WO2008110781A1 - Lateral flow assay and apparatus - Google Patents

Lateral flow assay and apparatus Download PDF

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Publication number
WO2008110781A1
WO2008110781A1 PCT/GB2008/000832 GB2008000832W WO2008110781A1 WO 2008110781 A1 WO2008110781 A1 WO 2008110781A1 GB 2008000832 W GB2008000832 W GB 2008000832W WO 2008110781 A1 WO2008110781 A1 WO 2008110781A1
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WIPO (PCT)
Prior art keywords
reaction zone
sample
zone
marker
base
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PCT/GB2008/000832
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French (fr)
Inventor
Paul Robert Edwards
Matthew Colin Partridge
Ian William Davidson
Omer Karim
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Mediwatch Biomedical Limited.
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Publication of WO2008110781A1 publication Critical patent/WO2008110781A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
    • G01N33/541—Double or second antibody, i.e. precipitating antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids

Definitions

  • This invention relates to an assay or test method and to an apparatus for performing the method.
  • the invention is expected to find its greatest utility in the field of immunoassay, and so the following description is directed primarily to that field, but the invention is not limited to that application.
  • Immunoassay is a method of diagnosis of certain diseases and conditions in humans, including testing for prostate cancer for example.
  • the method detects the presence of an analyte within a sample of blood or other bodily fluid by binding it to an antibody, the antibody being previously bound to a marker.
  • the analyte can be bound to the markeF and the marker can be used to indicate the presence of the analyte.
  • the analyte can be an antigen, a hormone, or another constituent of blood, urine, saliva or other appropriate bodily fluid.
  • a blood sample is taken, and delivered onto a blood separation zone of the apparatus.
  • a man suffering from prostate cancer will have a particular antigen present in his blood, namely PSA or prostate specific antigen.
  • the antigen is present in the blood serum rather than m the red blood cells, and it is first necessary to separate the blood so as to remove the red blood cells which would otherwise obscure the coloured marker.
  • the blood separation zone retains the red blood cells and allows the (substantially colourless) serum to continue to a reaction zone.
  • the reaction zone comprises a fibrous mat which has been impregnated with antibodies bound to a coloured marker. In the case of prostate cancer the coloured marker is most commonly gold sol.
  • any antigen which is present in the sample binds to the antibodies and carries the antibodies and bound coloured marker out of the reaction zone and into a test zone comprising a nitrocellulose membrane.
  • the sample flows along the nitrocellulose membrane past a test line upon which further antibodies have been immobilised.
  • the antigen in the sample binds to the further antibodies and since the further antibodies are immobilised upon the membrane the antigen also becomes immobilised and thereby also immobilises the coloured marker.
  • the further antibodies are arranged in a line across the membrane and the presence (or absence) of a line of coloured marker at the end of the test provides a visual indication of the presence (or absence) of the prostate specific antigen in the blood sample.
  • a line of coloured marker can be immobilised upon the membrane (downstream of the test line) as a control for the user to check the test line against the control line.
  • an absorbent zone adapted to absorb the sample as it passes out of the test zone.
  • the absorbent zone is often termed a "wicking pad" since its action is to ensure that the sample continues to flow from the blood separation zone to the absorbent zone, i.e. to ensure that the sample flows past the test line.
  • Such a method of immunoassay in which an antigen or other suitable analyte binds to two separate antibodies, is one of the many different types of assay and immunoassay methods in current use.
  • an arrangement such as that described above in which the sample is caused to flow through a first zone in which the analyte can combine with a first set of antibodies bound to a marker, then carry the antibodies and marker to a second zone in which the analyte can further combine with immobilised antibodies, is common to the known immunoassay methods.
  • Another immunoassay method is a pregnancy test, where the analyte is human chorionic gonadotropin, which is present in a woman's urine when she is pregnant.
  • the inventors have devised a different method of performing an immunoassay or similar test, and an apparatus for performing that method.
  • the first and second zones are combined, and there is no flow of sample from the first zone to the second zone.
  • the predetermined time is established as the time taken for a measureable quantity of any analyte which is present in the sample to combine or react with the reactant.
  • the method is used in immunoassay, and the reactant is antibodies.
  • the analyte is an antigen, a hormone, or another constituent of blood, urine or other bodily fluid appropriate to the specific analyte.
  • the reactant is a first set of antibodies and a second set of antibodies.
  • the first set of antibodies is bound to a marker such as a coloured marker. The use of coloured marker will enable the method to provide a visual result, but other markers can be used, such as chemical, biochemical, radioactive etc.
  • the method may be used in sandwich immunoassay, and one, two, three or more antibodies can be used if desired or required.
  • the first set of antibodies is distinct from the second set of antibodies.
  • the first set of antibodies can carry a marker such as a coloured marker or the like, and the second set of antibodies carry no marker but are immobilised in the reaction zone.
  • the first set of antibodies and the second set of antibodies may be initially physically separated by a soluble layer for example, which soluble layer is dissolved by the sample. Accordingly, little or none of the marker is initially immobilised in the reaction zone. If there is none of the specific analyte present in the sample which can become bound to the antibodies, the marker will be displaced from the reaction zone with the sample. The presence or absence of marker remaining in the reaction zone is therefore indicative of the presence or absence of analyte within the sample.
