CN107102136A - Analytical equipment - Google Patents

Abstract

The invention discloses the analytical equipment for the degree that whether there is and/or exist for analyte in the determination fluid sample in the concentration range of expansion, contain the first analysis and second of analysis, wherein the first analysis bag of analyte is containing first flow path, the flow path has the unique detection zone for the binding reagents for being capable of fixation mark, second of analysis bag with the analyte is containing second flow path, the flow path has the unique detection zone for being capable of fixation mark binding reagents, wherein binding reagents are marked present in detection zone, instruction is provided for the presence and/or degree of analyte in the fluid sample.

Description

Analytical equipment

The application be Application No. 200880011652.8, the applying date be on April 9th, 2008, it is entitled " analysis dress Put " Chinese invention patent application divisional application.

Technical field

The present invention relates to for determining the presence of analyte or analytical equipment, the reagent of degree in the concentration range of expansion Box and method.

Background of invention

In order to detect the analyte in fluid sample, simple sidestream immune analytical equipment has been developed and has been commercialized, See, for example, EP291194.Such device generally comprises porous carrier, and the carrier, which contains, to be combined with target analytes Drying, moveable, mark binding reagents, the binding reagents of immobilization can be also combined in mark binding reagents downstream The analyte provided in detection zone.The combination of mark in detection zone immobilization is detected, the presence for analyte in sample is provided Indicate.

Or, when target analytes are haptens, immunoassay apparatus can utilize competitive reaction, wherein mark The binding reagents of analyte or analyte analog and immobilization in the competition detection zone of analyte present in sample.Or, point Analysis apparatus can be using reaction be suppressed, wherein providing the analyte or analyte analog of immobilization, analysis dress in detection zone Put containing the moveable mark binding reagents for analyte.

When testing and analyzing thing, sandwich immunoassays are the analyses generally selected.But, sandwich assay is not always feasible , such as in the case of small molecule such as haptens, they are not big enough, it is impossible to thereon in combination with two kinds of different combinations Gametophyte.Shown using the dose response curve of the typical lateral flow devices preparation using sandwich assay method, the level of signal Increase with the increase of analyte, curve tends to be steady when the point of higher analyte level.In higher analyte During level, due to preferably capturing the analyte not combined with the reagent of mark also in detection zone, signal starts reduction.It is this existing As being referred to as hook effect (hook effect).Therefore, because the semaphore or strong observed under higher analyte level Degree may be identical or even lower with what is observed under relatively low analyte level, and sandwich immunoassays show limited point Analyse scope.

Competition or inhibition analysis method typically provide high RST in zero or low-level analyte.When analyte level liter Gao Shi, dependent on the ratio marked with reference to the amount of species and the amount of analyte of presence, signal level is still probably high.When When analyte level continues to increase, signal starts reduction, because uncombined analyte or the analyte with mark or analysis Thing analog competes the binding reagents of immobilization, or is combined with mark binding reagents, reduces mark binding reagents in detection The combination in area.

Therefore, the analyte in expanded scope, the problem of can be potentially encountered relevant hook effect are measured using sandwich assay. High analyte concentration starts to produce the reduction of signal Analysis.Competition or inhibition analysis cause analysis under high analyte concentration The loss of signal, so that the scope that analyte can be measured is restricted.

Therefore, above-mentioned analysis method is not suitable for measuring analyte level in the analyte ranges of expansion.

The method that US2005/0112780 discloses analytical equipment and the dynamic detection range for extensive diagnostic device, bag Include detection zone and the flowing through property porous carrier in the compensating basin in detection zone downstream is provided.Detection is related to the first binding reagents, should Reagent is combined with detection probe, produces the detection signal that intensity is directly proportional to the amount of analyte, compensating basin contains second of capture Reagent, the reagent is combined with detection probe, produces the signal being inversely proportional with the intensity of detection signal.Analysis can be included further The calibration areas of 3rd generation signal.The first binding reagents can be selected from antigen, haptens or streptavidin.The first and the Two kinds of binding reagents can be selected from many species, including antigen, haptens or streptavidin.

US2004/0197820 discloses the flowing through property porous carrier analysis for being used for containing detection zone reducing hook effect Device, wherein described device can include the calibration areas in downstream.

US2006/0019404 discloses the analytical equipment of the dynamic range with extension, contains lateral flow test strip, the survey Strip contains multiple detection zones, and they have the sensitivity gradually reduced to analyte concentration.Analytical equipment can contain two kinds Respectively there is the carrier of multiple detection zones.The amount of label/signal present in multiple detection zones is detected, to determine the dense of analyte Degree.

EP462376 discloses the analytical equipment that site is reclaimed containing capture site and conjugate, and wherein conjugate reclaims position Point is received and with reference to the conjugate or conjugate compound for migrating across the capture site, and wherein passes through detecting position The conjugate of the immobilization in site and capture site is reclaimed in conjugate, to determine the amount of target analytes.

