 overlies the filter 20 and is secured to the housing by pressure-sensitive adhesive or other adhering means and incorporates an application port or opening 22 and a reaction port or opening 24, said ports communicating, respectively, with the application zone 26 and the reaction zone 28 of the filter.

Alternatively, the entire structure may be inverted so that the bottom component 14 forms the top of the structure, with the cover or closure 16 being on the underside. When this inverted structure is employed, the application and reaction ports 22, 24 are formed in bottom component 14, rather than the closure 16. Small wells may be formed around the ports to receive the liquid and divert it into the port. This inverted embodiment is in all other respects identical to the embodiment of the invention having the closure 16 on top.

The test device 10 is designed for the performance of a single test, but, as best shown in Figure 2 of the drawings, a device 30 incorporating a multiplicity of application ports 22 and reaction ports 24 can be provided in a closure or cover 32 which overlies a suitably configured, as will be explained in greater detail below, lower housing portion 34 and filter 36. The ports 22 and 24 respectively overlie application and reaction zones 38 and 40 of the filter 36. The lower or bottom portion 34 of the housing of the test device 30 is configured, as best shown in Figures 4 and 5 of the drawings, to provide receptacles 56 for the application, separation and reaction zones of the filter 36 so that fluid flow is confined in the plane of the filter 36 because of the sandwich created between the closure 32 and the lower portion 34 of the housing. The receptacles are defined by integrally molded lobes 58 in the body of the lower portion 34 of the housing and stringently confine the relevant portions of the filter in the receptacles 56.

Juxtaposed to the receptacles 56 is a first elongated rectangular well 60 for the reception of the first absorbent means 44, and a corresponding well 62 is provided for the reception of the second absorbent means 48.

The closure or cover 32 of the device 30 can be fabricated from vinyl or other plastic sheet material and may be adhesively or otherwise secured to the bottom portion of 34 of the housing of the test device 30.

The application, separation and reaction zones are contiguous within the co-planar surfaces of the glass fiber matrix. A sample or samples applied to the sample application zones 38 will migrate laterally by capillary and chromatographic action. As will be described hereinbelow in greater detail, the fundamental result achieved by the test devices constructed in accordance with the teachings of the invention is bilateral flow of the fluid component of suspensions applied to the application zones 38.

During the bilateral migration, particulate matter present within the sample volume, i.e., cellular components of whole blood, salt crystals of urine or protein aggregates of serum or plasma, etc., are filtered from the fluid portion of the applied sample by particle size exclusion dictated by the mean pore size of the glass fiber matrix. Since the mean pore size of the glass fiber matrix is not an absolute value, but, rather, represents a Poisson distribution of a range of pore sizes, the length and width of the separation zone will be influenced and dictated by the mean porosity of the glass fiber matrix. Likewise, since the mean diameter of particulates within the sample will vary, a separation gradient will be realized within the body of the separation zone, with larger particulates remaining closer to the application zone, while smaller particulates will migrate some distance from the application zone. Therefore, the length and width of the separation zone between the reaction and sample application zones must be carefully established empirically in order to position the reaction zone at a proper distance from the sample application zone to prohibit an inhibitory quantum of particulates from entering the reaction zone. If the separation zone length is too short, some particulates may enter the reaction zone; if too long, the volume of filtered sample fluid containing the desired analyte to be detected may be insufficient for optimal detection.

Bilateral migration of the fluid portion of the applied sample is also channeled in a direction 180 degrees away from the separation zone and, subsequently, the reaction zone, by the tapered constriction in the lateral boundaries of the bulbous or lobular-shaped glass fiber matrix. This constriction of the glass fiber matrix favors migration of the sample through the separation zone in the direction of the reaction zone, yet still allows for some migration of fluid away from the separation and reaction zones, facilitating removal of unwanted or interfering debris (particulates, protein aggregates, unreacted test reagents) from the reaction zone upon subsequent application of wash solution and/or test reagents to the reaction zone. In essence, this design functions as a valve and reduces or eliminates back-washing of unreacted components into the reaction zone which may cause high background signals.

