FR2685956A1 - METHOD AND DEVICE FOR TESTING BY AGGLUTINATION OF PARTICLES. - Google Patents

Abstract

The device comprises a housing (20) containing a porous medium (10) and having at least one opening (21) which exposes to the exterior a sample area (2) of the porous medium for application of a sample of the. agent to be detected, the porous medium containing particles to which is attached a reagent specific for the agent to be detected, these particles being capable of migration through the porous medium. The particles are contained in a particle area (4) geometrically distinct from the sample area, or are contained within the sample area. They have a different color from the color of the porous medium.

Description

Method and device for particle agglutination testing The present

  The invention relates to a method and a device for detecting an agent to be recognized by analysis (hereinafter simply referred to as "agent") in

  a sample, based on a training reaction

  tion of a complex within the matrix of a medium

  fibrous The presence or absence of a specific agent

  fique is determined by the presence or absence of an agglutination-type reaction of a reagent attached to particles, which is specific to

the agent to be detected.

  Agent assay methods have already been proposed based on complex formation reactions, for example the formation of an antibody / antigen complex and the immobilization of these complexes in a support medium. For example, an analytical test device is described in international patent application WO-A-88/08534, where a labeled reagent becomes bound to a porous support in a certain

  area thereof when the agent sought is pre-

smells.

  One of the important criteria, when you

  The scope of such methods and devices, is the pore size and various other properties of the porous medium to be used in conjunction with the type of fluid or fluid sample to be studied. A finer pore size of the porous medium would be suitable for

  detection of antigens or antibodies in fluids

  purposes such as urine, while

  viscous lons such as serum or food samples would require a medium with

a larger pore size.

  It would be interesting to be able to develop test methods which are less dependent on the ability of the sample fluid to pass through the medium. It would also be interesting to be able to

  have a more uniform process and device

  Versels, capable of detecting different types of agents, for example bacteria, viruses,

  antigens, haptens or genetic material.

  One of the aims of the present invention is to pro-

  set up a simple and quick method to detect a

  agent in a sample Another purpose of the pre-

  sente invention is to provide a more universal device for detecting agents in a

  varied range of samples, especially vis-

  which is reliable and inexpensive to manufacture.

  The present invention provides a method and a

  device for detecting an agent in a sample

  The method comprises the steps of applying a test sample to a sample area of a porous medium and migrating particles to which is attached a specific reagent to the agent to be detected through the

  medium During migration, the particles pass through

  feels the sample area and, if the agent sought

  is present, there will be a reaction for-

  mation of a complex between the reagent and the agent The complex, if it forms one, is immobilized in the

  sample area A surface containing the parts

  immobilized cules is then observed in the area

  If the agent sought is not pre-

  feels, the particles will continue to migrate through

  towards the sample area and there will be no surface which can be distinguished It is possible to make the surface distinguishable by the fact that the complexes themselves make the surface optically observable, or else they make it

  optically observable by a subsequent reaction.

  In a preferred embodiment, the particles are given a color, so that the trapped complexes comprising the particles make the surface observable. One of the advantages of the method is due to the fact that the sample fluid, as such, does not need to have a very great ability to diffuse through the porous medium; it is simply necessary that the sample can pass through the small area of the sample area. Therefore, there are many types of

sample fluids.

  The migration of the particles is preferably carried out

  first by applying the particles to a

  second zone of the porous medium, geometrically

  tincture of the sample zone A washing solution is then applied to the medium, which entrains the particles out of this second zone over the entire extent of the porous medium When they pass at the place of the sample zone, the particles are immobilized due to agglutination or the complexing reaction, or

  continue to migrate through the trading area

  wash with washing solution when no agent

is not present.

  In one form of implementation, the par-

  the particles are applied to the porous medium in the sample area before applying the sample itself. The washing solution then passes through the

  sample area and wash away particles

  cules when the agent is not present, or otherwise

traps them after agglutination.