  • the amount of marker remaining in the reaction zone in most applications will be dependent upon the quantity of analyte in the sample, so that the method can be used quantitatively as well as qualitatively.
  • a qualitative test may be sufficient for some applications, whilst a quantitative test is required for other applications.
  • a controlled volume of sample is added to the reaction zone.
  • the volume of the sample is controlled by the user, means must be provided to ensure that any excess sample does not enter the reaction zone, i.e. any excess sample is kept out of the reaction zone rather than entering and then leaving the reaction zone. If any excess sample were to enter and then leave the reaction zone the analyte therein would also leave the reaction zone, and it could carry with it some of the reactant and thereby corrupt the result.
  • the present method has the additional advantage that a blood separation zone is not required.
  • the red blood cells take no part in the reaction between the analyte and the reactant and are displaced from the reaction zone with the sample. This has particular significance when the method uses a coloured marker, as red blood cells remaining in the reaction zone could obscure any coloured marker remaining therein.
  • An apparatus for performing the method set out above comprises: a base, a spacer engaging a part of the base, and a cover material covering the spacer and the base, the spacer separating the cover material from the base, and having a predetermined shape so that it does not engage a chosen area of the base, the base, spacer and cover together defining a volume corresponding to the chosen area, a reaction zone located within the chosen area, the reaction zone being provided with a reactant, and a displacement zone located within the chosen area, the displacement zone being connected to the reaction zone.
  • the sample When using the apparatus the sample is added to the reaction zone, retained there for a predetermined period of time, and then displaced out of the reaction zone and into the displacement zone. The reaction zone is then examined to determine the presence or absence (and if appropriate also the quantity) of the product of any reaction between the reactant and the analyte.
  • the volume of the displacement zone is greater than the volume of the reaction zone. This helps to ensure that substantially all of the sample is displaced from the reaction zone, and if the sample is blood then few if any red blood cells remain in the reaction zone to obscure and corrupt a visual marker.
  • the apparatus has an application hole through which the sample is added, and the application hole is connected to the reaction zone.
  • the application hole is connected to the reaction zone by a strip of the chosen area.
  • reaction zone and the displacement zone are connected by another strip of the chosen area.
  • the cross-sectional areas of the strips of the chosen area are desirably smaller than that of the reaction zone and the displacement zone, so that there is a restriction in the flow of sample from the reaction zone to the displacement zone. Such an arrangement helps to ensure that substantially all of the sample is retained in the reaction zone until the displacement step.
  • the cover material is hydrophilic. Such a material helps to draw a sample from the application hole into the reaction zone, and then from the reaction zone into the displacement zone. Preferably, the sample moves from the reaction zone to the displacement zone by way of capillary action.
  • the spacer is a double-sided adhesive layer, the layer adhering to the base and the cover.
  • a double-sided adhesive layer adhering to the base and the cover.
  • Such a material avoids the requirement for other means to be provided to secure the spacer and cover to the base.
  • the apparatus 10 comprises a base 12, a spacer 14 and a cover material 16.
  • the base 12 has a shallow depression 20 into which the spacer 14 and cover material 16 are located.
  • the cover material substantially fills the depression 20, whereas the spacer is shaped so as to engage only certain parts of the base and cover material, and not to engage a chosen area thereof, the chosen area corresponding to the removed central portion 22 of the spacer 14.
  • the spacer 14 has a predetermined thickness, for example in the desired range 0.08 mm - 0.2 mm, although thicknesses outside of this range may be useful in certain applications. It will be understood that when the spacer 12 and cover material 14 are located in the depression 20, they together define a free volume within the depression corresponding to the removed central portion 22.
  • the free volume provides the working volume of the apparatus, and the dimensions and form of the spacer 14 which provides that working volume can be determined according to the particular application.
  • the spacer 14 is made as a layer of double-sided adhesive tape with the central portion removed, the tape adhering to the base 12 and the cover material 16.
  • the base has a well 24, and at the other end it has a reservoir 26.
  • the chosen area of the base which is defined by the removed central section 22 of the spacer 14 comprises a first channel 30, a reaction zone 32, a second channel 34, a displacement zone 36, and a third channel 40.
  • the well 24 has a cut-out 42 which provides a continuation of the depression 20 beyond the cover material 16, providing an application hole through which the sample can flow into the first channel 30.
  • the first channel 30 connects the well 24 to the reaction zone 32.
  • the second channel 34 connects the reaction zone 32 to the displacement zone 36, and the third channel 40 connects the displacement zone 36 to the reservoir 26.
  • the cover material 16 is hydrophilic (or in alternative embodiments the underside thereof is coated with a hydrophilic material), which improves the flow of the sample along the channels 30, 34 and 40.
  • a hydrophilic cover material helps to ensure the flow of sample along the channel 30 into the reaction zone 32, and helps to ensure the flow of sample along the channel 34 out of the reaction zone 32 and into the displacement zone 36.
  • the reaction zone 32 is provided with a first set of antibodies bound to a marker, and a second set of immobilised antibodies (not shown).
  • the immobilised antibodies can be located by known means.
  • the immobilised antibodies can be located in a predefined pattern such as a line, a "+" symbol, or any other chosen pattern. If necessary in some applications, the antibodies bound to the marker can be initially separated from the immobilised antibodies, for example by a soluble layer, so as to ensure that the marker does not become bound in the reaction zone inadvertently.