Present inventors have demonstrated that, it is multiple for testing and analyzing thing for being provided wherein on same porous carrier For the analytical equipment of detection zone, the combination of upstream test section can change the binding characteristic of downstream test section, and upstream is examined Survey the linkage change that any change of area's combination can cause downstream test section to combine.Under compared with high analyte thing concentration level especially In this way, may simultaneously cause the poor accuracy of analysis.Furthermore, it has been found that in the running of test, present in detection zone May occur intersection between corresponding binding reagents to join, during the manufacture of device and they are preserved in the dry state, Also intersection is observed to combine.This shows has an impact to the accuracy and sensitivity level of analysis.In the prior art, these Seem not to be realized also before problem.

It is an object of the present invention to provide improved analytical equipment, kit and method, to expand the analyte of the analysis Scope.

The content of the invention

The on one side, and the invention provides for determining depositing for analyte in fluid sample in the concentration range of expansion And/or degree analytical equipment, comprising the first analysis and second of analysis, wherein the first analysis bag of analyte is containing the One flow path, with the unique first analysis detection zone for being capable of fixation mark binding reagents, second of the analyte Analysis bag is containing second flow path, with the unique second analysis detection zone for being capable of fixation mark binding reagents, wherein detecting There are mark binding reagents in area, instruction is provided for the presence and/or degree of analyte in the fluid sample.

The first analysis can provide instruction for the analyte level in the first concentration range, and second of analysis can be Analyte level in second of concentration range provides instruction.

The first and second of concentration range are different from each other.The first and second of concentration range can be overlapped, to carry For continuous concentration range.

Analytical equipment for analyte level can provide instruction relative to one or more threshold values.Analytical equipment can be low In or higher than multiple threshold values analyte level provide instruction.For example, the quantity of threshold value can be two, three, four, five Or more.Analytical equipment can contain the threshold value of one or more storages, and the value each stored both corresponds to the level of analyte.

The analysis of the first and second independently or can be combined to provide for the analyte level in the range of some and refer to Show.

The first analysis can provide instruction for the analyte level less than or equal to first threshold value, and second of analysis can To provide instruction for the analyte level higher than the 3rd threshold value.First and second analysis together can be more than or equal to First threshold value but the analyte level offer instruction for being less than the 3rd threshold value.

It can be provided in the detection in the form of immobilization for the binding reagents of analyte or analyte analog.Knot Closing reagent can select from for the binding reagents of target analytes, analyte or analyte analog, and this depends on analysis It is the analysis of sandwich-type or the analysis of type of competition.Similarly, mark binding reagents can include being directed to target analytes Mark binding reagents, mark analyte or mark analyte analog.

Or, reagent can be provided in the form of immobilization being capable of binding marker binding reagents-knot of analyte-second In the detection zone for the compound for closing reagent.For example, second of binding reagents may be provided in moveable form, with combining species For example biotin is combined or is otherwise attached to, and the reagent for being fixed on detection zone is complementary binding partners, for example chain parent With plain or anti-Avidin antibody so that in mark binding reagents-binding reagents of analyte-second of detection zone formation immobilization Compound-biotin-streptavidin compound.

Second of binding reagents may be provided in moveable form, and it fixation mark can combine examination in the detection Agent-analyte complex.For example, second of binding reagents can be combined with particle such as agarose or white latex, detection zone The filter of the granularity of particle but greater than flag binding reagents can be less than containing pore-size so that filter can intercept and capture presence The compound of any mark binding reagents-binding reagents of analyte-second, and any mark not being combined with capture agent Binding reagents can pass through filter.

The first and/or second of analysis can include mark binding reagents, and it is before use device, may move shape Formula is provided in the upstream of the analysis detection zone of the first and/or second of drying regime.

In the unlabelled combination for the immobilization that the analysis of the first and second can be provided on each comfortable each detection zone There is provided have moveable mark binding reagents for trip.

Analytical equipment can contain two or more analyses, every kind of to detect certain concentration range or be higher than or low In the analyte of one or more threshold values.

The analysis of the first and second can be alone or altogether whether the analyte or analyte of specified level are high In or less than the offer instruction of some threshold value.

Analytical equipment can have the common sample applying zone being in fluid communication with multiple flow paths.Therefore, it is applied to dress The fluid sample for the common sample applying zone put can be transported to corresponding detection zone along the flow path of corresponding analysis.

As the alternative solution that the first and second of analysis are provided in single analytical equipment, analysis may be provided as Separated analytical equipment, the result from each device, which is combined, can provide instruction or measure for the level of analyte.

Therefore, second aspect, the invention provides in the concentration range of expansion determine analyte presence and/or The assay kit of degree, containing first analytical equipment and second analytical equipment, wherein first and second analysis dress Put respectively containing first aspect present invention the first and second of analysis.

