GB2262986A

GB2262986A - Particle agglutination assay

Info

Publication number: GB2262986A
Application number: GB9200046A
Authority: GB
Inventors: Judith Margaret Parke; Deborah Lynne Morris; Eileen Margaret Ann Boulter
Original assignee: Pall Corp
Current assignee: Pall Corp
Priority date: 1992-01-03
Filing date: 1992-01-03
Publication date: 1993-07-07
Also published as: IT1257967B; CH684904A5; ITTO921060A1; GB9200046D0; ITTO921060A0; ES2052451A1; ES2052451B1; FR2685956A1; DE4244135A1

Description

2262936 A Method and Device for Particle Agglutination Assay The present

invention relates to a method and a device for detecting an analyte in a sample based on a complex formation reaction within the matrix of a fibrous medium. The presence or absence of a- specific analyte is determined by the presence or absence of an agglutination type reaction of a reagent attached to particles, which is specific to the analyte to be detected.

Analyte assay methods based on complex formation reactions, for example anti10 body/antigen complex formation and the immobilization of such complexes in a carrier medium have been advanced. For example, an analytical test device is disclosed in the international patent application WO 88/08534 by which a labelled reagent becomes bound in a certain zone of a porous carrier when the sought analyte is present.

is One important criteria for developing such methods and devices is the pore size and other properties of the porous medium to be employed in conjunction with the type of fluid or fluid sample to be investigated. A finer pore size of a porous medium would be appropriate for the detection of antigens or antibodies in fine fluids such as urine, while viscous samples such as serum or food samples would require a medium having a larger pore size.

It would be of advantage to develop assay methods which are less dependant upon the capability of the sample fluid to permeate through the medium. It would also be advantageous to develop a more universal method and device capable of detecting different types of analytes, for example bacteria, virus, antigen, haptans or genetic material.

An object of the present invention is to provide a simple and fast method of 3o detecting analyte in a sample. A further object of the invention is to provide a more universal device for detecting analytes from a range of samples especially viscose, which is reliable and inexpensive in manufacture.

Pall Corporation - 2 - P14542GB G/McG/M In accordance with the invention, a method and a device for detecting analyte in a sample are provided as defined in the claims. The present method comprises applying a sample to be tested to a sample zone of a porous medium and migrating particles having attached thereto a reagent specific to the analyte to be detected through the medium. Upon migration, the particles pass through the sample zone and if the sought analyte is present a complex formation reaction of the reagent with the analyte will occur. The complex if formed is immobilized in the sample zone. An area containing the immobilized particles is then observed in the sample zone. If the sought analyte is not present, the particles will continue to migrate through the sample zone and no distinguishable area is formed. T"he area becomes distinguishable in that the complexes either themselves make the area optically observable or through a subsequent reaction make the area become optically observable. In a preferred embodiment, the particles are provided with a color, so that the trapped complexes comprising the particles make the area observable.

An advantage of the method is that the sample fluid itself need not be highly diffusible through the porous medium, it is only necessary that the sample permeate into the small area of the sample zone. For this reason many types of sample fluids can be investigated.

Re migration of the particles is preferably performed by first applying the particles to a second zone in the porous medium spatially apart from the sample zone. A wash solution is then applied to the medium which carries the particles from this second zone throughout the porous medium. When passing the sample zone, the particles are either immobilized due to the agglutination or complex formation reaction or continue to migrate through the sample zone with the wash solution when no analyte is present.

In one embodiment, the particles are applied to the porous medium in the sample zone prior to applying the sample itself. Tle wash solution then passes through the sample zone and washes out the particles when the analyte is not present, or they are trapped after agglutination.

1 Pall Corporation - 3 - P14542GB G/McG/hl.

The present method has the advantages of being rapid and simple. The user must simply observe whether a discernable area develops. For example when colored particles are used, a colored area forms in the zone to which the sample has been applied.

The device of the present invention comprises a housing containing a porous medium. The housing is preferably of a moisture proof or nonabsorbent material such as plastic material. The housing further comprises at least one aperture which externally exposes a zone of the porous medium for the application of a sample.

The porous medium also contains particles coated with a reagent specific to the analyte to be detected. T"he particles are capable of migration through the mediurrL In a preferred embodiment, the particles are contained in a particle zone spatially distinct from the sample zone. The particles however can also be provided in the sample zone itself. It is particularly preferred that the particles have a color which is contrasts to the color of the porous medium.

