Device for determining at least one analyte capable of being contained in a liquid sample

Invention field

The invention relates to a method for determining at least one can be contained in an analyte in a liquid sample of the device.

The invention particularly relates to immunological chromatography through a lateral migration implementation of the technical device.

Background Art

Most used for determining the (one or more) of the analyte has been gradually developed to use more easily the device, allow the development of fast and low-cost conventional diagnostic method.

This change is very prominent in the medical field, through the "point of care" or "household test" in the diagnosis, wherein the patient's bedside or diagnosed at home, does not need to use the analytical laboratory automation technology.

Immunoassays is used for this kind of rapid diagnosis of the technical part.

These immunoassays cover members of the specific binding pair-based affinity between the diagnostic device and method of the reaction.

In of the whole, these immunoassays is divided into 2 main well-known method.

In the so-called "competition" method, the desired analyte and by marked detection reagent is a specific binding capture reagent and competition. Through the acquisition of the reagent (or measurable) lack of or presence of the signal, the expectations of respectively measuring in a sample the presence or absence of the analyte.

In the so-called "sandwich" method, the detection reagent by mark combine desired analyte, the latter through the capture reagent immobilized on a solid support. Through the acquisition of the reagent (or measurable) the presence of a signal or a lack of, separately measuring an analyte in a liquid sample for the presence of or lack of.

In in these immunoassays, the analyte to be particularly suitable for certain of the so-called "immunochromtography" device is known.

Immunochromtography is solid phase diagnostic method, the use of the inert film does chemistry and lateral migration.

Such immunoassays by use of strip-shaped form of the porous support, wherein the insertion and/or on the dry form needed by the test reagent.

The treatment porous support specific binding partner (usually antigen/antibody) at the capture zone recognition reaction between, and can be in the horizontal display.

In practice, when the analysis of the liquid sample is stored in the support, the along the membrane by capillary action. The desired analyte is normally fixed on the film of assigns the area specific capture reagent capture.

The principle according to the sandwich method, the reaction is by mark display of the specific detection reagent.

Also can use competition type of reaction. Usually in this case use is the detection of the analyte by mark reagent, it is desired to of the analyte with the fixed capture reagent specific binding competitors.

These chromatographic device is generally suitable for single and household. In fact, its use of simple and rapid, requiring only a minimum amount of operation, or because all reagent is inserted in the device.

However, in some special cases, these immunochromatographic device may provide is not completely reliable results.

In one aspect, when the through the "sandwich" shape when the test analyte determination, in the case of certain analyte observed that the "hook effect".

Hook effect amynologic test is known to be the role of the one does not want to. In the present in the sample of the analyte occurs at a very high concentration.

Hook effect then can lead to the false negative, caused by the conclusion of abnormality, that is, no analyte or analytes in a sample in a low concentration.

In the sandwich-type immunoassay analyte with a hook-shaped effect of generating Gaussian curve types of signal/concentration curve.

For a given signal, when the read time to confirm the results, for the desired analyte, two concentration therefore is possible, a low and another kind of high.

Competition test to be administered to allow for different two kinds of concentration of the analyte are respectively obtain two different signal.

Very quickly, however, the competition tests have also shown that the its limitations, because the target analyte concentration is relatively low there is a signal eradicating.

Usually these is used to overcome the shortcoming of the sandwich and competition test solutions is applied from a liquid sample of the analyte dilution range.

However, the use of such dilution range is not suitable for household. Furthermore, the use of the range of dilution of the sample requires additional operation and single use the test device because of the increase of the consumption of each sample at different dilution test.

For this purpose solutions for example, in WO   2007/023372 described in, its relates to a method for measuring an analyte in a liquid sample device, the device comprises the capillary diffusion tool, in the capillary diffusion vehicles to realize:

A) a sample storing or receiving area;

B) upstream release area, includes the visible and/or measurable marker conjugated analyte-specific detection reagent, through the capillary diffusion in the capillary diffusion tool freedom of movement in the wet state; and

C) two downstream capture zone, in the capillary diffusion direction thereof, comprising continuously, in one aspect, the analyte-specific capture reagent and, on the other hand, is the analogue of the analyte or analytes.

Analyte-specific detection reagents and capture reagent, allowing through the sandwich test measuring an analyte in a liquid sample; and then fixing the same analyte-specific detection reagents and analytes, or analyte analogue, through the competition test allows determining a target analyte in a liquid sample.

In such device, the detection reagent in excess of the complementary capture zone is formed upstream in the region of the single release.

Regardless of its benefits, inventors have found that the upstream capture excess detection reagent is not satisfactory, usually observed in non-specific reaction, strong background and migration.

In another aspect, the structure some chromatographic device is used for detecting a plurality of analytes at the same time.

Such chromatographic device, for example, EP-1657550 described in, advantageously contain capillary diffusion tool, in the capillary diffusion vehicles to realize:

A) a sample storing or receiving area;

B) upstream release area, which comprises the visible and/or measurable marker mixture of conjugated detection reagent, one of each type of the analyte is specific, its through the capillary diffusion in the capillary diffusion tool freedom of movement in the wet state; and

C) downstream capture zone, each comprising one of the specific capture reagent for an analyte, the downstream capture zone according to the direction of the migration of a continuous distribution.

Similarly, the detection reagent is stored in the single release area, the single release area is upstream of the at the capture zone, taking into account the capillary direction of migration.

However, the inventor found that once again different capture zone upstream of the mixture of the detection reagent is not satisfactory, because of the different detection reagent cross reaction between the reagent and the mutual ratio between the live inhibit.

Contained in the plurality of formed upstream capture (one or more) of a detection reagent release area of the chromatographic device therefore is not satisfactory.

Therefore, used for determining single kind of analyte and used for the determination of a number of analytes novel capillary diffusion measuring device a demand, the measuring device allows to reduce a plurality of in particular as a result of the report of the continuous capture zone upstream of (one or more) detection reagent to excessive and/or of the mixture.

Invention SUMMARY

The present invention therefore relates to a method for determining at least one can be contained in an analyte in a liquid sample the presence and/or amount of device, the device comprises the capillary diffusion tool, in the capillary diffusion tools according to the intention of the liquid sample capillary migration direction and way side to the migration.

In this capillary diffusion tool, to the capillary migration direction upstream to the downstream, realize a plurality of zones, in other words, at least:

-For storing liquid, a region of the sample,

-An upstream release area, comprising at least one of the visible and/or measurable marker conjugated detection reagent, the detection reagent can be the liquid sample in the capillary diffusion tool and the relocation of the movement, and

-At least two capture zone, each comprising at least one fixed in the capillary diffusion the capture reagent.

The device also includes at least one downstream release area, the downstream release area in the capillary diffusion on the tool is located in the capture zone and at least one of the in the downstream.

This downstream release area also comprises at least one of the visible and/or measurable marker conjugated detection reagent, the detection reagent can be the liquid sample in the capillary diffusion tool and the relocation of the movement.

Release area detection reagent and/or located in said release area for directly downstream of the (one or more) complementary capture area of the capture reagent then can be specifically bound to the analyte and/or specific to each other in the (one or more) complementary capture zone to form a complex, the to enable the determination of the analyte in a liquid sample.