  The present process has the advantages of being quick and simple The user can be satisfied

  to observe whether or not a surface is developing

  traceable For example, when using par-

  colored tiles, a colored surface forms in

  the area where the sample was applied.

  The device of the present invention comprises a housing containing a porous medium The housing is preferably made of a moisture-resistant or non-absorbent material, such as a plastic material The housing further comprises at least one opening which exposes to the outside an area of the porous medium for the application of a sample The porous medium also contains particles coated with a

  reagent specific to the agent to be detected

  cules are capable of migration through the medium In a preferred embodiment, the

  particles are contained in a particle area

  the geometrically distinct from the sample area-

  lon, although the particles may be in the

  Sample area proper Very advantageous

  the particles have a contrasting color

with the color of the porous medium.

  The housing of the device of the invention further comprises communication supply means

  with part of the porous membrane, for the app-

  plication of a washing solution These means of

  the supply comprises, in one of the embodiments, a second opening formed in the housing, which exposes to the outside the part of the medium to which the washing solution can be applied. Preferably, the supply means comprise a chamber capable of 'crushing,

  formed in one piece with the housing and which

  holds the washing solution When crushing a

  inner wall of this chamber, the feeding part

  tation of the porous medium communicates with the solution

of washing.

  The device can be packaged and stored in the laboratory in a simple way. For use, it can be held by hand, the sample to be tested can be applied by opening the zone.

  sample for example with a pipette.

  In another embodiment, the device comprises a plurality of strips of porous medium, each strip having particles coated with a

  different reagent specific for a different antibody

  rent In this way, we can test for the same sample the presence of different agents, by

  example different bacteria or other microorganisms

  nisms. Other details of the invention will appear on

  reading of the description below, made in reference to

Refer to the accompanying drawings.

  Figure 1 is a schematic drawing illustrating the basic principle of a method according to the present invention. Figure 2 shows an embodiment of the

  device of the present invention.

  Figure 3 shows another embodiment of the

  device, comprising a plurality of bands for

  reuses. The invention is very broadly applicable to the detection of bacteria, antigens, antibodies, viruses or other microorganisms. The reagent specific to the particular agent will normally be an antibody or an antigen. trade

  monoclonal and / or poly-

  clonal usable with the present invention.

  The process of the present invention can be ex-

  plicated using the illustration of Figure 1 A sample 1, for example a bacterial sample to be tested, is applied to a sample area 2 of a porous medium 10 Depending on the viscosity of the fluid sample, that -this penetrates more or less

  deep in the middle 10

  full is not necessary; it is only important

  as long as at least part of the middle matrix

  in the sample area contains the sample.

  Particles 3 coated with a specific reagent

  then migrate through the medium 10 In the example

  ple of Figure 1, the particles would migrate in a direction from a first end 5 to

  a second end 6 of the medium 10.

  When they pass through sample area 2,

  particles 3, with the reagent which is rat-

  stained, will react with the agent of sample 1 when this agent is present This reaction of formation of a complex produces complexes including the particles, which are then trapped in the porous medium 10 or in the vicinity of the zone of sample 2 A surface then develops at this location, which can be distinguished from the rest of the porous medium. The complexes themselves constitute the optically observable surface, for example by providing particles with a material which is optically active, or else they can be made optically active, for example by fluorescence or chemiluminescence reactions. In a preferred embodiment, particles which by themselves have a color other than that of the porous medium are used. It is also possible to make the optically observable surface by subsequent reaction of the complexes formed with an additional substance, for example a fluorescent or chemical material oluminescent or an enzyme that

  then generates fluorescence or chemilumi-

nescence.