The third aspect, the invention provides presence and/or the journey for determining analyte in the analyte ranges of expansion The method of degree, comprises the following steps：

A) fluid sample is added to offer and detection zone upstream is analyzed at unique first, containing moveable mark knot Close the first analysis of reagent and provide and detection zone upstream is analyzed at unique second, examination is combined containing moveable mark In second of agent analysis, the detection zone can fixation mark binding reagents, wherein detecting the reagent of mark in detection zone, be The degree of analyte and/or presence, which are provided, in fluid sample indicates.

B) analysis result is read.

In the case where the level of known analyte changes as the function of time, such as corpus luteum hormone hCG, analytical equipment Time-based instruction, such as pregnant degree by day or in units of week can be provided for user.

For purposes of the present invention, term " flow path " is to refer to liquid being transported to second position from first position Matrix, can be such as capillary channel, microfluidic pathways, or porous carrier such as lateral flow porous carrier.Porous carrier can be with Comprising one or more porous carrier materials, they are overlapped in the way of linear or stacked arrangement, or be in fluid communication.It is many Hole carrier material can be with identical or different.First and second flow path can be provided on separated matrix, or they can To provide on common matrix so that cannot pass through the stream of second of analysis along the liquid of the flow path conveying of the first analysis Footpath.For example, the first and second of analysis can be provided on same porous carrier so that first and second flow path that This isolation.This can for example, by be cut by laser the part of porous carrier make its become it is non-porous, so as to by first and second Flow path separates realization.Alternatively, first and second detection zone can substantially be arranged side by side, and carry For on same flow path so that either of which is provided without in another downstream.

Specifically, flow path can include lateral flow porous carrier.The mark binding reagents and detection zone of each analysis can be with It is respectively provided on different carrier materials.May be used as porous carrier includes nitric acid fibre to provide the suitable material of detection zone Tie up element, acetate fiber, cellulose or cellulose derivative, polyester, polyolefin or glass fibre.Porous carrier can include nitric acid Cellulose.It this have the advantage that binding reagents need not be chemically treated in advance and can be just firmly fixed.If porous solid Phase material includes such as paper, and fixation of the antibody in Two Areas needs to use such as CKBr, N,N'-carbonyldiimidazole or three fluoro second Alkanesulphonyl chlorides, are carried out by being connected chemically.

Analysis method can be provided in the form of analytical test strip, and wherein fluid sample flows along the test-strips.

Term " binding reagents " refer to combine to member, i.e., two kinds different molecules, one of which molecule passes through chemistry Or physics mode is combined with second of molecular specificity.Both molecules, are bonded to each other at them and are so that they can be by it Binding partners with similar quality other analysis group subregions separate in the sense that, be related.Specificity knot Close to member be referred to as part and acceptor (counter ligand), with reference to member and combining to gametophyte, etc..Molecule can also be The combination of molecular aggregate is to member；For example, the antibody that is produced for the immune complex of second of antibody and its corresponding Antigen is considered the combination of immune complex to member.Binding reagents can include antibody or can be with antigen binding Antibody fragment.

In addition to antigen and antibody binding are to member, others are combined to including, but not limited to, e.g. biotin and affine Element, carbohydrate and agglutinin, complementary nucleotide sequence, complementary peptide sequences, effector and acceptor molecule, the co-factor of enzyme and Enzyme, the inhibitor and enzyme of enzyme, peptide sequence and specificity are for the antibody of the sequence or intact proteins, the bronsted lowry acids and bases bronsted lowry of polymerization, dyestuff With protein conjugates, peptide and specific proteins conjugate (such as ribalgilase, S- peptides and ribonuclease S-protein), etc. Deng.In addition, specific binding is to that can include the member of the analog of original specific binding members.

" label " when used in mark binding reagents environment in when, be refer to generation can be by vision or instrument means Any material of the signal of detection.Being suitable for the various labels of the present invention is included by chemically or physically producing signal Label, can for example pass through optical detection.Such label includes enzyme and substrate, chromophore, catalyst, fluorescence chemical combination Thing, chemiluminescence compound, electroactive species, dye molecule, radioactive marker and particulate labels.Analyte in itself may be used Can inherently produce detectable signal.Label can be with binding reagents covalent bond.

Label can include particle such as gold, silver, colloid non-metallic particle such as selenium or tellurium, dyeing or coloring Grain is for example mixed with dyestuff or the polymer beads of dye sols.Dyestuff can have any suitable color, such as blue.Dyestuff Can be fluorescence.Dye sols can from commercially available hydrophobic dye such as Foron Blue SRP (Sandoz) and It is prepared by Resolin Blue BBLS (Bayer).Suitable polymeric marker thing can be selected from the category of synthetic polymer, example Such as polystyrene, polyvinyl-toluene, polystyrene-acrylic acid and polyacrolein.The monomer used be typically it is water insoluble, Emulsified in aqueous tenside to form monomer micelle, it is then poly- to induce by adding initiator in emulsifying agent Close.Produce made of substantially spherical polymer beads.According to exemplary embodiment, label is blue polymer particles.