In general, depend on the form is located in the release zone directly downstream of the (one or more) the capture of the capture reagent, such arrangement has the release area allows to adjust the amount of the detection reagent and/or specific special benefits.

Can be mutually combined or independent use of the display below other favourable:

-The two capture is inserted between the (one or more) downstream release area;

-Upstream region comprises directly upstream release zone located downstream of the upstream capture zone, advantageously suitable for the sandwich form test, after the upstream region group itself is MAX. one or a plurality, each comprising release area and one or a plurality of complementary capture zone; this feature allowed to be determined according to the (one or more) analyte detection threshold optimization (one or more) the concentration and downstream block specific.

In order to determine a single kind of analyte, release area of the (one or more) detection reagent and/or capture area of the (one or more) capture reagent advantageously can be specifically bound to the analyte.

In this case, each other releasing area preferably includes one or more of the same kind of detection reagent, each other and capture zone preferably includes the same one or more capture reagent.

Also in this case, the capillary diffusion tool advantageously includes:

-An upstream release area,

-With the upstream release zone complementary 1st capture zone or two 1st capture zone, and then

or

-Downstream release area, and

-And said downstream releasing area of at least two complementary 2nd capture zone,

Or at least two continuous area partner (  couples   of zones), each comprising:

-Release area, and

-With the associated complementary region of the release of the at least one capture zone.

At least two analytes in order to detect, then detection of the reagent and/or the release area of the adjacent downstream of the (one or more) complementary capture area of the capture reagent, advantageously can be specifically combined with one of the analytes.

Combination or independently of each other according to the still other advantages of the:

-In the capillary proliferation of vehicles to realize, respectively inserted into the downstream of the two capture zone between the quantity of the release area, is from 1 to 4 a, for example, 2 a, to the 25 mm the form of standard capillary support;

-In the capillary proliferation of vehicles to realize, and is then directly upstream of one of the number of the complementary capture, is from 1 to 10 a, preferably 5 a, to the 25 mm the form of standard capillary support;

-Releasing area in the detection of at least one of the reagent and the complementary capture in the capture of one of the reagent or they can be specifically binds the analyte or analytes sandwich in order to form at least one of the test patterns;

-Releasing area in the detection of at least one of the reagent and the complementary capture in the capture of at least one of the reagent or their, in one case, is the analogue of the analyte testing decides and, in another case, specific binding of said analysis is the analogue of the analyte the thing or states reagent, in order to form the competition patterns test;

-For storing liquid region of the sample (i) or merger with the upstream release (ii) is located upstream of an upstream release zone.

The invention also relates to a method for the quantitative determination of the stored in the device for measuring an analyte in a liquid sample of the method, the method comprises the following consecutive steps:

-In each capture zone measuring signal is the step of (intensity),

-In each area in the set, the measurement of the intensity of the added step,

-Calculating intensity proportion of steps, the intensity ratio of a region corresponding to the intensity of the sum of the group with another MAX. comparing the strength of the adding of the, and

-The calculated with the ratio of the intensity of the analyte in a liquid sample of the steps associated with the quantitative value, corresponding to the taken into account in a liquid sample based on the analysis of the intensity of the extractives calculated proportion of the standard curve.

Figure

The invention will be composed of the following according to the invention together with many kinds of devices with the description of the further description, but in any event is not the limit of this invention. The attached drawing wherein:

-Figure 1 diagrammatically described according to the present invention for determining the single kind of analyte 1st device, which comprises two MAX., by release area and a capture.

-Figure 2 is described more illustratively according to the invention for determining the single kind of analyte 2nd device, comprising a release area and 1st cell groups of a capture, the capture and then is connected with a plurality of cell groups 2nd;

-Figure 3 also diagrammatically described according to the present invention for determining the single kind of analyte 3rd device, comprising a number of cell groups thereof, a the release area and a capture zone;

-Figure 4 is described more illustratively according to the invention the two analytes used for determining the 4th device, which comprises two MAX., a capture by the release area and the, one of each for the analyte.

-Figure 5 still diagrammatically described according to the present invention used for measuring a number of analytes kind device, which comprises a plurality of cell groups, the two capture zone a and then, one of each for the analyte.

Invention details

The present invention therefore relates to a method for determining at least one can be contained in an analyte in a liquid sample of the device.

Through in the aforesaid Figure 1 to 5 a plurality of displayed in very diagrammatically described embodiment according to the present invention of the general structure of this device.

Generally, when the plurality of embodiments described, to retain the use of the reference number to indicate the same or similar structural element.

As shown in Figure 1 to 5 shown in the, each device 1 includes capillary diffusion tool 2, the liquid sample (not shown) intended to store in the capillary diffusion tool 2 is, and then to the upstream to the downstream capillary migration laterally and pointing direction of migration.

And pointing direction of migration by fig. 1 the point in the illustrative A by the arrow.

Generally, the concept "upstream" and "downstream" refer to the liquid sample along the capillary diffusion tool 2 of the length of and to the direction of migration.

In this capillary diffusion tool 2 to the upstream to the downstream capillary migration to realize different Serial A, in other words, at least:

-A zone 3 used for storing the liquid sample,

-An upstream release area 4, which comprises the visible and/or measurable marker conjugated at least one kind of detection reagent, the detection reagent because the liquid sample in the capillary diffusion tool 2 and the relocation of the to can move in, and

-At least two capture zone 5, each comprises a fixed in the capillary diffusion tool at least one capture reagent.

According to the present invention, the device 1 further comprises at least one additional release area 6, in the capillary diffusion tool 2 and is located on the capture zone 5 at least one of the in the downstream.

This additional downstream release area 6 also includes the visible and/or measurable marker conjugated at least one kind of detection reagent, the detection reagent because the liquid sample in the capillary diffusion tool can be the relocation of the mobile.

Capillary diffusion matrix 2 therefore comprises a plurality of Serial groups of 7, the same is composed of at least two Serial:

-A release area 4 or 6, the upstream, and

-One or a plurality of capture zone 5, referred to as "complementary", is located in the relevant release area 4, 6 directly downstream.

Selective release area 4, 6 and/or the detection reagent of (one or more) complementary capture zone 5 with the specificity of the capture reagent for analyte and/or bind to the specific binding to each other.

This method to ensure that the complementary capture zone 5 to form a (one or more) composite, the to enable the determination of the analyte in a liquid sample.

In particular, selection of each group region 7 detection and capture reagent used for implementing the sandwich shape and/or competition forms of immunology test.

Therefore, (one or more) additional release area 6 allow the presence of the (one or more) detection reagent in each district group of 7 the optimal distribution in, in order to then the relevant capture with respect to the different 5 arrangement way.

Therefore, with respect to the is located directly downstream of the (one or more) complementary capture zone 5 each release area 4, 6 of the detection reagent in the amount and specific.

Generally speaking, according to the device of this invention to enable the determination of low concentration in a liquid sample and a very high concentration of (one or more) analyte without obtaining false positive or false negative results.

Furthermore, according to the present invention can be constructed in a liquid sample of the device is used for one or more of the semi-quantitative measurement of analytes.