The housing of the claimed device further comprises supply means in communication with a portion of the porous membrane for applying a wash solution. Tlese supply means in one embodiment comprise a second aperture in the housing which externally exposes the portion of the medium to which the wash solution can be applied. Preferably, the supply means comprise a collapsible chamber integrally formed with the housing which contains the wash solution. When an internal wall of the chamber is collapsed, the supply portion of the porous medium communicates with the wash solution.

Tle device can be simply packaged and stored in the laboratory In use it can be hand held while a sample to be tested can be applied for example with a pipette through the aperture to the sample zone.

In a further embodiment the device comprises a plurality of strips of the porous medium, each strip having particles coated with a different reagent specific to a different antibody. In this manner the same sample can be tested for the presence of different analytes, for example 'different bacteria or other microorganisms.

Pall Corporation - 4 - P14542GB G/McG/h].

Further details of the invention will become apparent in the following description of embodiments by way of reference to the drawings.

Fig. 1 is a schematic drawing indicating the principle of a method of the present invention.

Fig. 2 shows one embodiment of the device of the present invention.

Fig. 3 shows a further embodiment of the device comprising a plurality of porous strips.

The invention is broadly applicable to the detection of bacteria, antigen, antibody, virus or other microorganisms. 'Me reagent specific to the particular analyte Will normally be an antibody or antigen. Suitable monoclonal and/or polyclonal. antibod- ies or antigens for use in the present invention are commercially available.

The method of the present invention can be explained on the basis of the iHustration in Fig. 1. A sample 1, for example a bacterial sample to be tested is applied to a sample zone 2 of a porous medium 10. Depending on the viscosity of the fluid sample, it may penetrate more or less deeply into the medium 10. Complete penetration is not necessary Important is only that at least a portion of the matrix medium in the sample zone contains the sample. The particles 3 coated with a specific reagent are then migrated through the medium 10. In the example of Fig. 1, the particles would migrate in a direction from a first end 5 to a second end 2s 6 of the medium 10.

When passing through the sample zone 2, the particles 3 with the reagent attached thereto will react with the analyte of the sample 1 when present. This complex formation reaction produces complexes including the particles which are then trapped in the porous medium 10 or in the vicinity of the sample zone 2. An area develops here which is distinguishable from the remainder of the porous medium. The complexes either themselves make the area optically observable, for example by providing the particles with material which is optically active or can be made optically active, such as by fluorescent or chemiluminescent reactions. In a 1 Pall Corporation P14542GB G/McG/lil preferred embodiment, particles are employed which themselves have a color other than the porous medium. It is also possible to make the area optically observable through a subsequent reaction of the formed complexes with an additional substance, for example a fluorescent or chemiluminescent material or an enzyme which subsequently leads to fluorescence or chemiluminescence.

In the embodiment shown in Fig. 1, colored particles are applied to a second zone 4 of the porous medium 10 spatially distinct from the sample zone 2. The migration of the particles is accomplished by applying a wash solution to the porous medium, for example at the first end 5 of the porous medium or directly to the second zone 4. The colored particles are then carried with the washed solution as it wicks along the medium toward the direction of the sample zone 2. The colored particles are preferably applied to the porous medium prior to application of the sample to be tested.

is In another embodiment, it is also possible to first apply the colored particles in the sample zone 2 and subsequently apply the sample to be tested thereon. The wash solution or wash buffer is then applied so as to wash out the sample zone 2. As before, if the analyte is present agglutinated complexes form and these would be retained in the sample zone and produce a visible colored area. If the analyte is not present, the sample zone is washed out by the buffer solution leaving no color develops and indicating a negative result.

Colored particles having two different colors can also be applied to the second 25 zone 4. In this embodiment, a reagent specific to one analyte can be applied to beads of one color and a second reagent specific to a second analyte can be applied to the beads of a second color. When washed through the sample zone, different situations can arise. If only one of the analytes is present in the sample, only the beads with the color associated to that analyte will be trapped and produce an observable colored area. It is also possible that both analytes are present in which case both colors of beads would be trapped in the sample zone.

