WO1997023781A1

WO1997023781A1 - Fecal test method and device

Info

Publication number: WO1997023781A1
Application number: PCT/US1996/020151
Authority: WO
Inventors: Mary Ann Childs; Mohammed A. Chowdury; Craig Chung; Diane Carter; Anjana Prakash; David Bernstein
Original assignee: Universal Healthwatch, Inc.
Priority date: 1995-12-22
Filing date: 1996-12-23
Publication date: 1997-07-03
Also published as: EP0868665A4; JP2000502452A; EP0868665A1; AU1336697A

Abstract

Methods are disclosed for determining the presence or amount of analyte in a fecal sample. In a preferred embodiment, a fecal sample is applied to an absorbent filter that contacts an immunochromatographic assay strip which contains a high concentration of detergent. The detergent extracts a surface antigen from an enteric pathogen and color is produced by a colloidal gold and selenium reaction system.

Description

FECAL TEST METHOD AND DEVICE Field of the Invention This invention relates to an improved method for analyzing fecal samples. In particular, this invention relates to simplified methods for preparing fecal samples for immunochromatographic assays and improved gold particle-based immunochromatographic assays that are carried out in strip formats.

Background of the Invention Fecal, or stool samples are routinely tested for the presence of viruses, bacteria, parasites, other organisms, and antigens shed from such organisms. Methods for conducting such assays begin with stool collection, usually involve a number of fecal sample manipulation steps and typically end with the development of a signal such as color formation within a signal development means to indicate the presence or absence of a test analyte.

Stool collection is usually non-invasive and is ideal for obtaining samples of certain digestive disease organisms such as Salmonella and Vibrio cholerae . Stool can be collected with a swab during examination and applied directly to a test surface or volume.

Traditional fecal examinations have used complex chemical and microbiological methods. These methods are being replaced with immunoassay methods. Immunoassay methods are highly sensitive and require only a small sample. Some immunoassay methods such as latex agglutination and enzyme immunoassays can be performed with test kits that contain vials and reagent solutions that are combined in a particular way to obtain a test result.

Although many immunoassay methods do not require electronic instrumentation or trained clinicians for their use, they are not easily carried out in underdeveloped areas of the world where modern devices and techniques are poorly understood. Even the simplest immunoassays usually require timed addition of reagents to test samples and the manipulation of plastic test parts that have to be brought together in a proper order.

The application of immunoassay techniques to fecal analysis in particular is difficult for several reasons.

Stool handling is disagreeable and hazardous. Sanitary and inoffensive procedures for processing stool are awkward and often complex. Such procedures may include weighing, centrifuging and storing, and are difficult except in a clinical laboratory equipped with a suitable apparatus, protective equipment, and a skilled technician. However, stool samples have to be processed before their use in an immunoassay in order to remove interferences. This processing can cause complexity within the test method and prevents more widespread use of cholera tests in rural and lesser developed regions where machinery and reagents for processing are non¬ existent.

Any reduction in the number of steps required to perform a fecal test and any reduction in contact between test operator and the test material could be a boon to testing in this area, particularly where a lethal disease agent such as Vijrio cholerae is involved. Such an advance would directly advance health by allowing earlier and more complete testing for cholera.

Attempts have been made to alleviate the methodology problem of handling stool specimens. For example, M.A. Grow et al . in U.S. Patent 5,198,365 describe a fecal samp_le handling method for a hemoglobin immunoassay that requires dilution of a stool specimen by 10 to 100 fold. Although dilution possibly can simplify the assay procedure, it lowers sensitivity by a dilution factor. A 10 to 100 fold dilution step is particularly unacceptable for many tests of infectious agents such aε Vijbrio cholerae because greater test sensitivity is desired to detect these agents at earliest clinical time periods. On the other hand, if a sample is tested without a significant dilution (i.e. more than 3 fold) then a centrifugation and/or filtering step is generally required as described by Vellacott et al . , Lancet (Jan. 3, 1981) and by Jikunen et al . in the Scand . J . Infect . Dis . 17: 245 (1985).

A recent attempt to eliminate the complexity problem in testing stool specimens was described in J . Clin . Micro . 32: 249 (1994) by J.A.K. Hasan et al . This reference cites a rapid colorimetric immunodiagnostic kit for the detection of the presence of Vijbrio cholerae 01 in clinical specimens.

In the procedure a stool specimen is passed through a filter that is separate from other kit components. Four drops of the stool filtrate are added to two drops of reconstituted gold labeled anti-Vi£>rio cholerae antibody. A swab is first added to the solution and then placed in an immunoassay testing device. Within the device, formed immune complexes are captured on a porous membrane that contains immobilized anti-VMJrio cholerae antibody.