The device according to the invention has remarkable especially suitable for the determination of the analyte of the hook effect, such as is pregnant hormone (hCG), prostate specific antigen (PSA) and hemoglobin (for example used for fecal hemoglobin in detection, also referred to as "fecal occult blood" (FOB)).

A plurality of release zone to the other benefits of this invention include:

-Depends on target density to be detection of the detection reagent and capture reagent optimization,

-More high levels of accuracy.

Because of the above-mentioned two points, the measuring device is obtained, and the visible and measurable explains the value of the signal to increase the sensitivity and specificity.

In different aspects of the present invention to display more detail below.

(One or more) and a liquid sample of an analyte

"Analyte" means to be detected in a sample of any chemical, biochemical, or biological entity.

The chemical entity is advantageously from life world, preferably, derived from plant or animal world, more preferably, in human entity.

According to the present invention in a device and method for the detection of analytes, can be especially mentioned protein, peptide, antibody, hormone, steroid, or tumor cells from infection dose of the antigen, such as infection dose bacterial, viral or parasitic, nucleic acid (DNA or RNA), therapeutic compound, drug or antibody.

In particular, the analyte is selected from known immunological chromatographic technique to measure the generated during those hook effect.

The "hook effect" through immunological chromatographic technique to obtain the mean of the false negative result, abnormal to the conclusions of the no in the sample analyte, when the analyte in order to very high in concentration is present in the sample.

Generally, the hook-shaped effect lead to visible and measurable weakening of a response signal, and to the contrary the increase of the density of the of the analyte, lead to (i) the two kinds of different concentration of the same signal (having the same strength), one of the two kinds of different concentration to the other of the high and low, and high (ii) inhibit the concentration of an analyte to the signal.

For example, the hormone hCG in most of the immunological chromatographic test, the hook effect in about 5000mIU/mL is started and will continue until the about 250000mIU/mL, involves especially for low (25mIU/mL) and high (200000mIU/mL) concentration of the same signal strength.

Therefore, analyte therefore advantageously selected from the group consisting of human chorionic gonadotropin (hCG), prostate specific antigen (PSA), hemoglobin, fecal occult blood (FOB), carcinogenic gene marker (such as ferritin, α-fetoprotein (AFP), CA15-3/CA27 . 29 (breast cancer), CA19-9 (pancreatic cancer), CA-125 (ovarian cancer)), C reactive protein (CRP), troponin I (TNI), cardiac marker (such as troponin T, CK-MB, myosin, B-type natriuretic peptide (t-BNP)), drug abuse (DOA), therapy monitoring biological marker, tumor necrosis factor (TNF-α), the circulation of other therapies of cancer related gene biological marker, other cell, cell or tissue-specific biological marker, and the like), luteinizing hormone (LH) or follicle stimulating hormone (FSH).

Dependent on its structure, the device according to the invention is capable of determining the single kind of analyte (single analyte) or measuring a number of analyte (number of (poly-) or more (multi-) or more (pluri-) kind of analyte).

"A number of analyte" is meant at least two analyte, more preferably 2, 3, 4 or 5 kind of analyte.

This multi-analyte method to study of autoimmune diseases ("autoimmune disease group"), allergy research (" allergic group "), the field of medicine therapy monitoring, toxicology, or to treatment of inflammatory markers, diagnostic tests for multiple infection (such as human immunodeficiency virus or HIV/hepatitis c/hepatitis b) may be interested.

For example, according to the device of this invention can be suitable for measuring a liquid sample a plurality of antibody, such as anti-HIV (human immunodeficiency virus or HIV), anti-HCV (hepatitis c virus), anti-HB (marker chronic viral hepatitis), anti-TB (tuberculosis).

"Liquid sample" is meant that the analysis of the solution or suspension in any sample.

In particular, the liquid sample can be any biological fluid or body fluid.

The liquid sample can also be already directly or indirectly obtained from the biological liquid or body fluid.

The sample can also be a solid sample of liquid extract.

Usually, the liquid sample is a urine, whole blood, blood plasma or serum.

According to the present invention in some of the method, when the liquid sample is a blood plasma, serum or whole blood for example, the use of diluents.

Diluent and sample stored together on the porous solid support. Alternatively, before or after the sample diluent is deposited in the porous solid support.

This diluent to migrate in the solid support, cause or assist in the sample with the labeled detection reagent to migrate in the porous support.

Typically, the diluent is the buffer salt solution, and may also comprise a detergent or reaction needs to be of any other component.

According to the apparatus of the present invention is interesting, is that it can be suitable for determining the presence of at least one analyte, and measuring an amount.

"Detecting" or "determining" is meant liquid sample to the one or more analytes in the qualitative assay (advantageously existence or non-existence).

"Detecting" or "determination", also meaning that measurement and quantitative sample in one or more of the analyte.

In fact, according to the present invention the performance of the apparatus and method also allows for the implementation of the liquid sample or an analyte in at least two different analytes quantitative or semi-quantitative measurement of the embodiment.

Capillary diffusion tool

According to the present invention, " capillary diffusion tool 2 the to constitute or to act as [...] continuous capillary diffusion units of any tools, by a lateral migration (in other words vertical to the implementation of the capillary diffusion of the (one or more) of the thickness of the capillary material).

Advantageously, the capillary diffusion tool is to allow liquid through the simple capillary diffusion and migration of the porous solid support.

The porosity of the holder the sample and/or reagent can be in a liquid or wet capillary diffusion (or lateral migration).

Such capillary diffusion tool is very widely used in all immunological chromatographic technique, in particular in lateral migration immunological chromatographic technique.

Such capillary diffusion tool is, for example, in the capillary diffusion (side to the migration) and/or in the direction of the upper extension of the support points.

This capillary diffusion tool consists in the following:

-A single capillary or porous material,

-A number of different capillary or porous element or material, conveniently positioned to each other (for example, by superposing), in order to obtain in the capillary diffusion direction or material from one element to another continuous capillary flow.

Such capillary diffusion tool determines the direction of any liquid and to the capillary proliferation, the liquid-receiving or deposited in the upstream end, and then move to the downstream end of the said tool.

Consider according to the present invention the capillary diffusion, also referred to as "side to the migration immunochromtography", in the implementation of the different filtering technique, the through the liquid in the filtering technique (one or more) of the thickness of the porous filtering material.

As an example, these capillary diffusion tool can be made of many kinds of chromatographic support composition, for example, cellulose, nylon, nitrocellulose, polyethylene or glass fiber.

Capillary diffusion tool can be composed of one or a plurality of individual parts. Support of the different part can be composed of a different material. When the capillary proliferation of different parts or tool consists of the different material, in order to allow the capillary proliferation of these components in the tool are arranged continuous capillary flow.

Generally, capillary spread tools in the capillary diffusion direction by extension of a porous solid support. Preferably, the apparatus according to the present invention the capillary diffusion tool includes chromatographic strip forms of porous solid support.

Capillary diffusion tool is, for example, is composed of superimposed or overlapping a plurality of objects in the form of a chromatographic strip.