Fluid sample can come from any source, for example industry, environment, agricultural or biological source.Sample can come from or Physiological sources are made up of it, including blood, serum, blood plasma, interstitial fluid, saliva, sputum, crystalline body fluid, sweat, urine Liquid, milk, mucus, synovial fluid, peritoneal fluid, percutaneous diffusate, pharyngeal diffusate, BAL fluid, tracheae suction out liquid, Cerebrospinal fluid, seminal fluid, cervical mucus, vagina or urethral secretions liquid and amniotic fluid.Specifically, source is the mankind, specifically, Sample is urine.

Analyte includes but is not limited to toxin, organic compound, albumen, peptide, microorganism, bacterium, virus, amino acid, core Acid, carbohydrate, hormone, steroids, vitamin, medicine (including applied due to therapeutic purposes and because violated purpose is applied Medicine), pollutant, the metabolin or antibody of insecticide and any of above material.Term analyte further comprises any Antigenicity substance, haptens, antibody, macromolecular, and combinations thereof.

Specifically, analyte is HCG (hCG).Analyte can have unique land or Epitope, or can have more than one land.For example, analyte hCG contains and luteinizing principle (LH), ovarian follicle Stimulin (FSH) and thyrotropic hormone (TSH) identical α subunits, and the exclusive β subunits of hCG.For the anti-of α and β subunits Body can be used for combining hCG in sandwich immunoassays form.

The analytical equipment of the present invention can be used for degree or the presence that hCG is measured in the concentration range of expansion.Scope can be with Change between about 10mIU to about 250,000mIU.

According to embodiment, device can measure the amount of hCG in fluid sample, and according to the reference value of storage with based on when Between unit indicate user pregnancy degree.Device can also be by determining that hCG level is above or below basic threshold Value, comes whether denoted object is pregnant.Reference value and threshold value are generally stored in the part as algorithm in device.Basic threshold one As can be in the range of 10-25mIU/ml.

According to embodiment, the first analysis can be above or below basic threshold according to the hCG levels detected To indicate respectively whether object is pregnant, and/or if pregnancy, indicate in first scope less than or equal to first threshold value Interior hCG levels, second of analysis provides finger for the hCG levels within the scope of second more than or equal to second threshold value Show, wherein the first and second of analysis is combined, for more than first threshold value but less than the 3rd model of second threshold value HCG levels in enclosing, which are provided, to be indicated.

The first and/or second of analysis can further include check plot, and the operation for indicating analysis test is order People's satisfaction, i.e., reagent is present in test device, and they are changed into mobile and along flow path during testing results Transport.Check plot can also indicate that immunochemistry interaction can occur for the reagent in device, it was confirmed that device it is chemical complete Whole property.When considering that device is stored and transported in certain temperature range in dry conditions, this is very important.Check plot The downstream of detection zone is normally at, the secure bond reagent for example for marking binding reagents can be included.Mark binding reagents Check plot and the upstream of detection zone can be present in moveable form.Mark binding reagents and the mark knot for analyte Closing reagent can be with identical or different.

Analytical equipment, which can be included, to be in fluid communication with first and second flow path and is located at the porous sample of their upstreams Recipient.Porous sample recipient can be shared for two kinds are analyzed.Therefore, the common sample for being applied to device is applied Plus the fluid sample in region can be transported in corresponding detection zone along the flow path of corresponding analysis.Porous sample recipient can be with There is provided in the inside of casing, or can at least partly stretch out the casing, can be used for for example collecting uroflow.Porous sample receives Device may be used as fluid reservoir.Porous sample receiving element can quickly absorb the bibulous many of liquid by any Hole or fibrous material are made.The porosity of material can be it is unidirectional (i.e. the trend in hole or fiber entirely or primarily parallel to The axle of element), or multidirectional (omnidirectional so that element has unbodied spongelike structure).Porous modeling can be used Expect material, such as polypropylene, polyethylene (preferably with very high molecular weight), Kynoar, ethylene vinyl acetate, propylene Nitrile and polytetrafluoroethylene (PTFE).Other suitable materials include glass fibre.

" groove " is absorbed it is possible if desired to be provided in the distal end of carrier material.Absorbing groove can be by such as Whatman 3MM Chromatographic paper is constituted, it should provide enough adsorption capacities, to allow any uncombined mark binding reagents to be eluted from detection zone Out.As the alternative solution of such groove, it is sufficient that length of porous solid phase material is extended to outside detection zone.