Unfortunately, the separate filtration step in this procedure prolongs the test. The test is too complex for many untrained people and requires separate manipulation of two vials, one vial cap, a swab and an immunoassay device. Furthermore, test kits and methods that require many manipulations have more sources of error which lead to higher error rates. Finally, manufacturing costs increase when multiple parts and separate reagents are added to test kits. Fecal tests are particularly important for testing antigens from pathogens such as Escherichia coli strain 0157, Salmonella , Shigella , Legionella , Helicobacter, Campy lobacter and other enteric microorganisms. Of these antigens, the lipopolysaccharide A chain of Vibrio cholerae 01, which is common to all serotypes of ViJbrio cholerae 01 has particular interest for cholera testing.

These immunoassay tests require that a number of reagents and parts be brought together in a particular way. Such manipulations are most reliably done by a trained operator. If a simpler test device could be fashioned to eliminate one or more parts and/or fluid addition steps then immunoassay tests for ViJbrio cholerae could be used more to monitor this disease.

Cholera, like many other enteric diseases, must be determined by testing of a fecal sample. This can be both disagreeable and hazardous. Sanitary and inoffensive procedures for processing stool specimens are awkward and frequently complex. Typically stool samples are processed via a combination of weighing, extracting, and centrifuging steps. Sample manipulations such as filtration (particularly by pressure means) and centrifugation often cause aerosols to form and concomitant contamination of the tester. Consequently many tests are limited to a clinical laboratory setting where a suitable apparatus and a skilled technician are available.

A recent attempt to eliminate the Vibrio cholerae test complexity problem was described in J . Clin . Micro . 32: 249 (1994) by J.A.K. Hasan et al . This reference cites a rapid colorimetric immunodiagnostic kit for the detection of the presence of Vibrio cholerae 01 in clinical specimens. In the reported procedure, a stool specimen is passed through a filter cap on a vial by squeezing the vial. Four drops of the stool filtrate are added to two drops of reconstituted gold labeled anti- ViJbrio cholerae antibody in a separate container. A swab is placed in the solution and then transferred to an immunoassay testing device. Within the device, formed immune complexes are captured on a porous membrane that contains immobilized anti-ViJbrio cholerae antibody. Unfortunately, however, the separate filtration step in this procedure prolongs the test. Furthermore the test is too complex for many untrained people and requires separate manipulation of two vials, one vial cap, a swab and an immunoassay device. Such test kits and methods that require many manipulations have more sources of error, and unless care is taken, can have high error rates. Finally, manufacturing costs tend to be higher for test kits that contain many pieces and reagents. One reason why extraction and other test procedures have not been incorporated fully into a convenient test format is because compounds such as detergents and protein often behave differently in a solid phase medium such as a nitro cellulose membrane then when in solution phase. Generally, when transferring a detergent or protein from a solution phase to a solid phase, the concentration of the detergent or protein should be decreased, particularly when the substance has to quickly re-dissolve during use in a test. One problem has been a lack of knowledge concerning reagents and reagent concentrations, useful for solid phase assays where water solubility is expected to limit the use of water soluble test reagents.

In sum, complexity and biohazard problems have prevented greater use of tests on fecal specimens. There exists a need for a sensitive enteric pathogen test device that is both safer and simpler to use.

Summary of the Invention

Accordingly, it is an object of the invention to eliminate or reduce the complexity of methods and kits for testing enteric disease from fecal samples.

A second object of the invention is to help limit spread of diseases present in fecal samples through the testing process itself. Yet a third object of the invention is to provide a relatively inexpensive fecal sample test that is relatively easy to use.

In accomplishing these and other objectives, one aspect of this invention provides a method for detecting analyte in a fecal sample. The method comprises the steps of: A) contacting a fecal sample with an extraction reagent comprised of at least one detergent and at least one buffer to form a mixture; B) applying the mixture to an absorbent filter that is in proximity to or in contact with a immunochromatographic assay strip such that analyte present in the sample is transferred to the immunochromatographic assay strip; and C) detecting the presence of analyte in the immunochromatographic assay strip. In one preferred embodiment of the method, the immunochromatographic assay strip comprises a porous strip that comprises a) a first group of particles having bound thereto a binding component capable of specifically recognizing an analyte; and b) a second group of particles having bound thereto a binding component capable of specifically recognizing said analyte, wherein the average diameter of the particles in the first group is larger than the average diameter of the particles in the second group. Particularly preferred is an embodiment wherein the larger particles are comprised of selenium and the smaller particles are comprised of gold.