According to the device of this invention, for example, the rigid support can be combined with a chromatographic strip.

Rigid support can be made of a variety of materials, such as cardboard, plasticized pasteboard or more preferably, plastic material. Preferably, the rigid support is made of polystyrene.

Advantageously, the tool can include capillary proliferation in the clamping support. The clamping support the capillary diffusion of tools are more easy to operate and also can make it from moisture.

The clamping holder can be partially or completely encase the capillary diffusion tool.

The clamping holder can be made of a variety of materials, such as cardboard, plasticized pasteboard or more preferably, plastic material. Advantageously, the clamping support is made of rigid and waterproof material.

The clamping holder or a sleeve, particularly in Patent WO-2007/023372, EP-0291194, EP -0560411, EP -0560410 and EP-1091808 described in.

Usually, the clamping support in the form of a sleeve.

Advantageously, the clamp of the support provided with at least one observation window to view the capture zone.

Advantageously arranged these observation window so as to provide only treat analysis of at least one analyte or when the determination of the appropriate, at least two kinds of determination of the analyte capture direct visual observation.

Storage

Storage area 3 corresponding to the capillary diffusion tool 2 upstream of the zone, in which is provided with the liquid sample.

Storage by the absorbent material can be made of the water system (catchment).

This water collecting system can be directly in contact with, for example, urine flow.

WO-00/00288 such as the described in the, water collecting system can move between two positions, one is used for collecting the liquid sample, is far away from the capillary diffusion tool, and the other one is connected with the capillary diffusion tool or capillary contact is connected with the storage.

In the present invention in one embodiment of, capillary diffusion tool may comprise sample used for storing or collecting area, said region extending from the clamp support in order to receive the liquid sample.

In the present invention in another embodiment, the clamping support or sleeve is provided with at least one opening is used for storing the liquid sample.

Detecting reagent and capture reagent

Capillary diffusion tool 2 comprises on its length, on the one hand, distribution in order to realize the release area 4, 6 of at least one kind of detection reagent, and on the other hand, distribution in order to realize the capture zone 5 of at least one capture reagent.

The more detailed description below "release area" or "capture zone" is meant the capillary diffusion tool 2 of the localization of the part and is delimited, in its are respectively store a certain amount of at least one kind of detection reagent or at least one capture reagent.

Release area 4, 6 and capture each of the 5 is advantageously a transverse lines or with (extending perpendicular to the direction of migration), the transverse lines or with a for example, range from 1 to 2 mm of width, and from the range 3 to 5 mm² surface area.

Generally speaking, "detection reagent" or "capture reagent" is any chemical, biochemical, or biological entity, which can specifically combine in order to form a complex, to enable the determination of the analyte in a liquid sample.

Detecting reagent and/or capture reagent in addition also referred to as "binding reagent" is a reagent.

In a liquid sample to enable the determination of one or more analyte binding reagent of this kind is well known and can be carried out on the choice of the present invention.

These binding reagents can be advantageously selected from the specific binding of the analyte and/or those of each other.

According to the implementation of the test patterns, the complementary binding reagent with intent to form different complexes:

-binding reagent can simultaneously binds the analyte to form a sandwich shape test,

One of the-binding reagent (the detection or capture reagent) other and capable of binding to the analyte binding reagent (separately capture or detection reagent), in order to form the competition patterns test.

In the context of the present invention, binding reagent selected from at least one advantageously can be specifically binds the analyte and/or analyte analogue chemical, biochemical, or biological entity.

"Binding" or "combined" is meant any strong binding, such as covalent, and with any weak, such as antigen/antibody or analyte/anti-analysis the combination of thing type.

"Anti-analyte" is meant can be specifically binds the analyte or the analyte competitive specific binding capture reagent of any chemical, biochemical, or biological entity, for example, antibody, antigen or nucleic acid.

"Suitable analyte analogue" advantageously is meant when suitable at that time, can the analyte competitive specific binding capture reagent or a detection reagent to any chemical, biochemical, or biological entity.

Binding reagent advantageously selected from the group consisting of antibody, antigen or nucleic acid.

Analyte and binding reagent specific to each other therefore usually capable of forming a pair of (couple) with, for example, ligand/anti-ligand pair, the antigen/antibody, or DNA/RNA the DNA/DNA.

Therefore, if the analyte is an antigen or hapten, binding reagent (detection reagent and/or capture reagent) in at least one of the analytes is advantageously specific antibody.

"Analyte-specific antibody" is meant in the antigen/antibody type can be combined with the antibody binds the analyte specificity.

It is usually to the analysis object is provided with a high affinity polyclonal or monoclonal antibody. Preferably, it is a monoclonal antibody.

If the analyte is antibody, in combination with at least one of a reagent is advantageously made of antibody recognition of the antigen.

If the analyte is a nucleic acid, in combination with at least one of a reagent is advantageously a complementary DNA probe.

(One or more) detection reagent advantageously with visible and/or measurable marker, advantageously particles conjugated marker.

"Visible and/or measurable marker" is meant because the capture area for the signal transmission allows naked eye or use of the instrument is directly or indirectly detects any marker.

The signal is, for example, a fluorescent, colored, isotope or magnetic signal exists.

Examples include fluorescent or colored particle markers such as colloidal gold, colored latex particles, fluorescent latex particles and Avidin and streptavidin Avidin conjugated particles.

Colored or fluorescent particle marker is therefore not soluble in water, and therefore the small size particles in a liquid phase to form a suspension, dispersion or solution.

Allow direct observation with the bare hole marker also includes dextran types of markers (Hansen   T.M., IVD   Technology   4, 35-40, 2003). Then the binding reagent of the glucan chains containing fluorophore of conjugated (derived from a polysaccharide).

The marker may also be enzymes (including alkaline phosphatase or AP, hrp or HRP), coloring agent (or dye) or chemiluminescent compound (including FITC fluorescein or thiocyanate).

In order to increase the sensitivity, can be used by the technicians of this field know technical labelled antibody (such as a biotinylated antibody), for example, used for indirect detection, so that the can by forming Avidin-biotin and marked by chain enzyme-streptavidin-biotin entities detected indirectly.

This pass mark and biotinylated antibodies also can be directly deposited in the capture zone in order to increase the sensitivity of the test line, or and specific detection antibody storage in order to increase the contact time and in addition specifically, increased sensitivity, for example because the number of binding sites.

On this part, by technical personnel in the field of the technical knowledge of the analyte-specific capture reagent immobilized on a solid support.

This capture reagent so that it can not move in the hygrometric state means is fixed.

Absorbed or can be covalently coupled, for example, by implementation of the fixed.

Composition of the Serial

Capillary diffusion tool 2 therefore comprises one or more capture zone 5, the capture zone 5 directly with the upstream (taking into account the capillary migration-pointing) the release area 4, 6 complementary.

The "direct upstream" meaning that capture zone 5 then upstream of a 1st 4, 6

"Complementary" is meant a release and capture of MAX., selecting the component binding reagent used for determining in order to allow for the implementation of the test target analyte, advantageously competition test and/or shape the sandwich form test.

Each of the cell groups thus release area, is connected with at least one complementary capture zone (when the appropriate when the release area before new block).