It is applied to after detection zone, remaining porous solid phase material can be handled by binding reagents, blocks Any remaining binding site.Blocking can be for example, by with albumen (such as bovine serum albumin(BSA) or lactoprotein) or using polyethylene Alcohol or monoethanolamine or its combination are handled to realize.In order to help to mark binding reagents when porous carrier is by sample wetness from By moving, porous carrier can also containing sugared such as sucrose or lactose, and/or other materials, for example polyvinyl alcohol (PVA) or Polyvinylpyrrolidone (PVP).Such material for example can be deposited on as aqueous solution will apply mark binding reagents In region.Such material can be applied on porous carrier as the first application thing, then apply label；Or, this The material of sample can also be mixed and be applied on porous carrier with label, or the two is combined.Such material can sink Accumulate at mark binding reagents or its upstream.

Or, porous carrier can not be blocked during fabrication；But be included in the instrument for blocking porous carrier porous In the material of carrier upstream.When test-strips are wetted, the instrument of porous carrier is blocked to be moved, blocking instrument is flowed into and through Porous carrier, is blocked when flowing and carrying out.Blocking instrument includes albumen such as BSA and casein, and polymer is for example PVP, PVA, and carbohydrate and detergent such as Triton-X100.Blocking instrument may reside in macropore carrier material.

Nitrocellulose porous carrier can have at least about 1 micron of pore-size, be greater than about 5 microns, And such as about 8-12 microns.

Nitrocellulose porous carrier can be lined with such as plastic sheet, to increase its manipulation strength.This passes through in a piece of back of the body Lining material is for exampleUpper formation a thin layer nitrocellulose, can easily be manufactured.

Dry binding reagents can be provided in the porous carrier material that the porous carrier materials upstream containing detection zone is provided On material.The porous carrier materials of upstream can be macropore.The carrier material of macropore should protein binding it is low or do not have, Huo Zheying This easily can be blocked by reagent such as BSA or PVA, to minimize non-specific binding and be easy in macropore main body The reagent that is marked after being soaked by fluid sample is moved freely.If desired, macropore carrier material can with surfactant or Solvent pre-treatment, to assign its more hydrophily and promote the quick intake of fluid sample.Suitable macropore carrier material bag Include plastic material such as polyethylene and polypropylene, or other materials such as paper or glass fibre.It can be examined in mark binding reagents use In the case of the particle marker of survey, macropore main body can have the hole chi bigger at least 10 times than the maximum particle size of particulate labels It is very little.Larger pore-size is capable of the reagent of preferably release mark.As the alternative solution of macropore carrier, binding reagents are marked In the nonporous matrix that can be provided in the offer of detection zone upstream, the nonporous matrix forms a part for flow path.

The analysis of the first and/or second can comprising provide the upstream of nitrocellulose porous carrier and distal end with The overlapping glass fibre macropore carrier of nitrocellulose porous carrier.

The degree of analytical equipment or kit also further containing one or more mark species for being used to determining to exist with/ Or the instrument of amount.For example, optical tooling include optical inspection tool, such as photodetector and one or more light sources such as LED, The position of light source allows to carry out illumination optical to detection zone, to determine the degree and/or amount of the mark species existed.Analysis Device can also include one or more power supplys, calculating instrument, signal transduction instrument, algorithm, show tools, storage instrument sum According to input/output end port.Analytical equipment can contain casing, for accommodating the other of the analysis of the first and second and device Part.Device can the threshold value containing storage.

Analytical equipment typically contains the casing for including analysis.Casing can be fluid-tight, and with suitable plastics material Expect that such as ABS is built.Analysis can also include the sample receiving element for being used for receiving fluid sample.Sample reception element can be from Extend in casing.

Casing can be built with fluid-tight material.Casing also suitably completely cuts off the light of surrounding.If fewer than 10%, preferably The visible ray incided outside device less than 5%, most preferably in less than 1% is penetrated into the inside of device, then casing or shell quilt It is considered substantially to completely cut off ambient light.For example poly- carbonic acid of light tight synthetic plastics material containing appropriate photoresistance off-color element Ester, ABS, polystyrene, general purpose polystyrene (polystyrol), high density polyethylene (HDPE) or polypropylene, are to be used to manufacture casing It is appropriately selected.Eyelet can be provided in the outside of casing, is connected with providing the analysis in casing internal space.Or, hole Eye can be used for allowing porous sample receiver to extend to the position outside casing from casing.

The analysis of the first and second can be with offers that be for example arranged side by side, or is arranged on separately with one of which analysis A kind of face-to-face arrangement above analysis is provided.Device can contain single photodetector, for detecting the detection zone of the two.

In addition to the check plot in the case of the detection zone for measuring corresponding analysis and presence, optical tooling can also be measured The part of binding reagents without drying regime in reference region, i.e. flow path.

The purpose of reference region is to provide signal value, and the signal value obtained in detection zone can be compared with it.Reference region Measurement can measure the reflection from flow path or the background level of transmitted light.Background level can be due to such as porous carrier Light reflection, the component of fluid sample or analysis for example mark the presence of binding reagents.Therefore, the light water measured in detection zone Flat to be corrected relative to the level of bias light, the mark existed to provide the signal designation detection zone compensated, which is combined, to be tried The amount of agent.Any deviation that measurement in reference region can also be directed between the fluid sample being applied on analytical equipment is mended Repay, such as urine sample possible difference in color is very big.