Another embodiment is a method for detecting Vibrio cholerae in a fecal sample. The method comprises the steps of: A) contacting a fecal sample with an extraction reagent comprised of at least one detergent and at least one buffer to form a mixture; B) applying the mixture to an absorbent filter that is in proximity to or in contact with a immunochromatographic assay such that analyte present in the sample is transferred to the immunochromatographic assay; and C) detecting the presence of ViJbrio cholerae in the immunochromatographic assay. In one preferred embodiment of the method, the immunochromatographic assay comprises a porous strip that comprises a) a first group of particles having bound thereto a binding component capable of specifically recognizing Vibrio cholerae antigen; and b) a second group of particles having bound thereto a binding component capable of specifically recognizing said ViJbrio cholerae antigen, wherein the average diameter of the particles in the first group is larger than the average diameter of the particles in the second group. The term "strip," as used in the present invention, means one or more solid phase materials in which sample flows by a wicking action. In preferred embodiments, the strip comprises two or more materials that are in physical contact with each other. The invention also provides a test kit for detection of Vibrio cholerae antigen comprised of A) an absorbent filter containing a freeze-dried extraction reagent comprised of at least one detergent and at least one buffer and that is in proximity to or in contact with an immunochromatographic assay strip comprising immunoreagents for detecting Vibrio cholerae antigen present in the sample.

In one preferred embodiment of the kit, the chromatographic strip comprises: A) gold particles coated with antibody against Vibrio cholerae antigen; and B) antibody against Vibrio cholerae antigen immobilized within a region of the strip.

Another preferred embodiment is a test kit for detection of Vibrio cholerae antigen comprised of: A) an extraction reagent comprised of at least one detergent and at least one buffer; B) an absorbent filter that is in proximity to or in contact with an immunochromatographic assay strip comprising immunoreagents for detecting ViJbrio cholerae antigen present in the sample.

In one preferred embodiment of the kit, the chromatographic strip comprises: A) gold particles coated with antibody against ViJbrio cholerae antigen; and B) antibody against Vijbrio cholerae antigen immobilized within a region of the strip.

In a preferred embodiment the extraction of a pathogenic antigen is carried out by virtue of a polyoxyethylenesorbitan-fatty acid compound such as Tween-20, that is applied to a solid phase used in the test at a concentration of more than 1.0 percent wgt/vol and dried down. Most preferred is Tween-20 at a concentration of more than 1.5 percent and less than 2.0 percent although other polyoxyethylenesorbitan-fatty acid compounds can be used at these high concentrations. The methods and kits of the present invention eliminate or reduce much of the complexity associated with prior art assay methods and, as a result, simplify operator training requirements and lower the cost of detecting analyte present in fecal samples. Furthermore, they help protect the test user from infection by accidental contact with fecal samples and treated fecal samples.

These advantages are made possible by simplifying the fecal test itself. In the inventive assay, the sample extraction step employs a fecal filter that is part of the immunochromatographic assay itself. This combination eliminates one or more tedious sample preparation and assay manipulation steps present in other tests. As a result of these changes the fecal assay is easier to carry out and fewer part are needed in the diagnostic kit. For example, only one sample container (e . g . , a tube) can be used for sample extraction. Further, a separate membrane filter tube, filter cap, and centrifugation step are not required. Furthermore, no additional fluid addition step such as a wash step is required.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

Figure l is an exploded view of one embodiment of the device in accordance with this invention. Figure 2 is an exploded view of another embodiment of the device in accordance with this invention.

Figure 3 is an exploded view of another embodiment of the device in accordance with this invention. Figure 4 is an exploded view of another embodiment of the device in accordance with this invention.

Figure 5 is an exploded view of another embodiment of the device in accordance with this invention.

Detailed Description of the Preferred Embodiments

The present inventors surprisingly discovered that a fecal sample test could be simplified by combining an immunochromatographic assay with a fecal filtering device. Moreover, the present inventors discovered that, when a suitable extraction reagent was used to contact a fecal sample, interfering substances and debris were retarded on the filter. As a result, additional sample preparation and manipulation steps were eliminated.

The inventors were surprised to discover accidentally during field trials with seven different detergents that Tween-20 at a concentration of more than 1%, and preferably at about 2% wgt/vol greatly improved test performance. The inventors were able to incorporate Tween-20 in the assay device itself in a dried form. In this preferred "one-step" embodiment liquid from the analyte, or from an analyte extraction fluid mixture, re- wets the Tween-20, and thereby allows the detergent to extract antigen from the test specimen.

The inventors also were surprised by their discovery that using a mixture of large selenium particles with smaller gold particles greatly improved assay sensitivity.

The term "immunochromatographic assay" means an assay that (1) uses antibody binding to specific epitope(s) to achieve selectivity and (2) allows test analyte and/or a test reagent to migrate within one or more solid phase materials to allow sequential reactions. In the context of the present invention "immunochromatographic assay" also includes, as one of the reactions, a binding reaction whereby a signal producing substance such as a gold or selenium particle becomes affixed within a zone of the solid phase assay device in response to the presence of test analyte.

The present invention is distinguished from other immunochromatographic assays by being in a strip format. This strip can be used as a dip-stick, in which case it is simply dipped into a sample to be tested. Alternatively, a sample can be brought to the strip by a pipette, test tube or other means. In practice, most fecal samples being tested for enteric diseases such as cholera will be watery and are applied directly to the device. Solid fecal samples, on the other hand, must be dispersed by mixing with water, or an extraction reagent. It is preferred to mix a small portion of solid sample with an approximately five fold excess of extration reagent comprised of detergent and buffer in water solution.