As previously discussed, this arrangement to allow the detecting reagent then the amount of adjustment and/or the specificity in a directly downstream of the (one or more) of the capture of the capture reagent to the nearest degree and is of particular benefit as accurate as possible.

Generally speaking, the capillary diffusion tool 2 can include:

Such MAX.-only, selected components of the binding reagent states the block in order to allow the implementation of competition patterns test; or

Such MAX.-only, selected components of states the block binding reagent of the sandwich shape in order to allow for the implementation of test; or

-At least one such MAX. of, the components of the binding reagent states the block in order to allow the implementation of competition patterns test, and at least one such MAX. of, the components of the binding reagent states the block in order to allow for the implementation of the sandwich form test; or

-At least one such MAX. of, the components of the binding reagent states the block in order to allow the implementation of competition patterns and sandwich shape test, and at least one such MAX. of, the components of the binding reagent states the block in order to allow the implementation of competition patterns and/or sandwich form test.

In the group in each region, can be mixed-release area and a capture zone, or a series of 1st capture zone.

Alternatively, capture zone, or a series of 1st capture zone, advantageously and release distinguish the distance of a few millimetres, for example range from 2 to 4 mm.

Furthermore, a series of capture zone advantageously separated from each other the distance of a few millimetres, for example, range from 1 to 4 mm.

More generally, an upstream release area can be located in a sample storing or receiving area.

However, this upstream release area also in the dry state can be stored in, or receiving area in the sample storage downstream, in order to prevent the deposited sample because the washing effect of the loss of any of the detection reagent.

In more general terms, in the capillary proliferation of vehicles to realize, with the releasing area directly upstream of the number of the complementary capture zone, is advantageously from 1 to 10 a, preferably from 2 to 5 a (still is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 the number of).

Such method is particularly useful for obtaining the results of semi-quantitative or quantitative type.

In fact, as shown in the construct, obtained on the continuous capture of the signal spectrum can be attributed to the concentration of the analyte in a liquid sample range. As shown in the construct, on the continuous capture signal spectrum obtained in a liquid sample also can be attributed to the accurate analyte concentration.

In addition also the embodiment of the display, stupendously, for implementing the sandwich test of the release and capture of the special arrangement of the, hook-shaped effect can be remarkably reduced.

In this regard, display the embodiment of interest comprising:

-Upstream MAX., comprising release area/capture zone or two capture zone, and then

- downstream block , which at least comprises the new release area, is connected with a series of capture zone.

This upstream MAX. advantageously based on a sandwich shape in order to ensure that in the upstream capture (one or more) analyte:

-(One or more) the prominent portion of an analyte,

-Or corresponding to part of the diagnosis value and the threshold value detected ("diagnosis value" referred to in the scientific that is to allow medical interpretation of the threshold of any analyte (or biological marker) concentration).

In particular, this predetermined upstream capture allow the liquid sample dogru through subsequent (one or more) the path of the zone controlled by the concentration in the group.

The existence of this upstream MAX. the presentation control (one or more) downstream area group of the benefits of the hook effect in.

Is still generally to, in particular, according to the International units or ng/mL (IU)/ mL in the expression of the desired nomenclature concentration of the diagnosis value concentration range, advantageously adjusting release the concentration of the detection reagent in the region and the concentration of the capture reagent in the capture zone.

The concentration is, for example, 0.1 and 1ng/porous support between the linear cm, depends on the specific binding reagent.

Furthermore, the quantity of the release zone, and therefore, the number of corresponding MAX., is from 1 to 4 a (still is selected from 1, 2, 3 or 4 of the digital). The preferred range is for example the length of the capillary support for the 25 mm of the two group.

Correlation to the choice of the binding reagent itself is conventional.

In the competition test, release area 4, 6 and complementary capture zone 5 advantageously comprises:

(I) as the analyte itself, or a suitable analyte analogue detection reagent of the mark, with visible and/or measurable marker conjugated, and

(Ii) fixed capture reagent, advantageously antibody-type anti-analyte, specific binding to the analyte in the sample and the aforesaid detection reagent.

This embodiment enables the analyte liquid sample through the test, the test in the competition (one or more) capture zone, in, on the one hand, the sample analyte and, on the other hand, the analyte or by mark by mark between analyte analogs.

Alternatively, still in the competition test, release area 4, 6 and complementary capture zone 5 advantageously comprises:

(I) as anti-analyte-labeled detection reagent, for example, the analyte or analyte-specific antibody, the visible and/or measurable marker conjugated, and

(Ii) as the analyte itself, or analyte analogue of the capture reagent, fixed in the capillary diffusion tool 2 of the capture zone 5 in at least one of.

This embodiment enables the liquid sample by a competitive test analyte, the competition test reagent can be detected in the sample analyte or capture reagent competition binding.

In these different competition test, in the absence of the target analyte in the sample under the condition of, by mark is the detection of the complex of reagent/capture reagent form. These fixed complex therefore in the absence of an analyte, such as the above defined the visible and/or measurable signal.

Under the condition of the presence of an analyte, the capture reagent is not fixed by marked detection reagent. Lack of signal at the capture zone thus corresponds to the presence of an analyte in a sample.

In the sandwich-type test, release area 4, 6 and complementary capture zone 5 advantageously comprises:

(I) with visible and/or measurable marker conjugated as anti-analyte-labeled detection reagent, its specificity binds the analyte, and

(Ii) fixed capture reagent, which specifically binds the analyte, and advantageously anti-analyte.

The mode of execution according to the sandwich form of the preferred embodiment, area group of 7 the release area 4, 6 comprising at least one of the analytes, or to at least one of the analyte specificity, with visible and/or measurable marker conjugated one or more antibody, the antibody is deposited in or on the capillary diffusion tool in a dry, but through the capillary proliferation of freedom of movement in the hygrometric state.

In this case, groups of the area 7 of the (one or more) complementary capture zone 5 contains a fixed known technology of one or more analyte-specific antibody. These antibodies can be fixed a way that they do not move in the hygrometric state.

Release area 4, 6 and (one or more) complementary capture zone 5 and respectively the antibody specifically binds the analyte, for example, on the two epitope site, the analyte the same or different.

The obtained complex MAX. an analyte/antibody is fixed at 7 of the (one or more) capture zone 5 is. These fixed complex therefore generates visible and/or measurable signal, such as the above defined.

In order to detect one analyte, release area of the (one or more) detection reagent and/or capture area of the (one or more) capture reagent advantageously can be specifically bound to the analyte.

Therefore continuous MAX. advantageously used for single kind of analyte.

In this case, then each other advantageously include one or more of the same kind of detection reagent, the same as each other and capture zone comprises one or more capture reagent.

Group all the different Serial implementation of the utility model is suitable for testing of the same morphology, the competition or sandwich.

In the alternative, each advantageously include releasing area specific detection reagent, and complementary capture zone include complementary capture reagent.

In this case, different Serial adapted to carry out the group and can be the same or different from each other of the test, in other words the competition or sandwich.