  • Channel 34 is of a smaller-cross-sectional area than the reaction zone 32 and the displacement zone 36, and represents a restriction on the flow of sample out of the reaction zone.
  • the channel 34 therefore helps to ensure that the sample remains in the reaction zone until the displacement step. However, it is desired that the volume of sample added to the well substantially equal the combined volume of the channel 30 and reaction zone.
  • the combined volume of the channel 30 and the reaction zone 32 is 4 ⁇ l, and so the sample volume is desirably 4 ⁇ l also. It will be understood that such a sample volume is considerably smaller than the sample volumes typically used in immunoassay methods, in which 10 ⁇ l represents the usual lower limit of sample volumes, with volumes up to 50 ⁇ l not being unusual. These volumes are necessary in prior art methods so as to ensure the required flow of sample from the first zone to the second zone, and it is a major advantage of the present invention that the absence of this required flow allows a reduction in the sample volume.
  • the present method and apparatus could be used with sample volumes lower than 4 ⁇ l in certain applications.
  • the volume of the reaction zone 32 should be maximised in relation to the volume of the channel 30, since any analyte present in the proportion of the sample which is retained within the channel 30 does not combine with the reactant.
  • the sample flows through the apparatus 10 by way of capillary action.
  • the sample volume does not exceed the combined volume of the channel 30 and the reaction zone 32 the majority of the sample will tend to remain in the reaction zone 32 until the displacement step, i.e. the sample will flow from the well 24 and into the channel 30 and the reaction zone 32 until the well 24 is empty.
  • the volume of sample added to the well 24 is therefore desirably chosen to match the combined volume of the channel 30 and reaction zone 32 so as to ensure that little or no sample flows into the channel 34 prior to the displacement step.
  • the apparatus could utilise absorbent materials, but capillary action is preferred as it is relatively easy to retain the sample within the reaction zone as above described, and subsequently to remove substantially all of the sample during the displacement step.
  • the volume of the displacement zone 36 in this embodiment is considerably greater than the volume of the reaction zone 32, and so when the sample is displaced substantially all of the sample which is not bound in the reaction zone flows along the channel 36 and into the displacement zone.
  • the reservoir 26 composes a deeper depression wfthin the depression 20, and the volume of the reservoir 26 is large in comparison to the displacement zone 36. The reservoir 26 therefore helps to ensure that all or substantially all of the unbound sample can be displacement out of the reaction zone.
  • the displacement zone can be small relative to the reaction zone.
  • the channel 34 can initially be blocked, for example by a plug of soluble material, and this will ensure that the sample cannot flow from the reaction zone to the displacement zone until the plug has dissolved. Ideally, the plug will take a chosen time to dissolve, the chosen time corresponding to the predetermined time required for the sample to remain within the reaction zone. If the channel 34 is initially blocked, then a sample volume greater than the volume of the channel 30 and the reaction zone 32 can be added to the well 24, but this is not preferred and instead it is preferred to add a controlled volume of sample as above described.
  • the base 12 is opaque and the cover material 16 is transparent, so that the apparatus is suitable for a visual examination of the reaction zone, and the marker can be a coloured marker.
  • the quantity of coloured marker which becomes bound in the reaction zone can be measured by reflectance.
  • the base is transparent and the cover material is opaque, or both are transparent in which case the quantity of a coloured marker can be measured by transmission.
  • a sample is placed into the well 24.
  • the sample flows, by capillary action, along the channel 30 and into the reaction zone 32.
  • the predetermined time during which the sample must remain within the reaction zone 32 can be determined empirically or by calculation, and will depend most significantly upon the reactant/analyte combination being utilised. Any analyte which is present in the sample will bind to the antibody and its marker, and also with the immobilised antibody, and so act to bind the marker in the reaction zone 32.
  • the apparatus can be calibrated so that the quantity of marker remaining in the reaction zone 32 (after the analyte has combined with the antibodies for the predetermined period of time), is indicative of the quantity of analyte in the sample.
  • the quantity of marker is measurable by its colour and/or its intensity in the case of coloured marker.
  • the displacement step is performed, in which water or a suitable displacement liquid is added to the well 24.
  • the volume of displacement liquid added to the well is considerably greater than the volume of the sample, so that the displacement liquid carries all (or at least substantially all) of the unbound sample, and all (or at least substantially all) of the unbound antibody and marker, through the channel 34 and into the displacement zone 36 (and if the volume of displacement liquid is sufficient also along the channel 40 and into the reservoir 26).
  • the reaction zone 32 will thereafter contain displacement liquid and bound marker, and since the marker will only be present there if it has been bound by antibodies, the quantity of marker remaining within the reaction zone 32 is representative of the quantity of analyte in the sample.
  • Much or all of the procedure may be automated, i.e. a machine may be provided to add the sample to the well 24, then add the displacement liquid after the predetermined time, and perform the reflectance (or transmission) test.
  • the marker used will be suited to the particular application. It may be a coloured marker such as gold sol for example which is typically used in prostate cancer immunoassay methods. Alternatively it may be a fluorescent marker or an enzyme marker, for example.