As used for the detection of analyte from stool samples, the method of this invention comprises the steps of: 1) contacting a fecal sample that is suspected of containing analyte with an extraction reagent to form a mixture; 2) applying the mixture to an absorbent filter that is in proximity to or in contact with an immunochromatographic assay such that analyte present in the sample is transferred to the immunochromatographic assay; and 3) detecting the presence of analyte by the immunochromatographic assay, for example, by development of color in the assay.

In a preferred embodiment, a single absorbent filter serves both to filter the fecal sample and provide a support for the immunochromatographic assay. In this case, the porosity of the absorbent filter can be chosen based on optimal separation of test analyte from interfering fecal substances during operation of the device. Porous plastic, plastic membrane, glass _fiber and the like are suitable for the absorbent filter. For example, where fiber filters are used, the particle retention size of the filter can be chosen to be between O.lum and 20um and preferably between 0.4um and 5 um. Skilled artisans readily will be able to determine acceptable filters for these purposes.

In another preferred embodiment, the absorbent filter is in proximity to, or attached to a separate porous strip for the immunochromatographic assay. The extracted analyte then passes through the filter into the immunochromatographic assay strip. The type of absorbent filter is chosen based on how well it separates fecal matter from analyte.

When used to detect cholera from stool samples, the method of this invention comprises the steps of: 1) contacting a fecal sample that is suspected of containing ViJbrio cholerae antigen with an extraction reagent comprised of at least one detergent and at least one buffer to form a mixture; 2) applying the mixture to an absorbent filter that is in proximity to or in contact with an immunochromatographic assay such that Vibrio cholerae antigen present in the sample is transferred to the immunochromatographic assay; and 3) detecting the presence of Vibrio cholerae antigen by the immunochromatographic assay, for example, by development of color in the assay.

Other combinations of immunochromatographic assays and absorbent filter are possible and readily will be recognized by skilled artisans.

The extraction reagent is any reagent that can extract analyte from the fecal sample. Acceptable extraction reagents comprise an aqueous fluid comprising at least one buffer and at least one detergent. Acceptable buffers can be in a concentration between lmM and IM and more preferably in a concentration of between 5mM and 250mM, and will have a pH between 3 and 13 and more preferably between 6 and 9. The buffer composition can be one or more chemical compounds which are known to skilled artisans. Advantageously, the volume of extraction reagent used will be at least about the same volume as the fecal sample. The detergent generally is an amphoteric compound, and many such detergents are known to skilled artisans. Examples of detergents include: deoxycholate, Tween-20, triton X-100, and sodium docedyl sulfate. In the case where the detergent comprises at least one chemical group that dissociates near neutral pH (pKa between pH 5 and 9) the buffer and detergent may be the same substance.

In a preferred embodiment, Tween-20 is the detergent and is provided at a high concentration of greater than 1.5 percent wgt/volume. Most preferred is the use of Tween-20 at a concentration of between 1.6 percent and 2.0 percent. To best facilitiate the movement of test substances, a preferred extraction composition is 1.8 percent Tween-20, 0.05% sodium azide, and 9 mM tris-Cl pH8.2. Concentrations of Tween-20 less than 1% as used in the prior art, and concentrations above 2.5% are not preferred.

An immunochromatographic assay suitable for the present invention will detect analyte from an aqueous sample that contacts it. Conveniently, detection can be carried out by the formation or disappearance of color from at least one part of the assay. Many other possibilities, however, will readily be recognized by skilled artisans.

A preferred immunochromatographic assay is a strip assay which may be conveniently combined with the absorbent filter that filters the extracted fecal sample. In a preferred embodiment, the immunochromatographic strip assay comprises: a) gold particles coated with antibody against analyte; and b) antibody against analyte immobilized within a region of the strip. The antibody- labelled gold particles are preferably dried on or within the chromatographic strip during its manufacture.

During operation of the one method embodiment in accordance with this invention, a fecal specimen reacts with the extraction reagent to release analyte (if present) into a small quantity of fluid (preferably lOOul to 500ul in volume) within a water impermeable container. A strip that comprises an absorbent filter and a strip detection means is placed into the container or already is present therein. Fluid containing antigen (if present) then wicks into the strip. The absorbent filter traps debris and solids while extracted antigen continues to diffuse into the strip with the liquid. The filtered extraction fluid which contains extracted antigen re- suspends antibody labelled particles that are present in the strip detection means. Immune complexes form between antibody-labelled particles and antigens. These immune complexes diffuse through interstices of the strip detection means, including a region which comprises immobilized antibody against the analyte. Some of the immobilized antibodies capture analyte that is bound to particles, and the capture of particles can be detected visually.