Also shown in Fig. 1 are absorption means 7 positioned in communication with the porous medium 10 at the end.6. In this embodiment, the absorption means promote the wicking action of the wash solution through the sample zone 2. In Pall Corporation - 6 - P14542GB G/McG/hi general, the absorption means, preferably an absorbing pad is located opposite the sample zone 2 from the second zone 4 containing the colored particles. In this manner, when applying the wash solution for example into or adjacent to the second zone 4, a directional flow of the wash solution arises towards the absorp- s tion means 7 and therefore through the sample zone 2.

The wash solution or wash buffer can be phosphate-buffered saline, physiologicalsaline or any appropriate physiological buffer. Phosphatebuffered saline is preferred. It has also been found suitable to include detergents such as 0.2% Triton& io X705, 0.3% Tween 20 and 0.1% TritonO X100.

The particles suitable for the present invention include latex beads also referred to as microspheres or microbeads. Beads with a diameter of 0.2 to 311m. have been used although, beads with a diameter of about 0.5 to about 0.8 Jim have been is found to be particularly suitable. The particles are available in various colors.

The particles or beads can be coated with the reagent in any suitable manner. The reagent material, proteins or molecules, can either be adsorbed to the latex bead or covalently bound by chemically coupling. The covalent coupling method is preferred. The beads are first washed by suspension in a phosphate- buffered saline (PBS) and centrifuged. The washed beads are then resuspended in PBS and an antibody containing solution of glutaraldehyde and subsequently incubated.

The porous medium 10 of the present invention will have the property that the particles can freely migrate through the pores of the material when the wash solution is applied. On the other hand, if the analyte to be detected, for example a bacteria is present in the example, the agglutinated complexes will be trapped in the matrix. The porous medium is preferably a fibrous hydrophilic: material of a polymeric fiber. In a preferred embodiment, the material is a polyolefin, prefera- bly polyethylene, polypropylene or an ethylene-lower olefin copolymer. LILDPE (linear low density polyethylene) is most preferred. Generally suitable polymers include those of olefins of 2 to 10 C atoms. More preferred is a copolymer of ethylene and 0.5 to 8 weight-% of a comonomer, more preferably 1 to 7 weight- ( 1 Pall Corporation - 7 - P14542GB G/McGffil %. Preferred comonomers are olefins with 4 to 10 C atoms, most preferably 4 to 8 C atoms. Most preferred is a copolymer of ethylene and 1-octene.

When polyethylene is used, it is made water wettable or bydrophilic by the s addition of a suitable amount of a wetting or surface active agent. Wetting agents can include a) an alkoxylated alkyl phenol along with a mixed mono-, di- and/or triglyceride or io b) a polyoxyalkylene fatty acid ester or c) a combination of (b) and any part of (a).

Such wetting agents are described in US 4 578 414. The alkoxylated alkyl phenol is preferably one where the alkyl group has 1 to 20 carbon atoms, most preferably is about 6 to about 12 carbon atoms. A polyethoxy chain is the preferred polyalkoxy chain. The mixed glyceride is preferably a glyceride of a fatty acid. The fatty acid may be saturated or unsaturated and is preferably a mixture of fatty acids having a carbon chain length in the range of about 12 to about 18 carbon atoms. Particularly preferred wetting agents include Atmer 645, a complex mixed glyceride with 20 a long chain fatty acid adduct, available from ICI America Inc. Another wetting agent is a monoester of Z-9-octadecenoic acid and 1, 2, 3 - propanetriol, available from Dow Chemical as XU 61518.10.

ne above described substances can be processed to form the porous medium of the present invention by any suitable method. The preferred fibrous bydrophilic material can be made for example from fibers, preferably from spun fiber, particularly from melt spun fibers. The most preferred form of the present material is one produced by melt blowing the above mentioned preferred ethylene copolymer combined with a wetting agent.

The formed fibrous material can be in the form of a web or mat and can be calendared. Uncalendered material has been found more effective however and is preferred.