  In the embodiment illustrated figu-

  re 1, we apply colored particles to a se-

  conde zone 4 of the geometrically porous porous medium

  tincture of the sample area 2 The migration of the particles is carried out by applying a solution

  washing in a porous medium, for example at the first

  first end 5 of the porous medium or directly to the second zone 4 The colored particles are then removed with the washing solution when the latter, by wicking effect, moves along the medium in the direction of the sample zone 2 The colored particles are preferably applied to the porous medium before application of the sample to be tested. In another form of implementation, it is

  also possible to apply first the

  colored particles in the sample area 2, then apply the test sample to this same area The washing solution or washing buffer is then applied so as to completely wash the sample area 2 As above, if

  the agent is present it forms agglomerated complexes

  which will be retained in the sample area

  lon and produce a visible colored surface If the agent is not present, the sample area is

  completely washed with the buffer solution, do not leave

  sant no color developing and thus indicating

a negative result.

  We can also apply to the second zone 4

  colored particles of two different colors

  In this embodiment, a reagent specific for a first agent can be applied to beads of a first color and a second reagent specific for a second agent to beads of a first color.

  second color When washing the sample area

  There are several situations that can arise If

  only one of the agents is present in the sample

  furrow, only the balls having the associated color

  ciée to this agent will be trapped and will produce a

  observable colored surface It is also possible

  ble that the two agents are present, in which case the two colors of beads will be trapped in the sample area FIG. 1 also shows absorption means 7 placed so as to communicate with the porous medium 10 at the end 6 In this form of implementation, the means

  absorption reinforce the wicking effect of the solu-

  washing through the sample area 2 In general, the absorption means, which preferably consist of an absorbent pad, are located opposite the sample area 2 with respect to the second zone 4 containing the colored particles In this way, when the washing solution is applied, for example in the second zone

  4 or in the vicinity thereof, a direct flow

  of the washing solution is established in the direction

  tion of the absorption means 7 and therefore through

to sample area 2.

  The washing solution or washing buffer can

  be a phosphate buffered saline

  a physiological saline solution or any other

  suitable physiological buffer Preference is given to using

  contains a phosphate buffered saline solution

  We also found that we could in-

  appropriately closes detergents such as Triton (trademark) X 705 at 0.2%, Tween 20 at

0.3% and Triton X 100 at 0.1%.

  Among the particles which are suitable for the present invention are latex beads, also

  designated "microspheres" or "microbeads" We used

  beads of 0.2 to 3 µm in diameter, although beads of about 0.5 to about 0.8 µm in diameter have been found to be particularly suitable These particles are

available in various colors.

  The particles or beads can be coated with the reagent in any suitable manner. The reactive material, formed of proteins or molecules, can either be adsorbed on the latex bead or covalently bonded by chemical coupling.

  covalent coupling is used preferentially-

  the The beads are first washed by suspension in a saline solution buffered with

  phosphates (PBS: Phosphate-Buffered Saline) and cen-

  The washed beads are then put back in

  suspension in PBS and a solution of glutaral-

  dehyde containing the antibody, which is incubated

after.

  The porous medium 10 of the present invention pre-

  will feel the property that particles can freely migrate through the pores of the material

  when applying the washing solution.

  On the other hand, if the agent to be detected, for example a

  bacteria, is present in the sample, the com-

  agglutinated plexes will be trapped in the matrix.

  The porous medium is preferably a hydrophilic fibrous material of a polymeric fiber. In a preferred embodiment, the material is a

  polyolefin, preferably polyethylene, poly-

  propylene or a lower ethylene-olefin copolymer

  A LLDPE (Linear Low Density Poly Ethylene) is preferred among all of the polymers which, generally

  rale, agree, we find those of olefins

  taking from 2 to 10 carbon atoms Very preferen-

  tially, it is an ethylene copolymer and from 0.5 to 8% by weight, preferably from 1 to 7% by weight.

  weight, of a comonomer The preferred comonomers

  tiels are olefins with 4 to 10 carbon atoms

  bone, very preferably 4 to 8 carbon atoms

  bone The most preferred copolymer is a copo-

  lymer of ethylene and 1-octene.

  When polyethylene is used, it is made wettable with water or hydrophilic by the addition of a

  appropriate amount of wetting or tensing product

  wetting agent Wetting agents may include

dre:

  a) an alkoxylated alkylphenol, with a monoglyc-

  wrinkle, diglyceride and / or triglyceride in me-

  diaper, or b) a polyoxyalkylene fatty acid ester, or

  c) a combination of (b) and some fraction

conch of (a).