Sandwich relationship between the filter 36 and the cover 32 with the bottom portion 34 of the housing of the filter and with the associated absorbent means 44 and 48 is illustrated in the cross-sectional view of Figure 5, which, of course, is equally applicable to the test device of Figure 1, as well as the test device of Figure 2. It will be noted from the showings of Figures 3 and 5 that a portion of each application zone 38 is in fluid communication with the corresponding first absorbent means 44, as best shown at 66 in Figures 3 and 5 of the drawings. Consequently, the absorbent means 44 is in fluid communication with the application zones 38 and causes a bilateral flow of fluid from the sample being applied to the application zones simultaneously with flow in the opposite direction from the application zones 38 into the separation zones 42.

The bilateral flow through the separation zones 42 is facilitated by the location of the reaction zones 40 adjacent to a relatively large area of the filter 36, shown, in the particular embodiment of the test device, as generally rectangular in configuration and overlying the second absorbent means 48. Alternatively, this relatively large area of the filter 36 may be sufficiently absorbent to serve as the second absorbent means by itself without providing any underlying absorbent material-

Consequently, bilateral flow established in this manner reduces the hydraulic pressure in the application zones 38 and causes rapid settling of particulates or other inclusions in the sample suspension, thus causing rapid settling out of the particulates or other detritus before reaching the reaction zones 40.

It will also be noted that the second absorbent means 48 is of much larger dimensions than the absorbent means 44, causing more rapid absorption of the excess fluid of the sample and causing the accentuation of the bilateral flow phenomenon achieved by the filter design and its association with the first and second absorbent means.

It will be readily apparent to those skilled in the art that the configuration of the application zones 38 can be readily altered to accommodate the needs of the particular samples being tested by the devices 20 and 30 and, furthermore, as specified hereinabove, the length and width of the separation zones be empirically established to conform to the bilateral flow patterns to be established for the particular sample.

Moreover, the relative dimensions and depth of the absorbent means 44 and 48 can be altered to establish greater or lesser fluid communication between the application zones 38 and reaction zones 40, respectively.

A typical glass fiber matrix filter has its source in Eaton-Dikeman Division of Filtration Sciences, Mount Holly Springs, Pennsylvania. The weight is 71 gm/m²; the depth is 0.43 mm; the mean pore size is 0.6 micron (u) ; the mean fiber diameter is 0.7u (0.25u to l.5u) ; and the composition is borosilicate glass.

The dimensions of the application zone are 8 mm in diameter, and the separation zone 4 mm X 9 mm.

These dimension are suitable to effect separation of cells, protein aggregates or other debris from a 30-40 ul sample of human whole blood or serum applied to the sample application zone followed by a wash volume of 50-60 ul applied to the same. Modification .of the preferred embodiment is indicated if the whole blood sample is from an animal other than a human, such as equine or bovine whole blood, which generally has smaller red blood cell diameters (5.5u or 5.9u, respectively) than human (6.9u-8.1u). In this instance, a glass fiber matrix of a smaller pore size would be desirable, or, alternately, a longer or shorter separation zone may be required.

Although the filters 20 and 36 are described as fabricated in accordance with the previously set forth specifications, it will be obvious to those skilled in the art that the filter means can be made of any porous material capable of drawing liquid through its structure by capillary action. The pores of the filter matrix should, obviously, be sufficiently small to accomplish filter separation of the non-solubilized components of the test sample from solubilized components.

The filter may be composed of such materials as glass fiber filter paper, nitrocellulose, plastic, synthetic polymer, cellulose, cellulose acetate, and various other equivalent materials having the qualities and characteristics described hereinabove.

Of course, it is desirable to utilize materials which are inert and chemically non-reactive with the analytes and washing solvents with which the test device is to be utilized.

The test devices 10 and 30, of course, have their respective reaction zones 28 and 40 treated with specific analyte reactants. Localized regions of the respective filters 20 and 36 are treated to provide the reaction zones 28 and 40 to prepare the test devices 20 and 30 for use with a predetermined test specimen without any preparatory additions to the test devices. For example, a binding protein could be placed in the reaction zones to which an antibody is bound, which antibody is immunologically reactive with a specific antigen.

An i muno-reagent, or chemical test reagent in the case of a biochemical test, complimentary to the test analyte or an analog thereof, conjugated with an enzyme or other suitable tracer, such as a radionuclide or fluorescent dye, may optionally be embedded in the application zones 38. This embodiment is useful in the simplified two-step method of use of the invention which is described hereinbelow. In this embodiment of the invention, for reasons to be described below, the conjugate is embedded in a manner which permits the conjugate to be hydrated and solubilized during the application of the sample.