1 4) Pall Corporation - 8 - P14542GB G/McG/H The preferred fibrous hydrophilic material according to the present invention has one or more of the following properties in combination in the following broad or preferred ranges.

s Property broad range preferred range Wetting agent content io (Olo by weight) in the polyethylene composi- 0.6 - 3.0 tion 0.5 - 5.0 particularly about 1.0 Fiber diameter (pm) 1.0 - 15 is CWST of the melt blown fiber (dynes/cm) 30 - 120 2 - 11 - 120 Pore size of the fiber material (microns) (ASTM F 316-80) 3 - 300 5 - 20 Retained water vol- ume/Volume of materi2s al (CM3 water/cm3 0.7 - 0.9 material) 0.6 - 0.95 particularly about 0.85 Material weight (g/&) 2 - 8 3 - 4 Treatment (calendared or uncalendered) - uncalendered preferred Thickness (cm) 0.01 - 0.07 0.02 0.06 particularly about 0.03 X Pall Corporation Ile reagent coated particles can be applied to the porous medium in any suitable manner. When the medium is to be stored before use, the particles can be applied in a sucrose solution. The following is an example of particle application.

P14542GB G/McG/hl s Using a Camag thin layer chromatography applicator, or by hand using a pipette,a 150 to 300 mg/ml sucrose solution is applied to the medium. In the present example, approximately 2 to 5 pl/cm of a 200 mg/ml sucrose was applied as a strip to the porous medium. The medium was then baked at 40T for 1 hour.

lo Ilie beads are applied to the strip in a 20 to 100 mg/ml sucrose solution. In the present example using a pipette, the latex beads in 50 mg/ml sucrose were applied to the sucrose strip. Approximately 5-10pl of latex beads/cm were employed. The medium was then allowed to dry in air at ambient temperature.

is As seen from this example, the particles can be applied to the surface of the porous medium or as indicated in Fig. 1 throughout the depth of the medium.

When the wash solution is applied adjacent to the fixed beads, they readily migrate out of the fixing zone and along the porous medium.

An embodiment of a device in accordance with the present invention is shown in Fig. 2. The device comprises a housing 20, preferably of a moisture proof or nonabsorbent material. The housing contains the porous medium 10 and has at least one aperture 21 which externally exposes a zone 2 of the porous medium for application of the analyte sample. The porous medium has a zone 4 cont g 2s particles coated with a reagents specific to the analyte to be detected. As mentioned above, the particle containing zone 4 may if desired be coincident with the sample zone 2.

As mentioned previously, the particles may have an additional material attached 30 thereto which makes the complex when formed optically discernable or observable, either directly or through subsequent reaction. The particles themselves can have a color which is distinct from the color of the porous medium which enables the trapped complexes to be observ&d visually. Colored particles are the preferred form.

Pall Corporation - 10 - P14542GB G/McG/hl It is also possible that the particles be supplied with at least two different colors. One colof of beads having a reagent specific to one analyte and another color of beads having a reagent specific to a second analyte. In this manner the presence or absence of two different analytes in a sample can be tested simultaneously.

lle housing further comprises supply means in communication with a portion of the porous medium for applying a wash solution. As shown in the embodiment in Fig. 2, these supply means comprise a second aperture 22 in the housing which externally exposes an end portion 5 of the porous medium. Alternatively, the supply means can comprises an absorbent pad as a feed pad (not shown in Fig. 2). The feed pad containing buffer solution can be located in the housing and placed in direct contact with the end 5 of the strip 10.

In another embodiment (not shown) the supply means can comprise a collapsible 1-5 chamber containing the wash solution formed integrally with the housing 20. 'llie chamber is formed for example surrounding the first end 5 of the porous medium. Upon external application of pressure, an internal wall of the chamber collapses allowing wash solution to contact the end portion 5 of the porous medium. In this embodiment, the device has only one external aperture.

Ile porous medium in the device of Fig. 2 is shown as an elongate strip of material. It is also possible that the device be provided with a circular disc-like piece of porous medium enclosed in a circular housing (not shown). In this embodiment, the opening 22 for applying wash solution is located at the center of 2S the circular housing to expose a central section of the fibrous medium. The bead zone 4 is arranged circumferentially about the central aperture 22 and comprises an annular region. The sample zone 2 is then displayed radially further outwardly from the bead zone 4. The outer periphery of the circular porous medium is then placed in contact with a ring shaped portion of absorbent material 7 to promote bead migration radially outward and through the sample zone.

As shown in Fig. 2 the porous medium is provided as an elongate strip having a first end 5 and a second end 6. Any suitable dimensions of the strip may be used, for example 1cm. by 6cm. Ile thickness is typically in the range of 0.02 to 0.06cm.