At least two analytes in order to detect, then detection of the reagent and/or the release zone downstream and proximate to MAX. common form (one or more) complementary capture area of the capture reagent, advantageously can be specifically combined with one of the analytes to be determined.

In this case, each of the cell groups is advantageously used for one of the analytes.

Different Serial adapted to carry out the group and can be the same as each other (competition or sandwich) or different (competition and sandwich) test.

According to the embodiment of interest, the group comprising each zone downstream of the labeling zone of states the block final capture zone.

This final capture zone includes a fixed reagent, the specific binding reagent can be derived from the then states the block of the labeled detection reagent.

This embodiment can be used as the reference zone; it allows also the same in such a manner that the end of the catch MAX. by mark of the benefits of the detection reagent, the stated manner such that the labeled detection reagent to MAX. is located downstream of and in order to prevent any interference phenomenon.

To more generally, area group of 7 the release area 4, 6 and complementary capture zone 5 advantageously comprises:

(I) as anti-analyte-labeled detection reagent, such as analytes or analyte analogue specific antibody, with visible and/or measurable marker conjugated,

(Ii) as the analyte itself, or analyte analogue of the capture reagent, fixed in the capillary diffusion tool 2 of the capture zone 5 in at least one of, and

(Iii) fixed capture reagent, analyte specific binding, also advantageously specifically bind anti-analyte, fixed in the capillary diffusion tool 2 another capture zone 5 in.

The latter embodiment allows for in the group in a single area at the same time form the sandwich form testing and competition test.

Special embodiments

According to the 1st embodiment of this invention in Figure 1 in the display.

This device is suitable for determining the qualitative manner that may be present in a target analyte in a liquid sample.

Capillary diffusion tool 2 includes two MAX. 7:

-Having 71, which is composed of complementary upstream release area 4 and an upstream capture zone 51 composition, and

- downstream group 72, which is composed of complementary downstream release area 6 and downstream capture zone 52 composition.

In each of the cell groups 7 in, the combination of the selection of respective reagent as described above for the competition test or sandwich test implementation purpose of the detection of the analyte.

These two MAX. 7 therefore is suitable for the implementation of:

-A single competition test or single sandwich test,

-A group competition test test of the sandwich and the other set.

In any case, each capture zone 5 upstream of the release area 4, 6 allows the presence of the closely adapt to the release area/capture the concentration of the in.

According to the present invention the multi-kind of device can also implement a single kind of analyte determination, the device has its specificity benefits.

Figure 2 and 3 display of such other device, its capillary diffusion tool 2 includes upstream MAX. 71, the upstream block 71 comprising:

-An upstream release area 4, and

-1st capture zone 51, and the upstream release area 4 complementary.

When appropriate, the two kind of variation for directly downstream of the (one or more) zone group 72 is feasible. .

In according to fig. 2 in the 1st variation, capillary diffusion tool 2 includes downstream area group of 72, the downstream area group 72 comprises:

-Downstream release area 6, and then

-A number of continuous 2nd capture zone 52, this is three, and the downstream release area 6 complementary.

This embodiment is provided with the special and reliable semiquantify as a result, the benefits of avoiding hook effect.

According to this embodiment, for measuring hormone hCG, concentration and its reagent can be expected to carry out the adjustment upstream block specificity 71 only during the period of detection corresponding to the missing 1st day of hCG hormone concentration, that is, about 20mIU/mL.

Downstream release area 6 then contain concentration is higher than the upstream 4 existing at the concentration of the detection reagent, allowing the downstream continuous capture a 2nd 52 is detect and distinguish between a plurality of analyte concentration.

In this shape, the period corresponding to the missing 1st day of hCG hormone concentration, so only in the 1st one capture zone 51 found that the detection signal of the visual and measurable.

For correspondent to one or more weeks of pregnancy hormone hCG concentration of, visual and measurable signal will be continuously appears in the downstream release area 6 downstream of the labeling zone 2nd 52 is, to provide the test sample the concentration of hormone hCG semi-quantitative analysis.

According to the same aspect, prostate specific antigen from the fit for the determination (or PSA) detection reagent can be of the "diagnosis value threshold concentration" (4ng/mL) and with the same analyte other more high concentration (up to 200ng/mL) distinguish, also allows semiquantify method.

Similarly, under the same shape, through being suitable for measuring blood (hemoglobin) (FOB or fecal occult blood test) detection reagent can be of the "diagnosis value threshold concentration" (40ng/mL) and with the same higher concentrations of analyte (up to 1000ng/mL and more) distinguish between, thereby providing a semi-quantitative results.

According to fig. 2 the 1st variation of options not statements, upstream block 71 includes 2nd capture zone, also with upstream release area 4 complementary.

The upstream region then capture 2nd 4 and 1st capture zone 51 downstream of, but still in downstream area group 72 of the upstream.

In this situation, the capillary diffusion tool 2 the group comprising upstream 1st region 7, the 1st group region 7 comprising:

-An upstream release area 4,

-Two consecutive upstream capture zone 51, and the upstream release area 4 complementary,

And then 2nd cell groups in the downstream is 7, comprising:

-Downstream release area 6, and

-A plurality of 2nd continuous capture zone 52, this is three, and the downstream release area 6 complementary.

In according to fig. 3 in the 2nd variation, diffusion tool 2 also includes a continuous downstream area group 72, this is three, each comprising:

-Release area 6, and

-Capture zone 52, with the associated release area 6 complementary.

The measurement under the condition of a single kind of analyte, with optimized this embodiment in continuous release and capture the benefits of the detection threshold.

In this variation of, is gradually increased from the upstream to the downstream continuous release area 4, 6 in the concentration of the detection reagent, while at the same time maintaining each capture zone 52 in the same capture reagent concentration may be interested.

This method has a plurality of concentration values diagnosis value (threshold) the detection of the analyte (such as hCG, PSA or hemoglobin) the usefulness of the environment.

According to another method of the present invention, can be for a number of analytes modified release area and the determination of the capture zone.

In this regard, according to fig. 4 the embodiment of the invention, capillary diffusion tool 2 includes a plurality of continuous MAX. 7, in this case, is two, each composed of release area 4, 6 and complementary capture zone 5 composition.

In this situation, upstream block 71 is suitable for through appropriate test (competition or sandwich) determining analyte 1st; downstream area group 72 is suitable for through appropriate test (competition or sandwich) determining analyte 2nd.

This kind of device will also includes one or more of the other additional MAX. 7, each suitable for a kind of analyte to be measured.

In the alternative, according to fig. 5 the embodiment of the invention, capillary diffusion tool 2 comprises a block 7, this is three, each composed of is connected with a plurality of capture zone 5 (two here) of the release area 4, 6 form.

Similarly, each of the cell groups 7 is suitable for through appropriate test (competition and/or sandwich) of a determination of analytes.

In each of the cell groups 7 exist in a plurality of capture zone 5 which makes it possible to obtain each of these analytes a semidefinite value.

These different embodiment are not in any way restrictive. A plurality of cell groups 7 and its respective composition can be adjusted in any way.

Control plot

In the preferred embodiment and as shown in Figure 1 to 5 is shown in the, device 1 also comprises control plot 10, the control plot in the downstream is formed and includes the control capture reagent.