In any embodiment of the invention, when a specimen is applied to the application zones 26 and 38 through the application ports 22, the fluid component of the suspension is immediately subjected to the bilateral action achieved by the specific construction of the test devices alluded to hereinabove.

The absorbent material utilized in the first and second absorbent means 44 and 48 may be of any suitable material, such as hydrophilic polymers, particulate absorbents, glass fiber, carbon fiber, cellulose fiber, wood pulp or sponge material.

As previously mentioned, the size and shape of the respective absorbent means 44 and 48 is dictated by the volumetric considerations applicable to the specific test for which the test devices 20 and 30 are designed, and corresponding diminishment or enhancement of the absorptive capacity of the first and second absorbent means 44 and 48 result from empirical calculations of the needs for the establishment of greater or lesser bilateral flow of the fluid components of the test specimen.

The test devices of the present invention may incorporate a variety of different control tests for use with methods of the present invention. Thus, for example, one or more of the reaction zones of a test device 30 having a multiplicity of application ports 22 may be provided which provide a positive control to verify that all reagents are performing correctly. JThis is especially important where false negative results are particularly troubling, such as a test for human chorionic gonadotropin (hCG) as an indication of pregnancy. An exemplary positive control may have the analyte to be tested immobilized to the filter matrix of the reaction zone 40.

Another use for a positive control zone is to provide a quantitative indication of the amount of analyte in the sample. Thus, reaction zones 40 may be provided which have the analyte being tested immobilized in known quantities. A comparison between the amount of tracer reagent bound to the control reaction zone and a test reaction zone will give an indication of the amount of analyte in the unknown. This technique is especially useful where a cut-off level of analyte indicates a positive test. For example, the level of antibody providing resistance to infection with rubella virus has been determined to be a hemagglutination inhibition titer of 1:8. Thus, a positive test result for rubella resistance would show an equal or greater amount of tracer reagent bound to the test reaction zone than to a positive control providing a level of tracer reagent bound for a titer of 1:8.

Moreover, negative controls can also be provided. An exemplary negative control may have a reaction zone 40 to which all of the steps of immobilizing the antibody are performed, however, without the presence of antibody. Such a negative control test would be useful in determining the amount of non-specific binding of analytes or interfering components to the filter matrix. Negative control tests guard against false positive test results. In many instances it may be desirable to provide both negative and positive tests to guard against both false negatives and false positives.

Method of the Invention In practicing the method of the invention, a suitable volume of sample is applied directly to the sample application zone of the glass fiber matrix. A suitable volume of wash reagent is then applied tp the same area of the sample application zone. Alternatively, no wash reagent may be necessary if a sufficient volume of sample is added to move sample components into the reaction zone.

Capillary and chromatographic forces within the body of the glass fiber matrix draw the fluid portion of the sample primarily in the direction of the separation zone but, also, secondarily in the opposite direction. The bilateral flow is defined by the lateral boundaries of the glass fiber matrix and the fluid communication of the application and reaction zones with their respective absorbent means. As the fluid migrates through the separation zone, particles larger than the mean pore size of the glass fiber matrix are restricted in their lateral migration toward the reaction zone, so that only the fluid portion of the sample reaches and flows into and through the reaction zone. The analyte, contained within the fluid portion of the sample reacts and binds with the specific complimentary immuno-reagent (antigen or antibody) or chemical test reagents, which have been immobilized to the glass fiber matrix in the area of the reaction zone.

Subsequently, a suitable volume of wash reagent is applied directly to the reaction zone. This washes away unreacted sample components which may interfere with subsequent steps, in bilateral directions, again defined by the lateral boundaries of the glass fiber matrix. Alternatively, this step of applying wash reagent may be omitted, with the required wash being accomplished by the subsequent application of a chromatic-eliciting substrate reagent or other solution in sufficient volume to wash away unreacted sample components.