1 Pall Corporation - 11 - P 14542GB G/McG/W The particle zone 4 is located adjacent to the first end 5, while the sample zone is locatedat an intermediate position between the particle zone 4 and the second end 6. As also shown in Fig. 2, absorption means, particularly an absorbing pad 7 is arranged in the housing in contact with the second end 6 of the strip. 71he suction of the absorbing pad supports the directional flow of the beads through the porous medium in the longitudinal direction toward second end 6. It is also preferred that the porous medium 10 be located on a non-absorbent base portion 23 of the housing 20.

is In some instances, it is also preferred to provide the device with a positive control zone x located near or at the second end 6 of the elongate strip 10 (see Fig. 1). The control zone verifies the beads 3 have indeed passed by migration through the sample zone 2. lle control zone x can contain the bacterial analyte sought, which would lead to the complex formation reaction with the beads and thus to a positive result in the zone x. Alternatively, glutaraldebyde can be placed in zone x which would bind at least a portion of the latex beads as they migrate through.

The present device can also be provided as a multi-test device. Such an embodiment is shown in Fig. 3. The porous medium is provided as a plurality of elongate strips encased in a housing 201. The strips are positioned within the housing in a manner to avoid fluid contact among the strips. A common aperture or well 221 exposes a first end 51 of each of the elongate strips for the application of wash solution. The particle zones 41 of the strips contain colored particles, where the particles of each strip are coated with a reagent specific to a different analyte.

As in the previous embodiment, each of the plurality of strips is arranged in communication with an absorbing pad 71. lle housing is also provided with apertures 212 for exposing the sample zone of the porous medium of each strip.

As shown in Fig. 3, the multi-test device comprises fiV6 separate elongate strips of the porous medium. llus the same sample could be tested simultaneously for five different types of analyte, for example five different bacteria. Also, five different samples can be tested simultaneously for one specific analyte, for example Uste Pall Corporation - 12 - P14542GB G/McG/M The multi-test embodiment of the present device can also be provided in a circular arrangement (not shown). The porous medium is provided as a circular layer enclosed in a housing with an aperture or opening at the center for application of the wash solution. A plurality of particle zones containing colored particles are arranged in the circular layer at a plurality of angular positions at a first radial distance from center. The wash solution then migrates radially outward from the center through the particle zones. Radially outwardly from each particle zone is then a sample zone positioned at a second radial distance from the center which is larger than the first radial distance. Apertures are arranged in the housing to expose each of the sample zones. The absorption means in this circular embodiment of the device comprises an absorption material in contact with the outer periphery of the circular layer of porous medium.

The operation of the present invention is illustrated by the following example.

is EXAWLE The example demonstrates the detection of a bacteria in a sample, where the reagent is an antibody specific to the bacteria. The bacterial sample contained Escherichia Coli strain HB101. Micrococcus. Sp. was used as the bacteria in a control run. Rabbit anti-Eggli, antibody was employed, available from Dako Ltd., Buckingham hi, UK.

The antibody was attached to latex beads of 0.55 micron diameter, which were colored blue. T'he beads were obtained from Polysciences Ltd., Northhampton, UK 1ml volume of latex beads (2.5% v/w) was transferred into an Eppendorf centrifuge tube. The tube was fIlled with a phosphate buffered saline (PBS) having a pH of 7.2 and containing 150mM NaCl, 5OmM Na2BPO4 and 8mM NaH2PO4. The bead suspension was centrifuged and the supernatant discarded. The beads were then washed twice by resuspending in PBS and again centrifuging.

The pellet of latex beads was resuspended in 1ml of 8% (v/v) glutaraldehyde of electron microscope grade in PBS. The resuspended latex beads were then left to incubate over night at room temperature with gentle mixing. Following incubation, I' ' 1 -1 Pall Corporation - 13 P14542GB G/McG/h], the beads were then washed by resuspension in PBS and again centrifuging them. The washed latex beads were resuspended in 1ml PBS and 400/ig of antibody solution. The suspension was incubated for four hours at room temperature with gentle mixing.