  • a line of coloured marker is immobilised as a control in the reaction zone and the immobilised antibodies are arranged in a perpendicular line. If no analyte is present in the sample only the control line will be seen after the test (as a "-" symbol or negative result), whereas if analyte is present both lines will be seen (as a "+" symbol or positive result).
  • the apparatus can be adapted to test for the presence of several different analytes at the same time.
  • it is advantageous to check for several analytes for example the different analytes present in response to different illegal drugs, or the different analytes present in response to certain diseases or medical conditions.
  • Variants of the device can be provided with a single well connected to separate reaction zones, each reaction zone having antibodies suited to a particular one of the various analytes.
  • each reaction zone has its own displacement zone; if multiple reaction zones were connected to a single displacement zone it would likely be difficult in practice to ensure that the sample is effectively displaced from each reaction zone, as the displacement liquid will flow along the path of least resistance which may be through only one of the reaction zones.
  • the cover 16 substantially fully closes-off the reservoir 26, i.e. little if any sample and displacement liquid which enters the reservoir 26 can leak from the device, and only air can pass out of the device as the sample moves along the device.
  • This is highly desirable for applications in which the sample may be infectious, or may otherwise present a health hazard for example.
  • This has the additional advantage that excess displacement liquid can be added to the well, the flow of displacement liquid from the well 24 and into the channel 30 stopping when the displacement zone 36 and reservoir 26 have been filled.
  • the spacer is integral with the base, perhaps being provided by a single piece of plastic or other suitable material.
  • a well or depression corresponding to the chosen area is formed in the base by etching, moulding, or other suitable process.
  • the area of the base surrounding the well or depression corresponds to the spacer of the embodiment shown in the figure, and enables a cover material to be fitted and define a volume corresponding to the chosen area.
  • a marker is not required in all applications and the presence or absence of analyte can alternatively be detected by a change in mass of the reaction zone (caused by the addition of analyte to the reactant).
  • the refractive index of a liquid within the reaction zone or one of many other physical or chemical properties of the product of a reaction between the reactant and the analyte, can be used as appropriate for the particular analyte.
  • the method and apparatus will find its greatest utility in immunoassay, but it is expected to be useful also in methods of chemical testing, organo-chemical testing and electro-chemical testing, for example.
  • the method and apparatus are expected to be useful in DNA testing, where a very large number of tests are to be undertaken on a particular sample.
  • a large number of reservoirs are filled with different gene sequences corresponding to one half of partial strands of DNA, and the sample material will combine with only those partial strands for which it has the same gene sequence. Knowing the reservoirs in which the DNA has combined with the gene sequence, and those in which no combination has occurred, allows the user to ascertain the gene sequences present in the sample DNA.
  • the large number of separate reservoirs could be replaced by a large number of reaction sites within the reaction zone, each reaction site having a respective gene sequence bound thereupon.

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Abstract

This invention relates to an assay method and to an apparatus for performing the method. The invention has particular utility in the field of immunoassay, for example sandwich immunoassay utilising two (or more) antibodies. The method comprises {i} providing a reaction zone with a reactant; {ii} adding a suitable sample to the reaction zone, by way of capillary action; {iii} retaining the sample in the reaction zone for a predetermined period of time; {iv} displacing the sample from the reaction zone; and {v} examining the reaction zone to determine the presence or absence of analyte. The apparatus comprises a base, a spacer engaging a part of the base, and a cover material covering the spacer and the base, the spacer separating the cover material from the base, and having a predetermined shape so that it does not engage a chosen area of the base, the base, spacer and cover thereby together defining a volume corresponding to the chosen area, a reaction zone located within the chosen area, the reaction zone being provided with a reactant, and a displacement zone located within the chosen area, the displacement zone being connected to the reaction zone.

Description

LATERAL FLOW ASSAY AND APPARATUS
FIELD OF THE INVENTION
This invention relates to an assay or test method and to an apparatus for performing the method. The invention is expected to find its greatest utility in the field of immunoassay, and so the following description is directed primarily to that field, but the invention is not limited to that application.
BACKGROUND OF THE INVENTION
Immunoassay is a method of diagnosis of certain diseases and conditions in humans, including testing for prostate cancer for example. The method detects the presence of an analyte within a sample of blood or other bodily fluid by binding it to an antibody, the antibody being previously bound to a marker. In this way the analyte can be bound to the markeF and the marker can be used to indicate the presence of the analyte.
The analyte can be an antigen, a hormone, or another constituent of blood, urine, saliva or other appropriate bodily fluid.
DESCRIPTION OF THE PRIOR ART
In a known immunoassay method and apparatus for prostate cancer, a blood sample is taken, and delivered onto a blood separation zone of the apparatus. It is recognised that a man suffering from prostate cancer will have a particular antigen present in his blood, namely PSA or prostate specific antigen. The antigen is present in the blood serum rather than m the red blood cells, and it is first necessary to separate the blood so as to remove the red blood cells which would otherwise obscure the coloured marker. Accordingly, the blood separation zone retains the red blood cells and allows the (substantially colourless) serum to continue to a reaction zone. The reaction zone comprises a fibrous mat which has been impregnated with antibodies bound to a coloured marker. In the case of prostate cancer the coloured marker is most commonly gold sol.