Unreacted sample components are carried away by the bilateral flow of the washing step, whether accomplished by a separate wash reagent or by subsequent application of another solution. When wash is applied to the reaction zone, the two wash directions of the bilateral flow are l) away from the separation and sample application zones, and 2) toward the separation and sample application zones, reversing the original direction of flow. Wash in the latter direction inhibits or prevents previously filtered particulates from reaching the reaction zone and actually acts as a "counter current" to back flush potential interfering particulates present in the original sample away from the reaction zone. The analyte present in the fluid portion of the sample is bound to the complimentary immuno-reagent or chemical test reagent immobilized to the glass fiber matrix at the reaction zone site. Subsequently, an immuno-reagent, or chemical test reagent in the case of a biochemical test, complimentary to the test analyte or an analog thereof, conjugated with an enzyme or other suitable tracer, such as a radionuclide or fluorescent dye, is applied directly to the reaction zone. Unbound immuno-reagent conjugate or chemical test reagent may optionally be washed from the reaction zone in the lateral bi-directional mode outlined above by the application of a suitable wash volume applied directly to the reaction zone. Alternatively, the complimentary reagent-tracer conjugate may be embedded in the application zone prior to application of the sample, as described above in connection with the description of fiber matrix of the test device. In this embodiment, the conjugate is embedded in a manner which permits the conjugate to be hydrated and solubilized during the application of the sample. Thus, when sample is applied to the sample application zone, the conjugate is hydrated and will react with any analyte found in the sample. Thus, if there was analyte present in the sample, an analyte-complimentary reagent-tracer conjugate will be formed. The bilateral flow of the test device will then carry the analyte-complimentary reagent-tracer conjugate and all other components through the separation zone and into the reaction zone. The reaction zone has an immobilized reagent complementary to the analyte present in the analyte-complimentary reagent-tracer conjugate. If the analyte was present in the sample, it will be sandwiched between the immobilized complimentary reagent and the enzyme conjugate immuno-reagents within the reaction zone. A suitable substrate or chromogen may then be added to the reaction zone if the tracer is an enzyme requiring such a substrate to develop color. The enzyme conjugated to the analyte bound immuno-reagent acts upon the substrate or chromogen to produce a colored product within the reaction zone which may be viewed or measured with an instrument. The tracer-complimentary reagent conjugate is applied to the application zone in molar excess of the sample to assure binding of the sample. Thus, not all of the tracer- complimentary reagent conjugate present in the reaction zone is bound to the immobilized complimentary reagent, even where analyte is present in the sample. This unbound conjugate should be removed prior to determining the results of the test. However, when applied in sufficient volume, the substrate solution serves also to wash away unreacted components from the reaction zone. Less preferred, a wash reagent may be added prior to addition of the substrate solution to wash away unreacted components. In either case, the unreacted components are washed from the reaction zone in a bi-directional mode.

Where a tracer requiring no substrate to be detected, such as a radionuclide or fluorescent dye is used, no substrate need be added. However, where no substrate solution is added, a suitable wash reagent should be added to wash away unreacted components from the reaction zone.

Thus, where a suitable conjugate is embedded in the fiber matrix of the application zone and where all unnecessary applications of wash reagents are eliminated by application of appropriate volumes of sample solution and/or substrate reagent, there is provided a simple two- step process for the detection of an analyte. In this simplified two-step process, the only steps required of the user of the test device are the application of the sample fluid to the application zone and the application of substrate or wash reagent to the reaction zone. Exemplary Filter Construction and Methods of Utilizing Same Example 1. Detection of Antibody to Rubella Virus in Whole Blood

Inactivated Rubella virus antigen is immobilized onto the reaction zone of the glass fiber matrix. This is followed by the addition of a blocking agent such as 2.0% bovine serum albumin or 0.5% non-fat milk suspension to the same area and allowed to dry.

The use of a blocking agent decreases the non-specific binding of extraneous proteins present in the fluid (serous) portion of whole blood to the reaction zone of the glass fiber matrix.

To perform an assay, approximately 30 microliters of whole blood is applied to the sample application zone of the device. This is followed by 60 microliters of a wash solution consisting of 0.5% non-fat milk in a phosphate buffered saline applied to the same area. Migration of the whole blood sample through the separation zone will filter and separate the cellular components from the sample within the area of the separation zone. Evidence of separation of the fluid portion of the whole blood sample is observed in the reaction zone by the appearance of serous fluids wetting the reaction zone area. 60 microliters of the wash solution is then applied to the wetted reaction zone. Upon absorption, the wash step is repeated. It will be noticed that the serous components and pigments contained therein will be eliminated via the lateral bi-directional mode described earlier from the reaction zone by this wash procedure.