Following incubation of the latex beads with the antibody solution, the beads were centrifuged and the supernatant discarded. The pellet of latex beads was then resuspended in 1ml of bovine serum albumin (BSA) and incubated for 10 mins. at room temperature. Latex beads were then centrifuged and the pellet of beads was io resuspended in 1ml of PBS solution. The pellet of latex beads was resuspended in Iml of solution containing 10mg/ml BSA, 0.1% NaN3 and 5% (v/v) glycerol.

The porous medium was prepared as a 1.5 cm by 5 cm strip of "hHDC' membrane material obtainable from Pall Corporation as P/N IMC 4G5. The material is is a fibrous hydrophilic medium based on an ethylene copolymer as described in US 4 578 414. An overnight culture of Escherichia Coli strain 1HE101 was diluted in PBS to give an approximate cell concentration of 106 bacteria/mL 10 pl of this bacterial suspension (approximately 104 bacteria) was applied directly to a sample zone of the hHDC strip. A number of strips were prepared in this way represent- ing the test strips. Control strips were prepared using PBS containing no bacteria, or containing Micrococcus.

After loading the hHDC strips, 101LI of the latex bead suspension containing beads coupled with the antibody was applied 1cm from the end of each strip opposite to that where the sample was applied. Absorbent pads were placed in contact with the strips immediately behind the end at which the sample was loaded. 200AI of PBS solution as the wash solution was applied to each strip adjacent to the point where the bead suspension had been loaded. The PBS then wicked along the strips in the direction of the sample zone. The presence or absence of an area of color developing after bead migration was recorded.

The test strips loaded with E.coli reacted with the 0.55 pm. blue beads coated with the anti--Ec-Q-li antibody producing-a distinctly observable zone of agglutination. A blue colored area resulting from immobilization of the complexes comprising the 4) Pall Corporation - 14 - P14542GB G/McG/W blue beads was visible in and around the zone where the E.coli sample had been applied. When control strips loaded with PBS or Micrococcus were tested, no agglutination reaction was observed, ie. no blue area developed. For the inventive run, a distinctively observable blue-colored area developed within one to three minutes. For the control runs, the beads had completely migrated through the sample zone and to the absorbing pad without complex formation within one to three minutes.

Numerous inventive runs to detect E,.coli and control runs with other bacteria were performed with no false positive or negative results. All of the positive or negative results were clearly distinguishable as being positive or negative. The test procedure illustrates that the method is rapid and simple and is capable of reliably detecting relatively small amounts of bacteria (approximately 104E.c Iiii).

W f - -11, 9.4 fl Pall Corporation - it) - P14542GB G/McG/W

Claims

C 1 a i m s

C) 1. A method of detecting an analyte comprising: a) applying a sample to be tested for said analyte to a sample zone of a porous medium, b) migrating particles having attached thereto a reagent specific to said analyte through said medium, where a complex formation reaction of the reagent with analyte will occur in said sample zone if the analyte is present, thereby immobilizing said particles, observing an area containing the complexes formed in said sample zone, wherein the complexes either themselves make the area optically observable or through a subsequent reaction have made the area optically observable.

is
2. Method of claim 1, wherein the step (b) of migrating the particles comprises applying said particles to a second zone of the porous medium spatially distinct from said sample zone, and applying a wash solution to the porous medium to carry the particles from said second zone through the porous medium, where the particles are either immobilized in the sample zone when the analyte is present or pass through the sample zone when the analyte is not present.
3. Method of claim 2, wherein the particles are applied to the porous medium prior to application of the sample to be tested.

m
4. Method of claim 1, wherein the step (b) of the migrating particles comprises applying said particles to the sample zone of the porous medium prior to applying the sample itself and applying a wash solution to the porous medium so as to pass through the sample zone, where the particles are either immobilized in the sample zone when the analyte is present or are washed out of the sample zone when the analyte is not present.
Pall Corporation. IQ - P14542GB G/McG/hl Method of one of the claims 1 to 4, wherein the area containing the complexes of -step (c) is optically observable by means of providing the particles with a color.
6. Method of claim 5, wherein the particles are provided in two colors, the reagent attached to the first colored particles being specific to a first analyte and the reagent attached to the second colored particles being specific to a second analyte.