Any antigen which is present in the sample binds to the antibodies and carries the antibodies and bound coloured marker out of the reaction zone and into a test zone comprising a nitrocellulose membrane. The sample flows along the nitrocellulose membrane past a test line upon which further antibodies have been immobilised. The antigen in the sample binds to the further antibodies and since the further antibodies are immobilised upon the membrane the antigen also becomes immobilised and thereby also immobilises the coloured marker.
The further antibodies are arranged in a line across the membrane and the presence (or absence) of a line of coloured marker at the end of the test provides a visual indication of the presence (or absence) of the prostate specific antigen in the blood sample.
A line of coloured marker can be immobilised upon the membrane (downstream of the test line) as a control for the user to check the test line against the control line. Also, downstream of the nitrocellulose membrane is an absorbent zone adapted to absorb the sample as it passes out of the test zone. The absorbent zone is often termed a "wicking pad" since its action is to ensure that the sample continues to flow from the blood separation zone to the absorbent zone, i.e. to ensure that the sample flows past the test line.
Such a method of immunoassay, in which an antigen or other suitable analyte binds to two separate antibodies, is one of the many different types of assay and immunoassay methods in current use.
Also, an arrangement such as that described above in which the sample is caused to flow through a first zone in which the analyte can combine with a first set of antibodies bound to a marker, then carry the antibodies and marker to a second zone in which the analyte can further combine with immobilised antibodies, is common to the known immunoassay methods.
Another immunoassay method is a pregnancy test, where the analyte is human chorionic gonadotropin, which is present in a woman's urine when she is pregnant.
SUMMARY OF THE INVENTION
The inventors have devised a different method of performing an immunoassay or similar test, and an apparatus for performing that method.
According to the invention there is provided a method of performing a test to locate a chosen analyte comprising the following steps:
{i} providing a reaction zone with a reactant;
{ii} adding a suitable sample to the reaction zone, by way of capillary action;
{iii} retaining the sample in the reaction zone for a predetermined period of time; {iv} displacing the sample from the reaction zone; and
{v} examining the reaction zone to determine the presence or absence of analyte.
In the present invention therefore the first and second zones are combined, and there is no flow of sample from the first zone to the second zone. The predetermined time is established as the time taken for a measureable quantity of any analyte which is present in the sample to combine or react with the reactant. This has the advantage that a reduced volume of sample and/or a reduced volume of reactant can be used as compared to the known methods.
Preferably, the method is used in immunoassay, and the reactant is antibodies. The analyte is an antigen, a hormone, or another constituent of blood, urine or other bodily fluid appropriate to the specific analyte. Desirably, the reactant is a first set of antibodies and a second set of antibodies. Preferably, the first set of antibodies is bound to a marker such as a coloured marker. The use of coloured marker will enable the method to provide a visual result, but other markers can be used, such as chemical, biochemical, radioactive etc.
The method may be used in sandwich immunoassay, and one, two, three or more antibodies can be used if desired or required.
In use in sandwich immunoassay with two sets of antibodies, the first set of antibodies is distinct from the second set of antibodies. Specifically, the first set of antibodies can carry a marker such as a coloured marker or the like, and the second set of antibodies carry no marker but are immobilised in the reaction zone. If desired in a particular application the first set of antibodies and the second set of antibodies may be initially physically separated by a soluble layer for example, which soluble layer is dissolved by the sample. Accordingly, little or none of the marker is initially immobilised in the reaction zone. If there is none of the specific analyte present in the sample which can become bound to the antibodies, the marker will be displaced from the reaction zone with the sample. The presence or absence of marker remaining in the reaction zone is therefore indicative of the presence or absence of analyte within the sample.
Furthermore, the amount of marker remaining in the reaction zone in most applications will be dependent upon the quantity of analyte in the sample, so that the method can be used quantitatively as well as qualitatively. A qualitative test may be sufficient for some applications, whilst a quantitative test is required for other applications.
Preferably, a controlled volume of sample is added to the reaction zone. Unless the volume of the sample is controlled by the user, means must be provided to ensure that any excess sample does not enter the reaction zone, i.e. any excess sample is kept out of the reaction zone rather than entering and then leaving the reaction zone. If any excess sample were to enter and then leave the reaction zone the analyte therein would also leave the reaction zone, and it could carry with it some of the reactant and thereby corrupt the result.
The present method has the additional advantage that a blood separation zone is not required. Thus, in applications in which the sample is blood, the red blood cells take no part in the reaction between the analyte and the reactant and are displaced from the reaction zone with the sample. This has particular significance when the method uses a coloured marker, as red blood cells remaining in the reaction zone could obscure any coloured marker remaining therein.
An apparatus for performing the method set out above comprises: a base, a spacer engaging a part of the base, and a cover material covering the spacer and the base, the spacer separating the cover material from the base, and having a predetermined shape so that it does not engage a chosen area of the base, the base, spacer and cover together defining a volume corresponding to the chosen area, a reaction zone located within the chosen area, the reaction zone being provided with a reactant, and a displacement zone located within the chosen area, the displacement zone being connected to the reaction zone.
When using the apparatus the sample is added to the reaction zone, retained there for a predetermined period of time, and then displaced out of the reaction zone and into the displacement zone. The reaction zone is then examined to determine the presence or absence (and if appropriate also the quantity) of the product of any reaction between the reactant and the analyte.