Analytical equipment for measuring analyte level in fluid sample, can be used comprising the optical inspection tool installed The intensity of the light reflected in measurement from the detection zone of analytical equipment, check plot and reference region.Optical tooling can comprising one or Multiple light sources such as LED and one or more photodetectors.

Analysis generally can mark binding reagents to be accumulated in test and check plot through occurring after a while, during this period On.The typical time period of analysis test for determining hCG in urine is 3 minutes.The analysis testing time can automatically begin to, for example When being measured to fluid sample by optical element and having had arrived at a part for flow path or porous carrier.

Suitable light source is LED.LED color is determined by the color of mark binding reagents.For blue markings thing, Suitable LED color is red.LED can be irradiated with specific frequency, to irradiate the specific region of analytical equipment. Light reflects or is sent on photodetector from the region, and photodetector records electric signal.The quantity of the electric signal of record will Depending on LED running frequency, therefore one or more signals can be recorded with the time.Signal is typically expressed as % absorbances (%A).Signal can be determined after the All Time of analysis test, or can be more early measured, such as more than specific After signal threshold value.

Each measurement zone is general by single LED illumination.Photodetector can detect the light from more than one measured zone, Therefore the reflected light from more than one LED can be detected.This can carry out irradiation process to realize by order so that device The light being able to know that from which region reflection reaches photodetector.During analyzing, the irradiation process of order can be with fixation Or the frequency of change repeated so that the level of signal level in each region with the time can be monitored.Analyzing bar can be with Place, photodetector and light source are located at the top of bar plane arranged in parallel so that detection control and reference region towards light source and Fluorescence detector.

Device can add the instrument of liquid stream comprising detection into analytical equipment.For example, can monitor from one or more The change for the light level that region detection is arrived, to determine whether and when fluid sample is applied on device.Analyze the timing of test It can automatically begin to, such as when fluid sample reaches specific region.

Device can comprising flowing control instrument, wherein from one or more region detections to light level change can use In it is determined that whether and when fluid sample is applied on device, and by measuring the stream between one or more measured regions Move the flow velocity to determine fluid sample along device.The determination of flow velocity may be used as another quality control inspection, if for example Flow velocity is more than or less than setting level, and analysis can cancel.Counting circuit can respond to signal, calculate fluid along load The flow velocity of body flowing, the flow velocity calculated and upper and lower bound is compared, if the flow velocity calculated is in upper and lower bound Outside, then analysis result is cancelled.

Typical Systems for optical inspection will include at least one light source and at least one photodetector (such as pole of photoelectricity two Pipe).It is preferred that light source be light emitting diode or LEDs.Reflected light and/or transmitted light can be measured by photodetector.For this The purpose of disclosure, reflected light refers to that the light from light source reflexes to light detection from porous carrier or other liquid transporting carriers On device.In this case, detector be usually provided in carrier with light source identical side.Transmitted light refers to through load The light of body, general detector is arranged on the side opposite with light source of carrier.In order to carry out reflection measurement, it can be carried to carrier For backing, such as white reflectivePlastic layer.Therefore, the light from light source will fall on carrier, it is some will be from Its surface is reflected, some to be penetrated into carrier and in the reflection of any depth, until and including be provided with the depth in reflecting layer.Cause This, the measurement of reflection type actually contains at least part thickness that light transmission crosses porous carrier.

Analytical equipment generally comprises one or more holes or window, and light can be by them from one or more irradiation sources On the specific region for being radiated at analysis or analysis bar.Window is used for the area for limiting light on the specific area, and determines to divide Which part of analysis or test-strips is illuminated.Each illuminated region can have corresponding window.Therefore, with 4 surveys 4 windows will be had by measuring the device in region.The light reflected from window is collected by one or more photodetectors.For containing with For the analytical equipment of the flow path in multiple regions, fluid sample can be measured and move the required time between zones.

Can light that periodically (such as substantially per second twice) measurement is reflected from each window, low-pass digital filter can be used Device excludes noise and makes data smoothing.Filtered value can be used for detection flowing and determine analysis result.

For each window, arrived when being dried by using the particular measurement region in flow path, in any fluid sample Measured value (" calibration value ") before up to the region, divided by when measured zone is wetted and line may have begun to show Measured value, can calculate ratio.The ratio is equal to causes the reflection of flow path as fluid sample along the result that flow path passes through After property changes, the ratio of the light of reflection.For example, when flow path includes porous carrier such as nitrocellulose, reflectivity The change of matter may be quite notable.

For each window, in the window ratio of reference, control and test window, t when being dried equal to porous carrier The measured value of=0 (before sample is added), divided by add the measured value of t times after sample：

For each time point t, the window ratio of each window can be assessed as follows：

The calculating of filtered %A values

For each time point t, these ratios of p-wire and control line can be used, using reference ratio as Either with or without in the window for showing line may occur background baseline, to calculate %A.