The control zone 10 can have the positive control to ensure that the liquid sample from the capillary diffusion tool 2 of the storage areas 3 to the capture zone 5 the effective capillary diffusion.

The control zone 10 by the permanently fixed in the capillary diffusion tool 2 downstream of the capture reagent.

It can be, for example, then a 4, 6 of the (one or more) detection reagent antibody. In this case, the control capture reagent allows the (one or more) detection reagent of migrating through to be checked capture zone 5.

In the alternative, the control capture reagent is independent of the analyte along and only allow the liquid sample to be checked the capillary diffusion matrix 2 diffusion.

Implementation

Then can be defined according to the method of use or implementation of this kind of device:

A) storing a liquid sample in the capillary diffusion tool 2 of the storage areas 3 in,

B) allowing a period of time spent, the time sufficient to make the liquid sample capillary diffusion migration to capture zone 5, when appropriate, to the control plot 10,

C) in the capture zone 5 achieve the following extent:

C. 1) detecting reagent to analyte composite of MAX. with its 7 of the complementary capture reagent (sandwich shape), and/or

C. 2) observation to MAX. detection reagent with its 7 capture reagent (competitive shape), then

D) from the obtained results of semi-quantitative and/or qualitative and/or quantitative determination (one or more) analyte.

In practice, in accordance with step c) in the capillary migration process, are connected in series to form a continuous through a liquid sample MAX. of 7 each.

In the implementation of the sandwich shape of each of the cell groups test 7 in, any of the analyte from its release area 4 or 6 of the detection reagent to form a composite material.

The complex so formed complementary capture zone 5, here they are corresponding capture reagent immobilized.

So retained in this capture the complex of providing visible and/or measurable signal, in the liquid allows detection of the presence of an analyte in a sample.

When appropriate, the signal intensity increased in the continuous capture area, in the (one or more) MAX. depleted on until the distribution of the complex by mark.

Advantageously through appropriate optical scanner measuring signal intensity. The signal strength, for example, in the all or part of the capture zone 5 is detected that the number of Image elements.

In this context, when it comprises a single kind of analyte a number of continuous capture zone to overcome the hook effect and obtaining semiquantify result, the device of this invention has a special interest.

In this regard, each of the signal intensity of the capture area with other continuous capture compare the intensity of the signal.

The detection reagent and the analyte then formed between a part of the complex by each of the continuous capture zone capture, until the migration of the analyte in a liquid sample is gradually depleted.

Each of the capture area relative to the signal strength of the capture of the surrounded before, until the intensity has reached its maximum value of the capture zone. Then the capture zone is connected with the signal intensity of the capture zone.

The same before the device can be of standard solution is implemented on an analyte in a liquid sample semiquantify result is assigned to the result table.

Can then by combining each of the assigned to pre-capture the concentration of the analyte in a liquid sample the range of change for semidefinite value of the result.

In the implementation of competition patterns MAX. test of the 7 in, prevent any analyte from its release area 4 or 6 and the detection reagent of (one or more) complementary capture zone 5 between the capture agent to form a composite material.

Under the condition in the absence of the analyte, and form the complex is generated at the corresponding capture can see and/or measurable signal.

Similarly, when it comprises a single kind of analyte a number of continuous capture and to overcome the hook effect when the semi-quantitative results, the device of this invention has a special interest.

With respect to the analyte concentration can be semi-quantitative interpretation of the results, such as for example, low, medium, high and very high.

Before the implementation of the same device again in this standard solution will be an analyte in a liquid sample semiquantify result spectrum assigned to the result.

Each capture zone through the pre-specified range of concentration of an analyte in a sample, then the result can be changed to a semidefinite value. Generally speaking, measurement device may also allow for quantitative measurement of the analyte.

Visual results thus obtained can be changed to, for example, in order to for example to be determination of an analyte in a liquid sample or ng/mL mIU/mL the concentration of the said.

For this purpose, the measuring device can be implemented in a standard solution of the analyte in a liquid sample to intensity spectrum designated quantitative value.

For example, in each of the capturing area measuring signal intensity (pixel number).

For each of the cell groups, will then be in these capture area adds measuring the intensity of the signal.

Respectively different MAX. the intensity of the sum of the changed into such a proportion, the proportion of an area corresponding to the intensity of the sum of the group relative to the other the intensity of the adding of the cell groups.

If necessary, to these proportional interpolation, for example linear interpolation, advantageously with each pre-measuring the concentration of the analyte associated with the proportion of the range of the coefficient, in order to obtain the corresponding to the intensity ratio (abscissa) as the concentration of an analyte in a liquid sample (ordinate) of the function of the standard curve.

Then the can be obtained through analysis of the intensity of the sample with the proportion of the analyte in a liquid sample associated with the quantitative value, taking into account the aforesaid standard curve.

In a particular embodiment, the application of the support so as to obtain detection and capture agent:

-Upstream MAX., which comprises two capture zone T1 and T2, then the 1st L1 later, and

- downstream block , comprising three capture zone T3, T4 and T5, in the 2nd release area L2 later.

In this situation, advantageously by the following formula (I) evaluation of the cumulative intensity ratio:

Cumulative intensity ratio = (three downstream capture zone T3, T4 and T5 of the sum of the intensity) / (two upstream capture zone T1 and T2 the sum of the intensity of)

Cumulative intensity ratio makes it possible to construct the signal curve (strength) as the liquid sample to be analyzed in the function of the concentration of the analyte.

Such quantitative analysis embodiment of below 2 further description.

Embodiment

Embodiment 1: used for the determination of analytes with a hook-shaped effect of lateral flow immunochromatographic device

Lateral flow immunochromatography by hCG semiquantify concentration of the test implementation hook effect the determination of the evaluation.

Venue, in order to offset the hook effect, usually in a single release region of the measuring device to provide excessive by the labelled antibody.

Excessive antibody meaning that the antibody concentration is higher than the requested diagnostic interest in determining the concentration of the threshold value.

Fitzerald from, for example, provided by the Company by "mating hCG antibody" (single/polyclonal antibody) constructing multi-kind of device.

According to the standard method (for example by Organon   Teknika Company has developed technology) (Aldrich   Sigma) with colloidal gold labeled anti-hCG antibody.

According to the invention the following details the different arrangements of the (test 1 to 5) in the anti-hCG antibody of the Company Millipore nitrocellulose film on the (NC).

Antibodies to adjust these stored so as to obtain a concentration of from 0.5 to 1 the concentration of g/mL.

The dilution in the urine hCG negative of the different concentrations of the hCG hormone (Sigma) test 4 mm slice (or slug). The test diluent is 0, 25, 50, 100, 1000, 5000, 50000, 100000, 250000 and 500000mIU/mL.

The concentration of these different from normal conditions corresponding to each stage of pregnancy (are pregnant 0, 1, 2, 3, 4, 5, 6, 9 to 12, 13 to 16 weeks).

Testing scheme is applied in the sheet storage zone 150 the each diluent   L; in 3 to 5 minutes, each of the (one or more) capture zone the presence of the observation coloring intensity.