  Such wetting products are described in the

  US-A-4,578,414 The alkoxylated alkyl phenol is pre-

  ference one of those in which the alkyl group has from 1 to 20 carbon atoms, very preferably about 6 to about 12 carbon atoms A chain

  polyethoxy is the preferred polyalkoxy chain.

  The mixed glyceride is preferably a glyceride of a fatty acid The fatty acid can be saturated or unsaturated and it is preferably a mixture

  fatty acids with a chain length of car-

  il bonée in the range from about 12 to about 18 carbon atoms Among the particularly preferred wetting products is Atmer 645, which is a complex mixed glyceride with adduction of a long chain fatty acid, available from ICI America, Inc Another product

  wetting agent is a monoester of Z-9-octadecenoic acid

  that and 1,2,3-propanetriol, available from

  Dow Chemical under the reference XU 61518 10.

  The substances described above can be treated in any suitable way to form the porous medium of the present invention. The preferred hydrophilic fibrous material can be produced.

  for example from fibers, preferably from

  shooting of spun fibers, in particular molten spun fibers The most preferred form of the material of the present invention is that produced

  by meltblowing of the preferred ethylene copolymer

  above mentioned with a wet product

lant.

  The fibrous material thus formed can be in the form of a fabric or a tampon and can be calendered. It has however been observed that a material not

  calendered was more effective, so this is the last

  deny material that will be used preferably.

  The preferred hydrophilic fibrous material according to the present invention has one or more of the following properties taken in combination with the

  wide and preferential ranges mentioned above

under.

  Property Wide range Preferred range Content of wetting product in the polyethylene composition (% by weight) Fiber diameter (pm)

0.5 to 5.0

1.0 to 15

0.6 to 3.0,

in part approx 1.0

2 to 11

  CWST of melt blown fibers (x 10-5 N / cm) Pore size of the fiber material, according to ASTM F 316-80 (pm) Volume of water retained / volume of material

0.6 to 0.95

0.7 to 0.9,

  in part approx 0.85 Specific weight of the material (g / m 2) lg / tiled foot Treatment (calendered or not) preferably not calendered Thickness (cm)

0.01 to 0.07

0.02 to 0.06,

  in part approx 0.03 The particles coated with reagent can be

  applied to the porous medium in any suitable way

  required When the medium must be kept before use, the particles can be applied

to 120

to 120

3 to 300

  at 20 to 90 l 2 to 8 l at 40 l 3 to 4 l

  in a sucrose solution We will give below

  under an example of particle application.

  A sucrose solution is applied to the medium.

  at 300 mg / ml using a chromatographic applicator

  Camag thin-layer graphic, or by hand using a pipette In this example, about 2 to Jl / cm of a sucrose solution at 200 mg / ml was applied in the form of a strip in the porous medium.

  medium was then baked at 400 C for 1 hour.

  The beads are applied to the strip in a sucrose solution at 20 to 100 mg / ml. In the example where a pipette was used, the latex beads in sucrose at 50 mg / ml were applied to the sucrose strip. ml Approximately 5-10 g of latex beads / cm were used The medium was then left

  air dry at room temperature.

  As can be seen from this example, the particles can be applied to the surface of the porous medium or, as illustrated in FIG. 1, over the entire depth of the medium. When the washing solution is applied in the vicinity of the fixed balls, it migrates easily outside the attachment area and

along the porous medium.

  In FIG. 2, an embodiment has been illustrated.

  tion of a device according to the present invention The device comprises a housing 20, preferably in

  a moisture-resistant or non-absorbent material.

  The housing contains the porous medium 10 and has at least one opening 21 which exposes to the outside

  zone 2 of the porous medium for application of the

  sample to be analyzed The porous medium has a

  zone 4 containing particles coated with a reagent

  tif specific to the agent to be detected As indicated above, the area containing the particles 4 can,

  if desired, coincide with the sample area

ion 2.