Analytical equipment can be containing the control threshold value (CLT) stored, if wherein the signal value arrived for blank determination< CLT, because control line does not show fully, as a result will be rejected, and if described value>CLT, will determine that control is satisfactory 's.

According to embodiment, analytical equipment can be containing two analytical test strips, and each contains porous carrier, wherein one Individual analysis is high sensitivity (HS) analysis, that is, it is sensitive to analyze to the analyte level under harmonic analysis thing concentration, and another is Muting sensitivity (LS) is analyzed, that is, is analyzed for being sensitive compared with the analyte under high analyte thing concentration.Specifically, analyte is hCG。

Analytical equipment can comprising two test (detection) areas, each analysis test-strips all contain test section, reference region and Check plot.The signal in HS and LS areas can be measured, it is possible to be defined as follows：

The filtered %A values in HS and LS areas can be defined as follows：

By using reference (ref.) window ratio and the window ratio of (control or test window) that is considered difference divided by Reference window ratio is simultaneously multiplied by 100%, gives normalized relative attenuation percentage (%A).

%A values generally will be in the middle value obtained of complete analysis developing time (FDT).

Flow detection and confirmation

Flow detection

The window ratio of each window can be used for detection by the flow of fluid of window.When ratio reduces flow detection threshold When being worth percentage (FDT%), flowing is classified as having arrived at window.This is same higher than its corrected value equivalent to filtered value Ratio.

For time t,

Or

For each window, the time when standard first fit is recorded, is confirmed for flowing.

Flowing confirms

Various parameters corresponding with mobile phase can be stored in a device, and for fluid sample along analytical equipment The flowing of porous carrier is classified.Device can show any flowing error caused due to use device.

When device can contain the minimal flow detection time (Min FDT) of one or more storages, maximum fluidity detection Between (Max FDT), minimum window passage time (Min MTT) and flow detection threshold value (FDT).

Device can include the threshold value of many storages, such as control line threshold value.Value greater than or equal to the threshold value can be by Effective control is determined as, and can be determined that it is invalid control less than the value of the threshold value, be i.e. test will be rejected.

The measured value that analytical equipment can also include one or more storages overflows parameter, if wherein any measured value is big In or much smaller than desired value, as a result it will be rejected.This can be such that analytical equipment repels in such as hardware fault, such as wiring board Disconnected or short circuit, dead battery, optical window are kept off, LED failure etc..

Analytical equipment can contain other threshold values, such as early stage decision threshold (EDT), if wherein signal is in test process The middle any time exceeds the threshold value, then provides earlier results, i.e., the time rating spent earlier than test run, (complete signal showed Between current).In the case where carrying out hCG measurements, the instruction of pregnancy will be provided by show tools.Analytical equipment can also contain Corresponding to minimum presentation time (MDT) storage values, wherein when MDT is when being exceeded, analytical equipment will only provide early stage knot Really.

The threshold value of various storages can be stored in a device, be used as the part of one or more algorithms.

According to embodiment there is provided the analytical equipment that hCG analytes are detected in urine, wherein described device is included：

Illumination optical and detection instrument for irradiating and detecting the mark binding reagents in detection zone；For calculating hCG The calculating instrument of level or value corresponding to hCG levels；Show tools for showing analysis test result；The basic threshold of storage The value instruction that value, wherein hCG levels correspond to less than the basic threshold of storage is not pregnant, and wherein hCG levels are corresponded to Or indicate pregnancy higher than the value of the basic threshold of storage；The threshold value of two other first and second storage, wherein hCG levels Indicate that pregnancy level is within the scope of first corresponding to higher than basic threshold but less than or equal to the value of first threshold value, hCG Level corresponds to higher than basic threshold and indicates that pregnancy level is within the scope of the 3rd higher than the value of second threshold value, hCG water It is flat to correspond to higher than basic threshold and indicate that hCG levels are in greater than or equal to first threshold value but less than the value of second threshold value Within the scope of 3rd, wherein show tools can indicate the degree of non-Pregnancy status or Pregnancy status and pregnancy.

The first analysis is different from second of analysis so that corresponding analysis can measure the analyte of varying level.

For example, the first and second of analysis can use different analytical structures, such as the first analysis uses sandwich Association reaction, second of analysis is using competition or suppresses reaction.The first analysis can include the pin provided in detection zone upstream To the moveable mark binding reagents of analyte, the detection zone contains the unlabelled immobilization combination examination for analyte Agent, second of analysis can there is provided for may move binding reagents comprising the moveable binding reagents for analyte The upstream of the unmarked binding reagents of immobilization.Or, second of analysis can include moveable labelled analyte or analyte There is provided in the upstream of the unmarked binding reagents of immobilization for analyte or analyte analog for analog reagent.For example, folder Heart analysis can be high-sensitivity analysis, i.e., it can measure the analyte in low concentration scope, and suppress or competition point Analysis can be muting sensitivity analysis, i.e., it can measure the analyte of higher concentration degree.