The use of gold particles in the immunochromatographic assay is a preferred embodiment because the particles can be seen directly without the need, for example, of a chromogenic enzyme reaction which can be employed in immunochromatographic assays in accordance with this invention. In the case of gold particles, the presence of analyte in the fecal sample is detected by the formation of a red to purple region within the region of the strip that contains immobilized antibody. If no analyte is present, then no color is detected. Advantageous devices and methods for performing particle assisted assays and fecal sample assays are provided in co-pending application Serial Nos. 08/577,108 and 08/577,128, both filed December 22, 1995 (identified as Foley & Lardner Docket Nos. 73294/103 "Particle Assisted Immunoassay" and 73294/108 "Device for Collecting and Testing Samples") , both of which are herein expressly incorporated by reference in their entireties. Priority documents US Serial Nos. 08/577,623 and 08/577,127, both filed December 22, 1995 also are herein expressly incorporated by reference in their entireties.

Referring now to the Figures, five physical embodiments in accordance with the present invention are provided.

Figure 1 depicts one embodiment of a device 102 in accordance with this invention. In this embodiment the absorbent filter and the immunochromatographic assay exist as a single unit, comprised of upper strip 104 and non-porous backing 101. In this embodiment, portion 106 serves to filter the extraction fluid and fecal sample mixture. Immunochromatographic assay portion 108 comprises reagents for detecting analyte. Antibody that binds analyte is directly and/or indirectly immobilized in region 110. Control antibody that binds mouse IgG is immobilized in region 112. Upper strip 104 is attached to non-porous backing 101 throughout its lower surface.

During operation, device 102 is placed into, or is already present in a container such as a test tube that contains an extraction fluid and fecal sample mixture.

The device 102 is positioned such that portion 106 of unit 104 contacts the mixture. The mixture enters portion 106 and filtered material passes through this portion to enter portion 108. A control line is formed at region 112 for a test, such as for cholera, that utilizes a mouse antibody reagent. Detection of analyte is completed by the formation of color in region 110 in response to the presence of analyte. Figure 2 depicts a variation of the device of Figure 1 in which absorbent pad 202 is interposed between non-porous backing strip 204 and immunochromatographic assay portion 206 comprising region 208 that has immobilized antibody that is specific for analyte. Control antibody that binds mouse IgG is immobilized in region 210. In this embodiment, immunochromatographic assay portion 206 is affixed to non-porous backing 204 along its lower surface except for the portion that overlies absorbent pad 202. Absorbent pad 202 is affixed to non-porous backing 204 along its entire lower surface.

The device of Figure 2 operates the same way as the device of Figure 1, except that absorbent pad 202 pulls aqueous fluid up the strip and through (from the top surface to the bottom surface) the region that comprises immobilized antibody.

Figure 3 depicts another variation of the Figure 1 device in which absorbent pad 302 is present above and in proximity to or in contact with a separate chromatographic immunochromatographic assay strip 304 comprising region 306 that has immobilized antibody that is specific for analyte. Control antibody that binds mouse IgG is immobilized in region 307. In this embodiment, absorbent pad 302 is affixed to non-porous backing 308 except for the portion that overlies the immunochromatographic assay strip 304. Immunochromatographic assay strip 304 is affixed to non- porous backing 308 along its entire length.

The device of Figure 3 operates the same way as the device of Figure l, except that absorbent pad 302 pulls aqueous fluid up the strip and up through (along the axis of the membrane surface) the region that comprises immobilized antibody.

Figure 4 depicts another device 402 comprised of absorbent filter 404 that physically contacts immunochromatographic assay strip 406. The immunochromatographic assay strip 406 comprises reagents for detecting analyte. An immobilized antibody reagent that is specific for analyte is present in region 408. Control antibody that binds mouse IgG is immobilized in region 409. Strip 406 is affixed to non-porous backing 410 along its entire lower surface. Absorbent filter 404 is affixed to non-porous backing 410 along its lower surface except for that part which overlies and contacts strip 406. During operation, device 402 is present in or placed into a test tube that contains an extraction fluid and fecal sample mixture. The mixture enters absorbent filter 404 and filtered fluid then enters strip 406. Detection of analyte is completed by the formation of color in region 408 in response to the presence of analyte.

Figure 5 depicts device 502 which comprises absorbent filter 504, immunochromatographic assay strip 506 and absorbent pad 508, which are affixed to non- porous backing 510. The ends of absorbent filter 504 and absorbent pad 508 that protrude towards the middle of the device can overlap on top of strip 506 or, alternatively, can abut the ends of strip 506. Absorbent filter 504, strip 506 and absorbent pad 508 are affixed to non-porous backing 510.

In this preferred embodiment, absorbent filter 504 filters the extraction fluid and fecal sample mixture. Strip 506 comprises reagents for detecting analyte. The immobilized antibody reagent that reacts with sample antigen is present in region 512 and control antibody that binds mouse IgG is immobilized in region 514.