* As previously mentioned, the particu-

  can wear additional material that

  is fixed and which makes, when formed, the com-

  optically discernible or observable plex, either

  directly either as a result of a subsequent reaction

  quente The particles can possess by them-

  same color distinct from the color of the porous medium, which makes it possible to visually observe the

  trapped complexes These colored particles are

kill the preferential form.

  It is also possible to plan for the par-

  at least two different colors, one

  ball colors with a specific reagent

  that of one of the agents and the other ball color

  having a specific reagent of a second agent.

  In this way, we can simultaneously test in a sample the presence or absence of two

different agents.

  The housing also includes means for supplying

  tation in communication with a part of the porous medium, making it possible to apply a washing solution As illustrated in the embodiment of FIG. 2, these supply means comprise a second opening 22 formed in the housing, which exposes to the exterior an end portion 5 of the

  porous medium As a variant, the means of feeding

  tion may include an absorbent pad such as a feed pad (not shown in Figure 2) The feed pad containing a buffer solution may be located in the housing and placed in direct contact with the end 5 of the

band 10.

  In another embodiment (not shown

  sensed), the supply means may include a chamber susceptible to crushing containing the washing solution, formed in one piece with the housing 20 The chamber is configured for example so as to surround the first end 5 of the porous medium the effect of the external application of pressure, an internal wall of the chamber crashes,

  allowing the washing solution to come into con-

  tact with the end portion 5 of the porous medium.

  In this embodiment, the device does not include

  door that an opening to the outside.

  The porous medium of the device of FIG. 2 has been illustrated in the form of an elongated strip of material. It is also possible to provide for

  this device a circular piece in the form of dis-

  that porous medium enclosed in a circular housing

  area (not shown) In such an embodiment

  sation, the opening 22 of application of the solution

  in the center of the circular housing

  laire, so as to expose a central part of the fibrous medium Zone 4 containing the beads is then configured circumferentially around

  the central opening 22 and includes an annular region

  laire The sample zone 2 is then presented radially even more to the outside, in a radial direction, relative to the zone of the balls 4 The external periphery of the circular porous medium is then placed in contact with a part of absorbent material in the form ring 7 in order to play the role of promoter of the migration of the balls radially

  outward through the sample area

lon 2.

  As illustrated in FIG. 2, the porous medium is in the form of an elongated strip with a first end 5 and a second end 6 Any dimension suitable for the strip can be used, for example 1 cm by 6 cm The thickness is

  typically between 0.02 and 0.06 cm.

  Zone 4 of the particles is located adjacent to the

  first end 5, while the sample area

  it is located in an intermediate position between

  the particle area 4 and a second end 6.

  As can also be seen in Figure 2,

  absorption means, in particular an absorption pad

  bant 7, are placed in the housing in contact with the second end 6 of the strip The suction of the absorbent pad assists in the directional flow of the balls through the porous medium in the longitudinal direction towards the second end 6 The medium

  porous 10 is located here again preferably

  on a non-absorbent base part 23 of the

housing 20.

  In certain situations, it may be advantageous to provide the device with a control zone

  positive x located near the second end

  mite 6 of the elongated strip 10, or at the place of

  this end (see figure 1) The control zone per-

  checks that the beads 3 have effectively migrated through the sample area 3 The control area x may contain the desired bacterial agent, which will produce the reaction for forming a complex with the beads and therefore indicate a positive result in zone x As a variant, glutaraldehyde can be placed in zone x, which would then bind at least to part of the latex beads when they pass through

migration this area.

  The device of the present invention can also be produced in the form of a device for multiple tests. Such an embodiment is illustrated in FIG. 3. The porous medium is in the form of a plurality of elongated strips.

  enclosed in a housing 201 The strips are placed inside the housing so as to avoid

  any fluid contact between the bands An open

  ture or common well 221 exhibits a first

  mite 51 from each of the elongated bands for the ap-

  washing solution plication Particle areas 41 of the strips contain particles

  colored, the particles of each strip being coated

  killed with a specific reagent from a different agent.