However, if the whole blood specimen contains antibodies to the Rubella virus, the antibodies in the serous portion of the blood sample will bind to the Rubella virus antigens immobilized within the reaction zone of the glass fiber matrix. Then, 60 microliters of an affinity purified rabbit anti-human IgG conjugated to alkaline phosphatase is applied to the reaction zone. This will bind to the antibody of the Rubella virus which may be present in the blood sample and will be trapped by the immobilized antigen located in the reaction zone of the glass fiber matrix. Unreacted enzyme conjugate is washed away as described above.

Finally, 60 microliters of a suitable substrate chromogen may be applied to the reaction zone. Appearance of a colored product at the reaction zone is evidence of enzyme activity and, therefore, indicative of antibody to Rubella virus present in the whole blood sample.

Example 2. De te rm i n at i o n o f Huma n

Choriogonadotropin in Urine by a "Sandwich" Technique

A polypeptide hormone, human choriogonadotropin (hCG) , is secreted into the maternal circulatory system by the trophoblasts of the developing fetus. Ultimately, this hormone is excreted in the maternal urine. Detection of hCG in the urine is presumptive evidence of pregnancy. The hCG is collected, concentrated and purified by well-known published methods. The purified hormone may be used to generate antibodies (polyclonal or monoclonal) in the appropriate species, i.e., rabbits or mice, respectively.

The antibody of hCG is immobilized to the reaction zone of the glass fiber matrix. A blocking protein is then applied to the reaction zone as described in the previous example.

To determine if a urine specimen contains hCG, a few drops of the specimen are applied to the sample application zone of the glass fiber matrix of the device. This is followed by a sufficient volume of a wash solution applied to the same area to cause the sample to migrate through the separation zone toward and through the reaction zone of the glass fiber matrix which contains the immobilized antibody to hCG. If hCG is present in the sample, it will bind to the immobilized antibody located within the reaction zone.

Alternately, sufficient volume of urine may be applied to the sample application zone to cause the sample to chromatograph through the reaction zone without the use of a wash. In either case, migration of the urine sample through the separation zone will filter out urine particulates which may interfere in subsequent testing steps.

Then a suitable volume of a washing solution is applied to the reaction zone. Lateral, bi-directional flow of the wash solution will carry unreacted urine components away from the reaction zone, i.e., away from the separation and sample application zones as well as toward the separation and sample application zones, reversing the original direction of flow. Movement toward the reaction zone of wash fluid in the latter direction prohibits further movement of unwanted particulates by counter flow forces. Indeed, subsequent addition of any wash or test reagent to the area of the reaction zone will force any trapped particulates or debris located within the separation zone away from the reaction zone.

Application to the reaction zone of an appropriate enzyme labeled antibody to hCG (either polyclonal or monoclonal) will bind to the hCG of the sample which has been trapped by the immobilized antibody bound to the reaction zone of the glass fiber matrix. Again, a wash solution is applied, as indicated above, to wash away, in a lateral, bi-directional mode, any unreacted enzyme conjugated antibody.

Subsequent addition of a suitable substrate chromogen solution to the reaction zone will indicate the presence of enzyme and, therefore the presence of hCG, by the development of a colored product at the reaction zone. The method described above in this example is typical of a "sandwich technique," whereby the analyte, hCG in this case, is sandwiched between two antibodies, one immobilized to the glass fiber matrix of the reactipn zone, the other conjugated to an enzyme or other suitable label. The presence of the hCG analyte is indicated by the development of color within the reaction zone.

Example 3. De te rminat i on o f Huma n Choriogonadotropin in Urine by

. Competitive Inhibition Immunoassay

The test devices of the invention are not limited to "sandwich" methodology, but may be applied to competitive inhibition techniques as described by the following example. Antibody immobilization, sample application and washing methods and separation/chromatographic principles are as described in the previous example. However, instead of application of an antibody enzyme conjugate, one may apply to the reaction zone an enzyme conjugate of the analyte, i.e., hCG coupled to an appropriate enzyme.