Preferably, the volume of the displacement zone is greater than the volume of the reaction zone. This helps to ensure that substantially all of the sample is displaced from the reaction zone, and if the sample is blood then few if any red blood cells remain in the reaction zone to obscure and corrupt a visual marker. Desirably, the apparatus has an application hole through which the sample is added, and the application hole is connected to the reaction zone. Preferably, the application hole is connected to the reaction zone by a strip of the chosen area.
Preferably also, the reaction zone and the displacement zone are connected by another strip of the chosen area. The cross-sectional areas of the strips of the chosen area are desirably smaller than that of the reaction zone and the displacement zone, so that there is a restriction in the flow of sample from the reaction zone to the displacement zone. Such an arrangement helps to ensure that substantially all of the sample is retained in the reaction zone until the displacement step.
Desirably, the cover material is hydrophilic. Such a material helps to draw a sample from the application hole into the reaction zone, and then from the reaction zone into the displacement zone. Preferably, the sample moves from the reaction zone to the displacement zone by way of capillary action.
Preferably, the spacer is a double-sided adhesive layer, the layer adhering to the base and the cover. Such a material avoids the requirement for other means to be provided to secure the spacer and cover to the base.
BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS
The apparatus of the invention will now be described in more detail, by way of example, with reference to the accompanying drawing, which shows an exploded perspective view of an apparatus according to the present invention. DETAILED DESCRIPTION
The apparatus 10 comprises a base 12, a spacer 14 and a cover material 16. In this embodiment the base 12 has a shallow depression 20 into which the spacer 14 and cover material 16 are located.
The cover material substantially fills the depression 20, whereas the spacer is shaped so as to engage only certain parts of the base and cover material, and not to engage a chosen area thereof, the chosen area corresponding to the removed central portion 22 of the spacer 14.
The spacer 14 has a predetermined thickness, for example in the desired range 0.08 mm - 0.2 mm, although thicknesses outside of this range may be useful in certain applications. It will be understood that when the spacer 12 and cover material 14 are located in the depression 20, they together define a free volume within the depression corresponding to the removed central portion 22.
The free volume provides the working volume of the apparatus, and the dimensions and form of the spacer 14 which provides that working volume can be determined according to the particular application.
In a preferred embodiment the spacer 14 is made as a layer of double-sided adhesive tape with the central portion removed, the tape adhering to the base 12 and the cover material 16.
At one end of the depression 20 the base has a well 24, and at the other end it has a reservoir 26. When the spacer 14 and cover material 16 have been secured to the base 12 the chosen area of the base which is defined by the removed central section 22 of the spacer 14 comprises a first channel 30, a reaction zone 32, a second channel 34, a displacement zone 36, and a third channel 40. The well 24 has a cut-out 42 which provides a continuation of the depression 20 beyond the cover material 16, providing an application hole through which the sample can flow into the first channel 30. The first channel 30 connects the well 24 to the reaction zone 32. The second channel 34 connects the reaction zone 32 to the displacement zone 36, and the third channel 40 connects the displacement zone 36 to the reservoir 26.
The cover material 16 is hydrophilic (or in alternative embodiments the underside thereof is coated with a hydrophilic material), which improves the flow of the sample along the channels 30, 34 and 40. Specifically, a hydrophilic cover material helps to ensure the flow of sample along the channel 30 into the reaction zone 32, and helps to ensure the flow of sample along the channel 34 out of the reaction zone 32 and into the displacement zone 36.
In applications in which the apparatus is to be used in sandwich immunoassay with two antibodies, the reaction zone 32 is provided with a first set of antibodies bound to a marker, and a second set of immobilised antibodies (not shown). The immobilised antibodies can be located by known means. The immobilised antibodies can be located in a predefined pattern such as a line, a "+" symbol, or any other chosen pattern. If necessary in some applications, the antibodies bound to the marker can be initially separated from the immobilised antibodies, for example by a soluble layer, so as to ensure that the marker does not become bound in the reaction zone inadvertently.
Channel 34 is of a smaller-cross-sectional area than the reaction zone 32 and the displacement zone 36, and represents a restriction on the flow of sample out of the reaction zone. The channel 34 therefore helps to ensure that the sample remains in the reaction zone until the displacement step. However, it is desired that the volume of sample added to the well substantially equal the combined volume of the channel 30 and reaction zone.
In this embodiment the combined volume of the channel 30 and the reaction zone 32 is 4 μl, and so the sample volume is desirably 4 μl also. It will be understood that such a sample volume is considerably smaller than the sample volumes typically used in immunoassay methods, in which 10 μl represents the usual lower limit of sample volumes, with volumes up to 50 μl not being unusual. These volumes are necessary in prior art methods so as to ensure the required flow of sample from the first zone to the second zone, and it is a major advantage of the present invention that the absence of this required flow allows a reduction in the sample volume.
It is envisaged that the present method and apparatus could be used with sample volumes lower than 4 μl in certain applications. Clearly, the volume of the reaction zone 32 should be maximised in relation to the volume of the channel 30, since any analyte present in the proportion of the sample which is retained within the channel 30 does not combine with the reactant.