  As in the previous embodiment, each

  one of the various strips is arranged so as to be in communication with an absorbent pad 71 The housing is also provided with an opening 212 for exposing the sample area of the porous medium of each strip As illustrated in FIG. 3, the device of multiple tests has five separate elongated strips of the porous medium. The same sample can thus be tested simultaneously in order to detect five different types of agent, by

  example five different bacteria We can also

  ment test the presence of the same particular agent,

  for example Listeria, in five different samples

  rents. The multi-test embodiment of the present invention can also be configured in circular form (not shown) The medium

  porous is in the form of a circular layer

  lair enclosed in a housing, with an opening in the center for the application of the solution

  washing A plurality of zones of particles contained

  colored particles are arranged in the circular layer in a plurality of positions

  angular, at a first radial distance from the center

  tre The washing solution then migrates radially outwards from the center, crossing the

  particle areas A sample area is available

  laid radially, outside each zone of particles, at a second radial distance from the center which is greater than the first radial distance. Openings are formed in the housing to

  to expose each of the sample areas The mo-

  absorption yen of this circ

  device ring include an ab-

  sorption in contact with the outer periphery of the

circular layer of porous medium.

  The example below will illustrate the

  implementation of the present invention.

  Example This example illustrates the detection of a bacterium in a sample, the reagent being an antibody specific for the bacterium The bacterial sample contained a strain HB 101 of Escherichia Coli Pour

  a control test, the Micrococ- bacteria were used

  cus Sp We used anti-E Coli antibodies

  rabbit, available from Dako Ltd, Buckinghams-

hire, United Kingdom.

  The antibody was bound to latex beads of

  0.55 µm in diameter, which were colored blue.

  The beads were obtained from Polysciences Ltd, Northhampton, United Kingdom A volume of 1 ml of latex beads (2.5% by weight) at 2.5% by volume a

  transferred to an Eppen- centrifuge tube

  dorf The tube was filled with tamine saline

  phosphated (PBS) with a p H of 7.2 and containing 150 m M of Na Cl, 50 m M of Na 2 HPO 4 and 8 m M of Na H 2 PO 4 The suspension of beads was centrifuged

  and the supernatant has been discarded The beads have been

  immediately washed twice, resuspending them

  sion in PBS and centrifuging them again.

  The granule of latex beads has been refilled

  board in 1 ml of 8% glutaraldehyde (in volu-

  me) of "electron microscopy" quality in

  PBS Latex beads re-added

  pension were then incubated overnight at

  room temperature, with moderate agitation.

  After incubation, the beads were washed by sus-

  again in PBS and centrifugation again The washed latex beads were resuspended in 1 ml of PBS and 400 Fg of solution

  antibody The suspension was incubated for

  for 4 hours at room temperature, with

moderate agitation.

  After incubation of the latex beads with the solution

  antibody, the beads were centrifuged and the supernatant was discarded The latex bead granule was then resuspended in 1 ml of bovine serum albumin and incubated for 10 min at room temperature The latex beads were was then centrifuged and the bead granule was

  resuspended in 1 ml PBS solution The gra-

  no latex beads were resuspended

  in 1 ml of solution containing 10 mg / ml of serumal-

  bovine bumine, 0.1% Na N 3 and 5% (by volume) of glycerol. The porous medium was prepared in the form of a 1.5 cm by 5 cm strip of membrane material "h HDC" available from Pall Corporation under the reference P / N HHDC 4 G 5 The material is a hydrophilic fibrous medium based on ethylene copolymer, as described in US-A-4,578,414 An overnight culture was diluted of HB 101 strain from Escherichia Coli

  in PBS to give a cell concentration

  approximate area of 106 bacteria / ml We applied

  that directly 10 ml of this bacterial suspension

  bot (approximately 104 bacteria) to a sample area of the HDC h band A number of bands were prepared in this manner, which represented the test bands Control bands were prepared using PBS containing no

  no bacteria, or containing Micrococcus.