If hCG is present in the sample, it will bind to a finite and limited number of available antibody binding sites located and immobilized within the reaction zone of the glass fiber matrix. If the sample contains substantial amounts of hCG, then all available antibody binding sites in the reaction zone will be saturated. Upon subsequent application of an enzyme conjugated to hCG (instead of enzyme conjugated to an anti-hCG antibody) , all available immobilized antibody binding sites are saturated with the hCG from the sample and will not bind to the enzyme-hCG conjugate. When a suitable wash solution is applied, the enzyme-hCG conjugate will be washed away from the reaction zone in a lateral, bi-directional fashion.

Application of a suitable substrate chromogen solution to the reaction zone will not develop a color in this instance since no enzyme is available. If, however, the sample contains no or insufficient quantities of hCG to saturate all immobilized antibody binding sites, then hCG enzyme conjugate will bind to the available immobilized hCG binding sites and will not be washed away with subsequent washing steps.

Therefore, in this instance, upon subsequent application of a suitable substrate chromogen solution to the reaction zone of the glass fiber matrix, some color will develop, indicating the sample had little or no hCG present. In a competitive inhibition assay as just described, the absence of color development in the reaction zone is indicative of the presence of the analyte (hCG) in the sample, while the presence of color development in the reaction zone indicates little or no analyte (hCG) in the sample. The device can also be used for competitive immuno¬ assays of low molecular weight analytes, such as thyroid hormones, therapeutic drugs, steroids and other low molecular weight analytes.

Example 4. D e t e r m i n a t i o n o f H u m a n Choriogonadotropin in Urine by a

Simplified Two-Step "Sandwich" Technique Incorporating a Positive Control

To each of two application zones of a glass fiber matrix, enzyme labeled antibody to an epitope on the β chain of hCG (anti-øhCG) is applied and allowed to dry, but not immobilized. The enzyme portion of the enzyme labeled antibody is alkaline phosphatase.

An antibody to an epitope on the α chain of hCG (anti- ochCG) is immobilized to one reaction zone of the glass fiber matrix and blocking protein applied as in Example 2. This* reaction zone, to which anti-αhCG has been applied, is the test reaction zone. To another adjacent reaction zone, the positive control reaction zone, antibody reactive with the anti-,5hCG embedded in the application zones (anti-anti- βhCG) is immobilized. Both reaction zones are included within a test device of the present invention.

To determine if a urine sample contains hCG, a few drops of the sample are applied to the sample application zone of both application zones of the device. The application of sample rehydrates the enzyme labeled antibody present in the application zone. If hCG is present in the sample, it will bind to the antibody portion of the enzyme labelled antibody. A sufficient volume of sample is applied to both application zones to cause the sample to migrate through the respective separation zones toward and into the reaction zones. Thus, any hCG-enzyme labeled antibody complexes are carried into the reaction zones.

If there are hCG-antibody complexes carried into the test reaction zone, these complexes will bind to the immobilized antibody found therein. However, enzyme labeled antibody which is carried into the control reaction zone will bind to the anti-anti_/3hCG found therein regardless of the presence of hCG in the sample.

An appropriate chromogen substrate is then added to each reaction zone in order to elicit color. The volume of chromogen solution applied to each application zone is sufficient so that lateral, bi-directional flow of the sample will occur to carry unreacted urine components and enzyme labeled antibody away from the reaction zones, i.e., away from the separation and sample application zones as well as toward the separation and sample application zones, reversing the original direction of flow. Movement toward the reaction zones of fluid in the latter direction prohibits further movement of unwanted particulates by counter flow forces, as in the step of applying wash reagent of Example 2. The chromogen solution indicates the presence of the enzyme at the reaction zone, and therefore the presence of hCG in the test sample, by the development of a colored product at the reaction zone.

The positive control reaction zone shows the strong presence of a colored product in all cases where the reagents are performing correctly. The test zone indicates a colored product only if hCG is present in the sample.

Example 5. Determination of Strep A antigen by a Simplified Two-Step "Sandwich" Technique

The antibody to Strep-A antigen is immobilized to the reaction zone of a glass fiber matrix and blocking protein applied. Enzyme labeled antibody is applied, without immobilization, to the application zone.

To determine if a throat swab extract sample contains Strep-A antigen, a few drops of the extract sample are applied to the sample application zone. Strep-A antigens will bind to the enzyme labeled antibody present in the application zone and be carried into the reaction zone.