Recording of results and follow the general applies for reading this type of qualitative test standard interpretation result, in other words:

0:no coloring

± : slight coloring, is not clear, -side boundary, ghost line (ghost   line), we can see that becomes

+ : Light colored but clearly visible

++ : Clearly visible line

+++ : Strong line coloring

++++ : Very strong line coloring

Test 1: having a single release area and a single capture zone (test wire 1) of the device

N° 1 in the table below the results obtained in the display.

Table 1

The use of lateral flow immunochromatographic rapid test display the 1000 and 5000mIU/ml between the hCG dose/response curve linear.

Because the hook effect ("high dose hook effect") have begun to emerge from this maximum concentration signal intensity loss

As a result, such testing cannot distinguish between certain of the outcome of the coloring intensity, whether it is the concentration of hCG 50mIU/mL or 100000mIU/mL.

Test 2: with a single release area and two continuous capture zone (board 1 and board 2) of the device

Producing containing (test wire 2) 2nd capture linepiece and of hCG concentration group is then used to test the same.

N° 2 in the table below the results obtained in the display.

Table 2

At the higher concentrations the hook effect. In fact, appears to be introduced into the hook-shaped effect capture line 2nd threshold relocated to the concentration of the higher concentration region.

Test 3: with a single release area and three continuous capture zone (test wire 1, test line 2 and holding wire 3) of the device

Production containing 3rd capture line (test wire 3) of the concentration of hCG piece and is then used to test the same group.

N° 3 in the table below the results obtained in the display.

Table 3

3rd capture line (test wire 3) no longer more migration hook effect. Then this may be due to the depletion of the antibody by mark.

Test 4: with a single release area and three continuous capture zone of the device

According to the test n° 3 conclusion, we maintain the three capture zone at the same time, the single release area of greater concentration of antibodies by mark.

Found that such a configuration tends to have strong background, so that the test is not readable and is not available.

Test 5: has two release area and four capture zone of the device

In the 1st (test wire 1) capture line introduced after then 2nd, capture antibody with 3 a supplementary line. This configuration corresponds to the such as the above with regard to Figure 2 of the embodiment described.

With the same hCG concentration set of test the new platform. N° 4 in the following table summarizes the results obtained in.

Introduced and 3 of a complementary continuous capture line in the 1st 2nd release area allows the assertion that capture line (test wire 1) in which a signal obtained in the plurality of concentration range and, therefore, the concentration range of announced, semiquantify pointing to the stage of pregnancy.

This method provides the hook effect of the solution to the problem.

Embodiment 2:is used in the quantitative measurement of analyte with a hook-shaped effect of lateral flow immunochromatographic device

Material

Chromatographic device comprises as nitration of porous support membrane, preferably, with 8 to 15 microns, and the size of the hole between the 2 to 3 cm wide between.

Sample sheet, and an eyepiece of the conjugated and absorbent porous material is selected from a plurality of selected cellulose and glass fiber paper supplier, thickness of between 0.2 and 1.0 mm depending on application between, porosity in the 100 and 200 microns.

BiosPacific provided by the Company from the pairing of hCG antibody (single/polyclonal antibody) constructing antibody.

In the PBS buffer solution and the anti-hCG monoclonal β-hCG and polyclonal antibody concentration is preferably 1 mg/mL.

According to the standard method (for example by Organon   Teknika Company has developed technology) (Aldrich   Sigma) with colloidal gold labeled anti-hCG antibody.

The anti-hCG antibody of the Company Millipore nitrocellulose film on the (NC).

Antibodies to adjust these stored so as to obtain a concentration of from 0.5 to 1 the concentration of g/mL.

Releasing reagent for the preparation of the colloidal gold (marker) concentration is preferably from 10 to 15OD/mL, particle size is 20 to 60 microns.

Preparation and calibration (NIBSC according to International standards) hCG (Sigma) compared with the range in order to obtain the range from 0mIU/mL to 1000000mIU/mL the hCG concentration, the concentration of the test is to assess the performance and its quality control required. The test diluent is 0, 25,250, 2500, 25000, 250000, 500000 and 1000000mIU/mL.

Method

The nitrocellulose support application detection and capture reagent in order to obtain:

-1st release area L1, advantageously inserted in the PVC sample piece and the membrane on the support between the conjugated substance of the on-chip,

-Two upstream capture zone T1 and T2, then the 1st L1 later,

-2nd release area L2, and

-Three downstream capture zone T3, T4 and T5, in the 2nd release area L2 later.

2nd release area L2 is disposed in the 2nd upstream capture zone T2 and 1st downstream capture zone T3 between between them in order to advantageously ensure that at least 2 mm distance.

Adjusting release area and a capture zone of reagent concentration to ensure that the detection of a given concentration (0 to 1000000mIU/mL) of the desired analyte hCG.

After the administration of reagent, in programming the temperature and the humidity of the dry nitrocellulose support under the conditions of (usually the 37 [...] 1h).

After drying, the fiber support with the nitration of the reagent to the construction of a master form test ("bande   bandelette   testàcouper   de   en"), the support comprising a PCV, printing nitrocellulose membrane, conjugated tablets (then 1st), sample piece and the absorbent sheet.

Sample sheet, conjugated eyepiece and the absorbent sheet with printed is arranged in the membrane the nitration of PVC support of porous material, adjust and optionally immune chemical processing to the actual receive the sample (sample piece), filtration of the sample, with the same releasing area of 1st that the reagent in the reaction (conjugated tablet), is provided on the membrane and the relocation of the final absorption of excessive reagent and termination of migration (absorbent sheet).

Then cut a master in order to obtain from 2 to 6 mm wide, preferably, from 4 mm or 5 mm wide sheet.

Then in the plastic sleeve of the rectangular shape (box shape device) packaging pieces, prepared to use.

Test procedure

Preparing different hCG concentration in order to obtain the crossing from 0 to 1000000mIU/mL hormone hCG concentration range of the control range of the

Each concentration of test two.

Generally speaking, the different test on the unit using each hCG concentration of 100 to 200 micro-liter sample between.

Observing reaction a maximum of ten minutes.

Assessment of the results obtained with the bare hole (concentration range).

Furthermore, with the optical reading tool (instrument or reader) recording the results obtained, the stated tool allowing the measuring of the intensity of each capture line (expressed in pixels).

The quantitative result by the reader provides (the digital result) is calculated from this algorithm, the algorithm of the basic calculated taking into consideration according to the following formula (I) the ratio of the cumulative intensity:

Cumulative intensity ratio (CSR) = (three downstream capture zone T3, T4 and T5 of the sum of the intensity) / (two upstream capture zone T1 and T2 the sum of the intensity of)

Formula (I)

In this experiment the results obtained under the conditions in the following table 5 in the display.

With the liquid accumulated strength ratio of the concentration of an analyte in a sample to increase.

Through interpolation, allowing the accumulated intensity ratio (strength) construct the signal curve in the liquid sample as a function of the analyte concentration.

Then can be from 0 to 1000000mIU/mL on the range of concentration of hCG in a liquid sample, the sample in the presence of the liquid under the conditions of measurement in the measuring device for the signal curve and the accumulated intensity ratio.