  After having thus loaded the HDC bands, 10 μl of the suspension of latex beads, containing beads coupled to the antibody, were applied 1 cm from the end of each strip opposite to that where the sample had been applied We have placed

  absorbent pads in contact with the immersion bands

  diat behind the end at which the sample

  tillon was loaded 200 ml of PBS solution was applied as washing solution to each strip in the vicinity of the point where the suspension of the beads had been loaded The PBS then moved by wicking along the strips in the direction of the area

  The presence or absence of

  sence of a colored surface developing after

migration of the beads.

  Test strips loaded by E Coli reacted

  with 0.55 gm blue beads coated with anti

  anti-E Coli body, thus producing an ag-

  glutination distinctly observable An over-

  blue colored side, resulting from the immobilization of the complexes comprising the blue beads, was visible in the area where the sample of E Coli

  had been applied, as well as around this area.

  When testing control strips loaded with

  PBS or by Micrococcus, no agglutina-

  tion was observed, that is to say that no sur-

  blue side has only developed For the test according to

  the teachings of the invention, a colo-

  rée en bleu, distinctly observable, developed in 1 to 3 min. For the control tests, the beads had completely migrated through the sample zone and up to the absorbent pad without

  formation of a complex in 1 to 3 min.

  Numerous tests according to the teachings of the invention were carried out to detect E Coli, with control tests and other bacteria, without giving any false positive or false negative.

  positive or negative results could be clear-

  Mentioned as positive or negative The test procedure clearly illustrates that this process is quick and simple, and that it is capable

  reliably detect relative quantities

  slightly weak bacteria (about 104 E Coli).

Claims (26)