With a spacer having the dimensions indicated above, and with the cover material presenting a hydrophilic surface to the sample, it will be understood that the sample flows through the apparatus 10 by way of capillary action. Provided that the sample volume does not exceed the combined volume of the channel 30 and the reaction zone 32 the majority of the sample will tend to remain in the reaction zone 32 until the displacement step, i.e. the sample will flow from the well 24 and into the channel 30 and the reaction zone 32 until the well 24 is empty. The volume of sample added to the well 24 is therefore desirably chosen to match the combined volume of the channel 30 and reaction zone 32 so as to ensure that little or no sample flows into the channel 34 prior to the displacement step.
Instead of relying upon capillary action, the apparatus could utilise absorbent materials, but capillary action is preferred as it is relatively easy to retain the sample within the reaction zone as above described, and subsequently to remove substantially all of the sample during the displacement step.
The volume of the displacement zone 36 in this embodiment is considerably greater than the volume of the reaction zone 32, and so when the sample is displaced substantially all of the sample which is not bound in the reaction zone flows along the channel 36 and into the displacement zone. The reservoir 26 composes a deeper depression wfthin the depression 20, and the volume of the reservoir 26 is large in comparison to the displacement zone 36. The reservoir 26 therefore helps to ensure that all or substantially all of the unbound sample can be displacement out of the reaction zone.
In some applications it is not necessary that all of the sample is displaced from the reaction zone prior to examination of the reaction zone, and in such applications the displacement zone can be small relative to the reaction zone.
In some embodiments, the channel 34 can initially be blocked, for example by a plug of soluble material, and this will ensure that the sample cannot flow from the reaction zone to the displacement zone until the plug has dissolved. Ideally, the plug will take a chosen time to dissolve, the chosen time corresponding to the predetermined time required for the sample to remain within the reaction zone. If the channel 34 is initially blocked, then a sample volume greater than the volume of the channel 30 and the reaction zone 32 can be added to the well 24, but this is not preferred and instead it is preferred to add a controlled volume of sample as above described.
In this embodiment the base 12 is opaque and the cover material 16 is transparent, so that the apparatus is suitable for a visual examination of the reaction zone, and the marker can be a coloured marker. The quantity of coloured marker which becomes bound in the reaction zone can be measured by reflectance. In other embodiments the base is transparent and the cover material is opaque, or both are transparent in which case the quantity of a coloured marker can be measured by transmission.
When it is desired to utilise the device, a sample is placed into the well 24. The sample flows, by capillary action, along the channel 30 and into the reaction zone 32.
The predetermined time during which the sample must remain within the reaction zone 32 can be determined empirically or by calculation, and will depend most significantly upon the reactant/analyte combination being utilised. Any analyte which is present in the sample will bind to the antibody and its marker, and also with the immobilised antibody, and so act to bind the marker in the reaction zone 32.
The apparatus can be calibrated so that the quantity of marker remaining in the reaction zone 32 (after the analyte has combined with the antibodies for the predetermined period of time), is indicative of the quantity of analyte in the sample. For example, the quantity of marker is measurable by its colour and/or its intensity in the case of coloured marker.
After the predetermined time the displacement step is performed, in which water or a suitable displacement liquid is added to the well 24. It will be understood that the volume of displacement liquid added to the well is considerably greater than the volume of the sample, so that the displacement liquid carries all (or at least substantially all) of the unbound sample, and all (or at least substantially all) of the unbound antibody and marker, through the channel 34 and into the displacement zone 36 (and if the volume of displacement liquid is sufficient also along the channel 40 and into the reservoir 26). The reaction zone 32 will thereafter contain displacement liquid and bound marker, and since the marker will only be present there if it has been bound by antibodies, the quantity of marker remaining within the reaction zone 32 is representative of the quantity of analyte in the sample.
Much or all of the procedure may be automated, i.e. a machine may be provided to add the sample to the well 24, then add the displacement liquid after the predetermined time, and perform the reflectance (or transmission) test.
The marker used will be suited to the particular application. It may be a coloured marker such as gold sol for example which is typically used in prostate cancer immunoassay methods. Alternatively it may be a fluorescent marker or an enzyme marker, for example.
Variations upon the embodiment described can be provided as desired. In one variant, a line of coloured marker is immobilised as a control in the reaction zone and the immobilised antibodies are arranged in a perpendicular line. If no analyte is present in the sample only the control line will be seen after the test (as a "-" symbol or negative result), whereas if analyte is present both lines will be seen (as a "+" symbol or positive result).
The apparatus can be adapted to test for the presence of several different analytes at the same time. Thus, in some immunoassay methods it is advantageous to check for several analytes, for example the different analytes present in response to different illegal drugs, or the different analytes present in response to certain diseases or medical conditions. Variants of the device can be provided with a single well connected to separate reaction zones, each reaction zone having antibodies suited to a particular one of the various analytes.
In embodiments with multiple reaction zones it is preferred that each reaction zone has its own displacement zone; if multiple reaction zones were connected to a single displacement zone it would likely be difficult in practice to ensure that the sample is effectively displaced from each reaction zone, as the displacement liquid will flow along the path of least resistance which may be through only one of the reaction zones.