is lo
7. Method of one of the preceding claims, wherein the step of applying the particles to the porous medium comprises applying a sucrose solution to the porous medium and thereafter drying the med' applying the reagent coated particles in a sucrose solution to the porous medium and again drying the medium.
8. Method of one of the claims 2 to 7, wherein the porous medium is provided as an elongate strip and the wash solution is applied to provide particle migration along the longitudinal direction toward one end of the strip.
9. Method of claim 8, wherein the step of migrating the particles further comprises providing an absorption means in communication with the strip of porous medium, said absorption means being located at said end of the strip and adjacent to the sample zone so as to promote flow of the wash solution 25 through the sample zone.
10. Method of one of the preceding claims wherein said analyte is a bacteria, antigen, antibody or hapten, in particular wherein the analyte is a bacteria.
11. Method of one of the preceding claims, wherein said wash solution is selected from the group of phosphate-buffered saline, physiological saline and deionized water.

I- Pall Corporation - II. P14542GB G/McG/hl
12. Method of one of the preceding claims, wherein the particles are plastic beads with bL diameter in the range of about 0.2 to about 3.0pm, in particular about 0.5 to about 0.8 pm.

s
13. Method of one of the preceding claims, wherein the porous medium comprises a fibrous hydrophilic material with an average pore size of about 5 to about 20pn.L is
14. A device for detecting an analyte comprising a housing containing a porous medium and having at least one aperture which externally exposes a sample zone of the porous medium for application of an analyte sample, the porous medium containing particles having attached thereto a reagent specific to the analyte to be detected, said particles being capable of migration through the porous medium.
15. Device of claim 14, wherein the particles are contained in a particle zone spatially distinct from the sample zone or wherein the particles are contained in the sample zone.
16. Device of claim 14 or 15, wherein the particles are provided with a color distinct from the color of the porous medium.
17. Device of one of the claims 14 to 16, wherein the housing further comprises supply means in communication with a portion of said porous medium for 25 applying a wash solution.
18. Device of claim 17, wherein the supply means comprise a second aperture M the housing which externally exposes said portion.
19. Device of claim 17, wherein the supply means comprise a collapsible chamber containing said wash solution, said portion of the porous medium communicating with said chamber when collapsed.

1 \ Pall Corporation -(4. P14542GB G/McG/lil
20. Device of one of the claims 14 to 19, wherein the porous medium is provided as at least one elongate strip and the portion for applying wash solution is located at a first end of the strip.
21. Device of claim 20, wherein the particle zone and the sample zone are arranged between the first end and a second end of said at least one strip of porous medium, the particle zone being closer to said first end.
22. Device of claim 21, wherein absorption means, particularly an absorbing pad is arranged in said housing in contact with the second end of said at least one strip.

is
23. Device of one of the claims 20 to 22, wherein the porous medium comprises a plurality of elongate strips, said housing being formed to prevent fluid contact among the strips, where the particle zones of the respective strips comprise particles having attached thereto reagents specific to respectively different analytes to be detected.
24. Device of claims 14 to 19, wherein the porous medium is provided as a 20 circular layer and the portion for applying wash solution is located at the center of the layer.
25. Device of claim 23, wherein a plurality of particle zones containing particles having reagents specific to different analytes are arranged in said circular layer 2S at a plurality of angular positions at a first radial distance from said center.

M
26. Device of claim 25, wherein a sample zone is associated with each of said plurality of particle zones, each sample zone being positioned at a second radial distance from said center, the second radial distance being greater than the first radial distance.
27. Device of claim 26, wherein absorption means are provided in said housing, the absorption means being iii contact with the outer periphery of the circular layer.

1-11 Pall Corporation - 1 - P14542GB G/McG/hl,
28. Device of one of the preceding claims, wherein the particles are plastic beads with a diameter in the range of about 0.2 to about 3.0pm, in particular about 0.5 to about 0.8 pm.

s
29. Device of one of the preceding claims, wherein the porous medium comprises fibrous hydrophilic material with an average pore size of about 5 to 20 pm and a ratio of retained water volume to volume of material of about 0.7 to about 0.9 cm:3 water/cm:3 material.

io
30. Device of claim 29, wherein said fibrous bydrophilic material comprises polyethylene fiber containing a wetting agent, in particular when said wetting agent is selected from the group of: a) an alkoxylated alkyl phenol along with a mixed mono-, di- and tri-glyceride or b) a polyoxyalkylene fatty acid ester or c) a combination of (b) and any part of (a).

is 4 a