  1 A method for detecting an agent by analysis,   this process being characterized by the stages consisting   both to: a) apply to a sample area (2) of a porous medium (10) a sample (1) for which the presence of the agent is to be tested,   b) migrate through this environment   cells (3) to which a reagent is attached   agent specific, a formative reaction   tion of a reagent complex with the agent occurring in the sample area if the agent is present, so as to immobilize said particles, and c) observe a surface containing the complexes   formed in the sample area, the   plexes making the surface optically observa-   ble either by themselves or under the effect of a subsequent reaction.   2 The method of claim 1, wherein step (b) of migrating particles comprises: applying these particles to a second   zone (4) of the porous medium, geometrically   tincture of the sample area, and applying a wash solution to the   porous medium, in order to transport the particles   them through the porous medium from the second zone, the particles being immobilized in the sample zone when the agent is   present or crossing the sample area   lon when the agent is not present.   3 The method of claim 2, wherein the particles are applied to the porous medium   before application of the test sample.   4 The method of claim 1, wherein step (b) of migrating the particles comprises: applying these particles to the area   sample of the porous medium before applying   tion of the actual sample, and the application of a washing solution to the porous medium so that it can pass through the   sample area, the particles being immobile   biliized in the sample area when the agent is present or being washed away completely from the sample area when the agent is not present.   5 The method of one of claims 1 to 4,   in which the surface containing the complexes of step (c) is made optically observable by a color given to the particles.   6 The process of claim 5, in which the particles are present in two colors, the reagent linked to the first colored particles being specific for a first agent and the reagent linked to the second colored particles being specific for a second agent.   7 The method of one of the preceding claims   teeth, in which the step of applying the particles to the porous medium comprises: applying a sucrose solution to the porous medium, then drying the medium, and applying the reagent-coated particles in a sucrose solution in the porous medium and then drying the medium again.   8 The method of one of claims 2 to 7,   in which the porous medium (10) is in the form of an elongated strip and the washing solution is applied so as to ensure the migration of the particles in the longitudinal direction towards   one of the ends (6) of the strip.   9 The method of claim 8, wherein the step of migrating the particles further comprises communicating absorbent means (7) with the strip of porous medium, these absorption means being located at said end   (6) of the strip and in the vicinity of the exchange zone   till to act as a promoter of the flow of the wash solution through the sample area.   The method of one of the preceding claims   teeth, wherein said agent is a bacterium, a   antigen, antibody or hapten, any particular a bacteria.   11 The method of one of the preceding claims   teeth, becoming dependent on one of the claims   tions 2 or 4, in which the washing solution is chosen from the group comprising phosphate buffered saline solutions, physiological solutions saline and deionized water.   12 The method of one of the preceding claims   teeth, wherein the particles are plastic beads having a diameter between about 0.2 to about 3.0 µm, in particular about 0.5 to about 0.8 gm.   13 The method of one of the preceding claims   teeth, wherein the porous medium comprises a hydrophilic fibrous material having a dimension   average pore of about 5 to about 20 gm.   14 A device to detect an agent by ana-   lysis, characterized in that it comprises a housing (20) containing a porous medium (10) and comprising at least one opening (21) which exposes to the outside a sample area (2) of the porous medium for application of '' a sample of the agent (1), the porous medium containing particles to which   is attached a specific reagent of the agent to be de-   tecter, these particles being liable to migrate tion through the porous medium.   The device of claim 14, in which the particles are contained in a particle zone (4) geometrically distinct from the sample zone, or in which the particles   are contained in the sample area.   16 The device of one of claims 14   or 15, in which the particles have a   their different from the color of the porous medium.   17 The device of one of claims 14 to   16, wherein the housing further includes   supply means in communication with a par-   tie of porous medium, for the application of a solution washing.   18 The device of claim 17, wherein the supply means (20) comprise a second opening (22) in the housing, which   exhibits said part outside.   19 The device of claim 17, wherein the supply means comprise a   crushable chamber containing the solu-   washing part, said part of the porous medium comprises   providing with this room when the latter is overwritten.   20 The device of one of claims 14 to   19, in which the porous medium is in the form of at least one elongated strip and the part for applying the washing solution is   located at a first end (5) of the strip.   21 The device of claim 20, wherein the particle zone (4) and the sample zone (2) are arranged between the first end (5) and a second end (6) of the strip or strips of porous medium , the closest area   of said first end being the area of the   ticles. 22 The device of claim 21, in which absorption means (7), in particular   an absorption pad, are arranged in the box   tier in contact with the second end (6) of the band or bands.   23 The device of one of claims 20 to   22, in which the porous medium comprises a plurality of   elity of elongated strips (101), the housing (201) being shaped so as to prevent fluid contact between the strips, the areas of the particles (41) of the respective strips comprising particles   to which are attached specific reagents   of the respective agents to be detected.   24 The device of claims 14 to 19, in   which the porous medium is in the form of a   circular layer, the part intended for the applica-   tion of the washing solution being located in the center of this layer.   25 The device of claim 23, in   which a plurality of particle areas contain   of particles with specific reagents   that different agents are available in the   circular layer in a plurality of positions an-   gular at a first radial distance from the center.   The device of claim 25, wherein a sample area is associated with each   of said particle zones, each sample zone   spindle being disposed at a second radial distance from the center, this second radial distance being   greater than said first radial distance.   27 The device of claim 26, in which absorption means are provided in the housing, the absorption means being in contact   with the outer periphery of the circular layer laire.   28 The device of one of claims 14 to   27, wherein the particles are plastic beads having a diameter in the range of from about 0.2 to about 3.0 µm, in particular about 0.5 to about 0.8 gm.   29 The device of one of claims 14 to   28, wherein the porous medium comprises a material   hydrophilic fibrous line having an average pore size of approximately 5 to 20 gm and a ratio between volume of water retained and volume of material of approximately 0.7 to about 0.9.   The device of claim 29, in which the hydrophilic fibrous material comprises a polyethylene fiber containing a wetting product, this wetting product being in particular chosen from the group comprising:   a) an alkoxylated alkylphenol, with a monoglyc-   wrinkle, diglyceride and / or triglyceride in me-   diaper, or b) a polyoxyalkylene fatty acid ester, or   c) a combination of (b) and some fraction conch of (a).