During operation, device 502 is present in or placed into a test tube that contains an extraction fluid and fecal sample mixture such that portion 504 contacts the mixture. The mixture enters portion 504 and filtered material passes through this portion to enter strip 506. Absorbent pad 508 draws aqueous fluid up the strip and up through (along the axis of the membrane surface) the region that comprises immobilized antibody. Detection of analyte is completed by the formation of color within region 512 in response to the presence of analyte. For cholera tests and other tests that use mouse antibody, completion of the test is shown by the formation of color within region 514.

The following examples are presented by way of illustration and not by way of limitation.

EXAMPLE ONE

This example illustrates the formation of test reagent particles and the use of these particles in a cholera test.

Preparation of particles: One hundred milliliters of a 0.03% w/v selenium dioxide solution were heated to boiling. Then, 2.25 milliliters of a freshly prepared solution of 2% ascorbic acid were added to the 0.03% Se0₂ solution. The admixture was boiled until its volume evaporated to 50 milliliters and cooled to room temperature. The optical density of the cooled solution at 520 nm and 580 nm was 1.457 and 1.167 respectively. The colloidal selenium that was prepared by this treatment was centrifuged at 10000 x g for 15 minutes. The supernatant was discarded and the pellet was resuspended in 100 ml of distilled water.

A 4% solution of gold chloride was prepared by dissolving 360 mg of gold chloride (tetrachloroauric acid trihydrate) into 9 milliliters of deionized water. A 1% solution of sodium citrate was prepared by dissolving 1.0 gram of sodium citrate into 100 milliliters of deionized water. Three liters of deionized water were placed into a 4 liter beaker and brought to a boil on a hot plate. Then 7.5 milliliters of the 4% gold chloride solution were added to the boiling water. Seventy two milliliters of the 1% sodium citrate solution were added to the beaker and the solution was boiled until its volume was reduced to 2.2 liters. A colloidal gold solution waε removed from the hot plate and allowed to cool to room temperature. The cooled colloidal gold solution was filtered through a 0.2 micron cellulose acetate filter unit into a clean amber bottle. Optical densities at 520 nm and 580 nm of the resultant filtrate were 1.54 and 0.455 respectively.

A one milliliter aliquot of colloidal selenium was mixed with an equal volume of colloidal gold. The pH of this mixture was 5.0. The combined selenium-gold mixture was centrifuged at 10000 x g for 1 minute to remove any aggregated material. The pH of the supernatant was adjusted to 8.0 by the careful addition of 0.2 M potassium carbonate.

Preparation of mouse monoclonal Vibrio cholerae antibody: Purified Vibrio cholerae antibody was commercially obtained from Intelligent Monitoring Systems, (Gainesville, Fl.) The antibody was dialyzed against 0.002 M borax buffer pH 8.2, filtered through a 0.2 micron cellulose acetate filter, and diluted in the same buffer to a final concentration of 100 ug per milliliter.

Labelling of selenium-gold mixture with antibody The colloidal selenium and colloidal gold solutions were mixed in a 50/50 volume to volume ratio. The pH of the mixture was adjusted to 8.0 with 0.2M potassium carbonate. Two hundred microliters of the anti Vibrio cholerae monoclonal antibody (lOOug/ml) were added to 2.0 milliliters aliquots of the colloidal selenium-colloidal gold mixture. Then 200 microliters of 20% bovine serum albumin were added to each aliquot. Each mixture was incubated for 5 minutes at room temperature. The antibody treated mixtures were centrifuged at 16,000 x g for 5 minutes. The supernatants were removed and each pellet was resuspended in 0.02M Tris buffer pH 8.2 containing 1% bovine serum albumin. Each preparation was washed twice. After the last wash the pellet was resuspended in 100 microliters of 0.02M Tris buffer pH 8.2 containing 1% bovine serum albumin.

Application of antibody to nitrocellulose strips Non-laminated five micron nitrocellulose (Millipore Inc. , New Bedford, Mass.) was cut into 22mm x 4mm strips. Rabbit anti-ViJbrio cholerae affinity purified antibody (Louisiana State University, Baton Rouge) was diluted in 0.05M sodium borate buffer pH 8.2 to a concentration of 1.6 mg/ml. 1.5 microliters of antibody were spotted near the center of the nitrocellulose strip. The strips were dried in a vacuum desiccator.

A vinyl strip with acrylic adhesive was cut into 4m mx 70mm portions. The prepared nitrocellulose strips were affixed onto these cut vinyl strips. One absorbent paper cut to 4mm x 30mm (Type III, Gelman Sciences) was affixed to overlap the top of each nitrocelluloεe strip and one glass fiber pad (Gelman Sciences) was affixed to overlap the bottom of each nitrocellulose strip. Ten microliters of the selenium-gold antibody mixture were pipetted onto each glass fiber pad and dried in a desiccator at room temperature overnight.