Preferably, the cover 16 substantially fully closes-off the reservoir 26, i.e. little if any sample and displacement liquid which enters the reservoir 26 can leak from the device, and only air can pass out of the device as the sample moves along the device. This is highly desirable for applications in which the sample may be infectious, or may otherwise present a health hazard for example. This has the additional advantage that excess displacement liquid can be added to the well, the flow of displacement liquid from the well 24 and into the channel 30 stopping when the displacement zone 36 and reservoir 26 have been filled.
In an alternative embodiment the spacer is integral with the base, perhaps being provided by a single piece of plastic or other suitable material. A well or depression corresponding to the chosen area is formed in the base by etching, moulding, or other suitable process. In such embodiments the area of the base surrounding the well or depression corresponds to the spacer of the embodiment shown in the figure, and enables a cover material to be fitted and define a volume corresponding to the chosen area.
It will be understood by those skilled in the art that a marker is not required in all applications and the presence or absence of analyte can alternatively be detected by a change in mass of the reaction zone (caused by the addition of analyte to the reactant). Alternatively again, the refractive index of a liquid within the reaction zone, or one of many other physical or chemical properties of the product of a reaction between the reactant and the analyte, can be used as appropriate for the particular analyte.
As above indicated, it is envisaged that the method and apparatus will find its greatest utility in immunoassay, but it is expected to be useful also in methods of chemical testing, organo-chemical testing and electro-chemical testing, for example.
In addition, the method and apparatus are expected to be useful in DNA testing, where a very large number of tests are to be undertaken on a particular sample. Specifically, in the known DNA testing methods a large number of reservoirs are filled with different gene sequences corresponding to one half of partial strands of DNA, and the sample material will combine with only those partial strands for which it has the same gene sequence. Knowing the reservoirs in which the DNA has combined with the gene sequence, and those in which no combination has occurred, allows the user to ascertain the gene sequences present in the sample DNA. With the present invention, the large number of separate reservoirs could be replaced by a large number of reaction sites within the reaction zone, each reaction site having a respective gene sequence bound thereupon.

Claims

1. A method of performing a test to locate a chosen analyte comprising the following steps: {i} providing a reaction zone with a reactant;
{ii} adding a suitable sample to the reaction zone, by way of capillary action;
{iii} retaining the sample in the reaction zone for a predetermined period of time; {iv} displacing the sample from the reaction zone; and
{v} examining the reaction zone to determine the presence or absence of analyte.
2. The method according to Claim 1 used in immunoassay, in which the reactant is antibodies, and in which the analyte is an antigen, a hormone, or another constituent of blood, urine or other bodily fluid.
3. The method according to Claim 1 or Claim 2 in which the reactant comprises a first set of antibodies and a second set of antibodies.
4. The method according to Claim 3 in which the first set of antibodies is bound to a marker.
5. The method according to Claim 4 in which the marker is a coloured marker.
6. The method according to Claim 4 or Claim 5 in which the marker is a fluorescent marker.
7. The method according to any one of Claims 4-6 in which the marker is an enzyme.
8. The method according to any one of Claims 3-7 used in sandwich immunoassay, in which the first set of antibodies is distinct from the second set of antibodies, in which the first set of antibodies carry a marker, in which the second set of antibodies are immobilised in the reaction zone, and in which the presence or absence of marker remaining in the reaction zone during the examination step is indicative of the presence or absence of analyte within the sample.
9. The method according to any one of Claims 1-8 in which a controlled volume of sample is added to the reaction zone.
10. An apparatus for performing a test to locate a chosen analyte comprising: a base, a cover material covering the base, a spacer to separate the cover material from the base, the spacer having a predetermined shape to define a chosen area of the base, the base, spacer and cover thereby together defining a volume corresponding to the chosen area, a reaction zone located within the chosen area, the reaction zone being provided with a reactant for the analyte, and a displacement zone located within the chosen area, the displacement zone being connected to the reaction zone.
11. An apparatus according to Claim 10 in which the spacer is a layer of material lying between the base and the cover material.
12. An apparatus according to Claim 10 in which the spacer is integral with the base.
13. The apparatus according to any one of Claims 10-12 in which the volume of the displacement zone is greater than the volume of the reaction zone.
14. The apparatus according to any one of Claims 10-13 having an application hole through which the sample is added, the application hole being connected to the reaction zone.
15. The apparatus according to Claim 14 in which the application hole is connected to the reaction zone by a strip of the chosen area.
16. The apparatus according to Claim 15 in which the reaction zone and the displacement zone are connected by another strip of the chosen area.
17. The apparatus according to Claim 16 in which the cross-sectional area of the strips of the chosen area are smaller than the cross-sectional areas of the reaction zone and the displacement zone.
18. The apparatus according to any one of Claims 10-17 in which, the cover material is hydrophilic.
19. The apparatus according to any one of Claims 10-18 in which the sample moves from the reaction zone to the displacement zone by way of capillary action.
20. The apparatus according to any one of Claims 10-19 in which the spacer is a double-sided adhesive layer, the layer adhering to the base and the cover.
21. The apparatus according to any one of Claims 10-20 in which there is a plurality of reaction zones and a plurality of displacement zones, with each reaction zone being connected to a respective displacement zone.
22. The apparatus according to Claim 21 in which the reactant in one of the reaction zones differs from the reactant in another of the reaction zones.
PCT/GB2008/000832 2007-03-09 2008-03-07 Lateral flow assay and apparatus WO2008110781A1 (en)

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