After an appropriate period of time to allow any Strep-A antigen-enzyme labeled antibody complexes to bind to the antibody in the reaction zone, a few drops of a suitable chromogen solution is applied to the reaction zone. Unreacted sample components and enzyme labeled antibody will be carried away by the bi-directional flow of the chromogen solution through the glass fiber matrix. Thus, the appearance of a colored product in the reaction zone indicates the presence of Strep-A antigen in the sample extract.

Example 6. Detection of Glucose in Whole Blood The device may be used to perform assays to indicate the presence or quantitation of analytes without employing immunological methods and principles. For example, one may detect the presence of glucose in whole blood by standard enzyme analytical techniques. In this instance, a mixture of the enzymes glucose oxidase and horseradish peroxidase is immobilized to the reaction zone of the device. Whole blood is then applied to the sample application zone.

A suitable wash solution is then applied to the sample application zone to effect the bi-directional lateral chromatographic separation of the fluid portion of the sample from the cellular components as described previously to introduce the fluid portion containing glucose into the reaction zone. The immobilized oxidase acts upon the glucose of the sample to produce D-glucono-δ-lactone and hydrogen peroxide.

The horseradish peroxidase, also immobilized within the reaction zone, catalyses the hydrogen peroxide in situ as it is generated. Subsequent addition to the reaction zone of a suitable chromogen test reagent will react with the products of catalysis to produce a colored product, the intensity of which is proportional to the amount of glucose present in the original sample. The intensity of color development may be observed visually or detected by the use of suitable instrumentation. It is evident from this example that the device of the invention accomplishes other than immunoassays with equal effectiveness.

It will be readily apparent that the utilization of the test devices manufactured in accordance with the teachings of this invention provides both more effective and less time-consuming testing of various suspensions in the field by relatively inexperienced personnel. The bilateral migration of the fluid components of the various samples applied to the application zones attributable to the unique construction of the test devices prevents the contamination of the reaction zones by the particulates in the suspension samples and also facilitates the migration of the fluid component of the sample to the reaction zones.

It is also contemplated by the invention that a plurality of test devices manufactured in accordance with the teachings of the invention and incorporating single application, separation and reaction zones may be snapped together or otherwise associated on a mounting board or the like to permit a series of different tests to be accomplished by juxtaposition of the single test devices.

It will be obvious to those skilled in the art that various modifications of the test devices of the invention may be made without departing from the scope of the claims.

Claims

WE CLAIM :

1. A method of determining the presence or absence of an analyte in a sample fluid comprising: immobilizing a specific reagent complimentary to at least one antigenic site of said analyte to a reaction zone of a fiber filter matrix, said fiber filter matrix also having a separation zone and a sample application zone, said separation zone separating said application zone from said reaction zone; applying a tracer reagent which binds to said analyte to said application zone, said tracer reagent being a reagent conjugated with a tracer; applying a volume of said sample fluid to said sample application zone; permitting said tracer reagent to bind to any analytes present in said sample fluid; permitting said fiber matrix to draw at least some of the fluid portion of said sample into said reaction zone; permitting any analytes bound to tracer reagent contained within said fluid which is drawn into said reaction zone to react and bind with said specific reagent; washing said reaction zone of any unreacted sample components or tracer reagents; and determining the presence of said analyte in said sample by the presence or absence of said tracer in said reaction zone.

2. The method of Claim 1 wherein said tracer is an enzyme which will react with a chromogen to create a colored product, additionally comprising the step of applying chromogen solution after the step of permitting any analytes bound to tracer reagent to react and bind with said specific reagent.

3. The method of Claim 2 wherein the step of washing said reaction zone of any unreacted components or tracer reagents comprises said step of applying chromogen solution.

4. The method of Claim 2, wherein the step of determining the presence of said tracer comprises applying chromatic-eliciting substrate to said reaction zone, said substrate being acted upon by said tracer to produce a chromatic reaction.

5. The method of Claim 1 wherein said tracer is a radionuclide.

6. The method of Claim 1 wherein said tracer is a fluorescent dye.

7. The method of Claim 1, wherein sufficient volume of said sample fluid is applied so that some of said fluid portion of said sample is drawn into said reaction zone without the application of additional fluid.

8. The method of Claim 1, additionally comprising the application of additional fluid after the step of applying a volume of said sample fluid.