Preparation of Extraction reagent Extraction reagent was prepared by adding 1% Triton X-100 (vol/vol) and 1% bovine serum albumin (wgt/vol) to 0.02M Tris buffered saline at pH 8.0. Testing of selenium-gold antibody conjugates ViJbrio cholerae 01 (ATCC strain #11628) and ViJbrio cholerae non 01 (ATCC strain #14547) were grown in alkaline peptone water overnight at 35 degrees C. An aliquot of each sample was centrifuged at 5000 x g for ten minutes. The supernatants were removed and each pellet was resuspended in a saline solution to an optical density at 650nm of 0.040 (approximately 10₈ organisms per milliliter) .

Fifty microliters of each sample were pipetted into 10mm x 75mm test tubes. One hundred microliters of extraction reagent were added to each tube. A test strip was placed in each tube such that fluid diffused up and through each nitrocellulose strip. After 10 minutes each strip was examined for the presence of color in the region where antibody had been immobilized. The results were as follows:

ATCC #11628 ATCC #14547 Vibrio cholerae 01 Vibrio cholerae nonOl

IO⁸ 10⁷ 10⁶ 10⁵ 10⁸ 10' IO⁶ 3+ 2+ 1+ N N N N

4+ very strong color development

3+ strong color development

2+ moderate color development

2+ weak color development

N no reaction

Stool specimens were obtained from the Hospital San Juan de Dios by the Ministerio de Salud Pubica y Asistencia Social, Direccion General de Services de Salud, Guatemala City, Guatamala CA. Each sample was cultured as follows: lOul added to 10ml of alkaline peptone broth and incubated at 30 degrees C overnight.

A sample of each broth was then subcultured onto TCBS

(thiosulfate, citrate, bile salts and sucrose) agar selective media and incubated at 37 degrees C for 18 hrs.

Stool samples were tested using the prepared test strips. Fifty microliters of each sample were pipetted into 10mm x 75mm test tubes. One hundred microliters of extraction reagent were added to each tube. A test strip was placed in each tube such that fluid diffused up and through each nitrocellulose strip. After 10 minutes each strip was examined for the presence of color in the region where antibody had been immobilized.

Control tests were performed on the same stool samples using a commercially available cholera test SMART lot 120 (New Horizons Diagnostics Corp. Columbia, MD) according to the manufacturer's package insert.

Test results were:

TCBS CULTURE POSITIVE FECAL SAMPLES SAMPLE DESCRIPTION STRIP CONTROL

1 liquid pos pos

2 clear pos neg

TCBS CULTURE NEGATIVE FECAL SAMPLES

3 liquid neg neg 4 liquid neg neg

5 semi liquid neg neg

6 brown, semi liquid neg neg

In each test, the strip assay result agreed with the culture assay results.

EXAMPLE TWO

This example illustrates a simplified procedure in which the extraction reagent is provided in dried form within the strip itself and a separate extraction fluid is not required. Alternative detergents were compared at various concentrations for their effects on cholera tests performed in the field.

Reagents and strips were prepared as described in Example One except that extraction reagent comprising 1.8% Tween-20, 0.05% sodium azide and 9mM Tris in water at a pH of 8.2 was dried (120 microliters) onto the sampling portion end of each strip. These strips were used, as described above, to test suspected cholera samples in Guatamala. More than two hundred wet stool samples were tested directly without adding extraction reagent and more than forty solid samples were tested after dispersing them by mixing with approximately a five fold volume of 7:10 diluted extraction reagent.

Each specimen was tested using the device of the present invention and also using a standard culture method. All samples that tested positive for cholera by the culture method also tested positive using the device of the present invention. All samples that tested negative by the culture method also tested negative with the device of the present invention. The following detergents were separately tested at in the extraction reagent:

Tween-20

Triton X-100

Igepal Zwittergent

Brij cetyl triammonium bromide polyethylene glycol

The improvement in assay performance with high Tween-20 concentration was not seen using Tween-20 concentrations of 1% or below, or above 2%. Furthermore, this effect of Tween-20 was not seen with tests performed in the laboratory on organisms prepared from broth culture.

EXAMPLE THREE

In this example, the inventive methods and device from Example Two were used in a one step Escherichia coli 0157 strip test. An affinity purified goat anti-£. coli serotype 0157 antibody was obtained and took the place of the V. cholerae antibody used in this example. Cross reactivity of this antibody preparation to other E . coli strains had been minimized through extensive adsorption using non-0157:H7 serotypes of E . coli .

A pure culture of E. coli 0157:H7 cells was grown in LB broth at 37 degrees centrigrade for 18 hours. The cells were serially diluted 10 fold into sterile fresh LB broth and then tested directly. For each test, five drops (approximately 200 microliters) of the diluted culture in a test tube were applied to a test strip and the result was interpreted after five minutes. A positive test result was obtained from samples that contained 2000 bacteria (colony forming units) .

It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and processes of this invention. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents.