9. The method of Claim 1, additionally comprising permitting said fiber matrix to draw the fluid portion of said sample primarily in the direction of the separation zone, but, secondarily in the opposite direction.

10. The method of Claim 1, additionally comprising the application of additional fluid after the step of permitting said fiber matrix to draw some of said fluid portion of said sample into said reaction zone.

11. The method of Claim 1 wherein sufficient volume of said tracer reagent is applied so that a bilateral flow of the test reagent continues after the reagent contacts the reaction zone without application of additional fluid.

12. The method of Claim 1, additionally comprising applying a blocking agent in order to decrease non-specific binding of extraneous components of said sample after the step of immobilizing said reagent onto said reaction zone.

13. The method of Claim 1, wherein said sample is a bodily fluid from a mammal.

14. The method of Claim 13, wherein said bodily fluid is selected from the group consisting of blood and urine.

15. The method of Claim 1, wherein said immobilized reagent is an antibody specific to said analyte, wherein said tracer reagent is a tracer conjugated to an antibody specific to said analyte, and wherein the presence of said tracer upon the completion of the assay indicates the presence of said analyte in said sample.

16. The method of Claim 1, wherein said immobilized reagent is an antibody specific to said analyte, wherein said tracer reagent is a tracer conjugated to a molecule having an antigenic site reactive with said antibody, and wherein the absence of said tracer upon the completion of the assay indicates the presence of said analyte in said sample.

17. The method of Claim 1, wherein said immobilized reagent is an antigen specific to a first antibody, wherein said analyte to be determined is a first antibody, wherein said tracer reagent is a tracer conjugated to a second antibody, said second antibody being specific to said first antibody, and wherein the presence of said tracer upon the completion of the assay indicates the presence of said first antibody in said sample.

18. The method of Claim 1, wherein said immobilized reagent is an antigen specific to a first antibody, wherein said analyte to be determined is a first antibody, wherein said tracer reagent is a tracer conjugated to a second antibody, said second antibody being specific to said antigen, and wherein the absence of said tracer upon the completion of the assay indicates the presence of said first antibody in said sample.

19. The method of Claim 9, wherein the step of causing said fiber matrix to draw fluid portion of the sample primarily in the direction of the separation zone, but, secondarily in the opposite direction is performed by providing a first absorption means contiguous to said sample application zone and a second absorption means contiguous to said reaction zone.

20. A test device for performing solid phase assays of an analyte, comprising: a substantially planar fiber filter matrix, said matrix including a test sample application zone, a separation zone and a reaction zone, said separation zone separating said application zone from said reaction zone; a tracer reagent which binds to said analyte, said tracer reagent being embedded in the filter matrix of said application zone and being solubilizable by application of sample fluid to said application zone; and a specific reagent complimentary to said analyte immobilized in said reaction zone.

21. The device of Claim 20, wherein said separation zone is sufficiently long to inhibit migration of particulates in said sample into said reaction zone

22. The device of claim 20, additionally comprising: first absorbent material in communication with said sample application zone, and second absorbent material in communication with said reaction zone for causing bilateral flow of the liquid component of said test sample

23. The device of Claim 20, wherein said application zone is of bulbous configuration and said separation zone is an elongated relatively narrow shank portion integral with said application zone and with said reaction zone to permit fluid flow from said application zone through said separation zone into said reaction zone.

24. A method of determining the presence or absence of an analyte in a sample fluid comprising: immobilizing a reagent which will react with said analyte to a reaction zone of a fiber filter matrix, said fiber filter matrix also having a separation zone and a sample application zone, said separation zone separating said application zone from said reaction zone; applying a volume of said sample fluid to said sample application zone; permitting said fiber matrix to draw at least some of the fluid portion of said sample into said reaction zone; permitting any analytes contained within said fluid portion which is drawn into said reaction zone to react with said immobilized reagent to create reaction products; and determining the presence of said analyte in said sample by the presence of said reaction products in said reaction zone.

25. The method of Claim 24, additionally comprising the step of applying a solution of a chromogen agent after the step of permitting any analytes to react with said immobilized reagent, said chro agen agent reacting in the presence of any of said reaction products to produce a colored product, wherein the step of determining the presence of said analyte comprises determining the presence of a colored product.