Claims

What is claimed is:

1. A method for determining the presence or amount of analyte in a fecal sample comprising the steps of:

A. applying the fecal sample to an absorbent filter that is in proximity to or in contact with animmunochromatographic assay strip such that analyte present in said sample is transferred to said immunochromatographic assay strip; and

B. detecting the presence of analyte by said immunochromatographic assay strip.

2. The method of claim 1, wherein said absorbent filter comprises a dried detergent that can extract said analyte from said fecal sample upon rewetting of said detergent.

3. The method of claim 2, wherein said detergent is a polyoxyethylenesorbitan-fatty acid.

4. The method of claim 3, wherein said polyoxyethylenesorbitan-fatty acid is Tween-20 and comprises at least 0.5% of the absorbent weight.

5. The method of claim 3, wherein said polyoxyethylenesorbitan- atty acid comprises at least 1% of the absorbent weight .

6. The method of claim 1, wherein said immunochromatographic assay strip comprises a porous strip and said detecting step comprises detecting a color produced within said strip.

7. The method of claim 6, wherein said color is produced by gold particles.

8. The method of claim 6, wherein said absorbent filter and said porous strip are part of the same material .

9. The method of claim 6, wherein said absorbent filter and said porous strip are separate materials in proximity to or in contact with one another.

10. The method of claim 1, wherein said fecal sample is contacted with at least an equal volume of extraction reagent prior to applying the sample to said absorbent filter.

11. The method of claim 1, wherein said analyte is a surface antigen obtained from an enteric organism.

12. The method of claim 11, wherein said surface antigen is selected from the group consisting of Vibrio cholerae antigen, Es cheri chi a col i serotype 0157 lipopolysaccharide and Salmonella lipopolysaccharide.

13. The method of claim 1, wherein said immunochromatographic assay strip comprises :

A. a first group of particles having bound thereto a binding component capable of specifically recognizing the analyte, and

B. a second group of particles having bound thereto a binding component capable of specifically recognizing said analyte, wherein the average diameter of said particles in said first group is larger than the average diameter of the particles in said second group.

14. The method of claim 13, wherein said larger particles comprise selenium and said smaller particles comprise gold.

15. The method of claim 13, in which the total volume of smaller particles exceeds the total volume of larger particles.

16. The method of claim 15, in which the mean diameter of said smaller particles is less than 50 nm and the mean diameter of said larger particles is greater than 50 nm.

17. The method of claim 16, in which said smaller particles are gold particles.

18. The method of claim 13, wherein step B is carried out by determining the presence or amount of analyte- particle complexes as a measure of the amount of analyte in the sample by chromatographic movement through the immunochromatographic assay strip.

19. The method of claim 18, wherein at least a portion of said strip is coated with antibody.

20. A test kit for detection of analyte comprising:

A. an extraction reagent comprising at least one detergent; and

B. an absorbent filter that is in proximity to or in contact with an immunochromatographic assay strip, said immunochromatographic assay strip comprising immunoreagents for detecting said analyte.

21. The test kit of claim 20, wherein the chromatographic strip comprises:

A. gold particles coated with antibody against said analyte; and

B. antibody against said analyte in which said antibody is immobilized within a region of the strip.

22. A method for determining the presence or amount of analyte in a fecal sample comprising the steps of:

A. contacting said fecal sample with an extraction reagent comprised of at least one detergent to form a mixture; B. applying the mixture to an absorbent filter that is in proximity to or in contact with an immunochromatographic assay strip such that analyte present in said sample is transferred to said immunochromatographic assay strip; and

C. detecting the presence of analyte by said immunochromatographic assay strip.

23. The method of claim 22, wherein said immunochromatographic assay strip comprises a porous strip and said detecting step comprises detecting a color produced within said strip.

24. The method of claim 23, wherein said absorbent filter and said porous strip are part of the same material .

25. The method of claim 23, wherein said absorbent filter and said porous strip are separate materials in proximity to or in contact with one another.

26. The method of claim 1, wherein said fecal sample is contacted with at least an equal volume of extraction reagent .

27. The method of claim 1, wherein said extraction reagent comprises an aqueous solution of detergent that is present in a concentration that equals or exceeds its critical micelle concentration.

28. The method of claim 27, wherein said detergent is Tween-20 and said concentration is greater than one percent .

29. A method for determining the presence or amount of analyte in a fecal sample comprising the steps of: A. contacting said sample with at least an equal volume of extraction reagent comprising at least one detergent to form a mixture;

B. applying the mixture to an absorbent filter that is in contact with a porous immunochromatographic assay strip such that analyte present in said sample is transferred to said immunochromatographic assay strip said immunochromatographic assay strip comprising

(i) a first group of particles having bound thereto a binding component capable of specifically recognizing analyte, and

(ii) a second group of particles having bound thereto a binding component capable of specifically recognizing said analyte, wherein the average diameter of said particles in said first group is larger than the average diameter of the particles in said second group; and

C. determining the presence or amount of analyte- particle complexes to detect the amount of analyte in the sample.