Analytical equipment can be with for example, contain the first and second of analysis, the unmarked combination examination of wherein the first analysis Agent is different from the unmarked binding reagents of second of analysis, and/or the mark binding reagents of the first analysis and second of analysis Mark binding reagents it is different.For example, this can be that concentration is different, or to analyte, analyte analog or binding reagents Compatibility is different.The analyte sensitivity that high-affinity binding reagents will compare the binding reagents of low compatibility is high.Similarly, it is low Concentration binding reagents are low by the analyte sensitivity than high concentration binding reagents.The first analysis and second of analysis can pass through This mode changes so that they can determine the analyte of various concentrations degree.

Therefore, analytical equipment can be analyzed containing the first of high analyte thing sensitivity, and it, which contains, is provided with detection zone Second of analysis of trip, the removable marking binding reagents with finite concentration or compatibility, and harmonic analysis thing sensitivity, it Containing being provided with detection zone upstream, the moveable mark binding reagents with low concentration or compatibility.For choosing or additionally, The first analysis contains finite concentration or the immobilization binding reagents of compatibility in detection zone, and second of analysis can be in detection zone Contain the immobilization binding reagents with low concentration or compatibility.

Sensitivity for analysis can be manipulated by changing the ratio of binding reagents and label.If being used as mark using particle Remember thing, then the amount for the binding reagents being applied on label can be changed.Another lever for manipulating sensitivity for analysis is to change Become the amount of the label used in analysis is determined.For example, can be by reducing binding reagents and for marking binding reagents The ratio of species is marked, to reduce sensitivity for analysis.Therefore, analytical equipment can include point of the first high analyte thing sensitivity Analysis and the analysis of second of harmonic analysis thing sensitivity, wherein the first analysis contain removable provided in detection zone upstream Grain mark binding reagents, they have the ratio of binding reagents and particulate labels, and wherein second analysis bag exists containing offer The removable particle marker binding reagents of detection zone upstream, they have the binding reagents and particle marker less than the first analysis The ratio of thing.

Another means of manipulation sensitivity for analysis are the optical density for changing label.By using the low mark of optical density Thing, sensitivity for analysis can be reduced.This can be for example, by providing the polymer beads label or logical with low concentration dyestuff Cross and realized using to the relatively low colored labels of fluorescence detector sensitivity.Therefore, analytical equipment can be high containing the first The analysis and the analysis of second of harmonic analysis thing sensitivity of analyte sensitivity, wherein the first analysis bag are containing offer in detection zone The removable particle marker binding reagents of upstream, the label has optical density, and wherein second analysis bag exists containing offer The removable particle marker binding reagents of detection zone upstream, wherein the label has the optical density lower than the first analysis.

The another way of measurement high analyte thing level is to use non-particulate mark binding reagents.When passing through sandwich combination During analysis measurement, high-caliber analyte needs high-caliber binding reagents.Label is the situation of particulate labels wherein Under, high-caliber analyte is provided within or on porous carrier may cause steric hindrance, cause sensitivity for analysis poor.Phase Instead, may be because optical density is low and produces low signal using non-particulate mark binding reagents under relatively low analyte level.But It is that under high analyte thing level, non-particulate label can exist with easily detected enough high levels.Therefore, analysis can So that comprising the first high analyte thing sensitivity analysis, it, which contains, is provided with detection zone upstream, optically detectable particle marker knot Close reagent, and second of harmonic analysis thing sensitivity analysis, it, which contains, is provided with detection zone upstream, optically detectable non-particulate Mark binding reagents.The example of optically detectable non-particulate label can be dyestuff.Dyestuff can be fluorescence.

Sensitivity for analysis may be influenceed by the flow velocity of porous carrier.The method of reduction sensitivity for analysis is to use to have The porous carrier (such as nitrocellulose) of high flow velocities.Therefore, analytical equipment can contain the first high analyte thing sensitivity Analysis, it contains the porous carrier with certain flow rate, and second of harmonic analysis thing sensitivity analysis, and it contains velocity ratio the One kind analyzes higher or faster porous carrier.

For choosing or additionally, the speed that can be discharged by changing mark binding reagents to be originated from it is sensitive to manipulate analysis Degree.Another method of reduction sensitivity for analysis is from many during being contacted with fluid sample there is provided mark binding reagents The quick release of hole carrier.Mark the release of binding reagents can be by providing sugar, albumen or other polymeric materials in a device For example Methyl cellulose usually changes.Such material can be provided in the trip near or on of binding reagents.

The use of scavenger reagent

Another method of reduction analyte sensitivity is to provide the scavenger binding reagents combined with analyte.Scavenger Binding reagents can be provided in the upstream of detection zone, can be immobilization, it is moveable or the two.Scavenger binding reagents can With provide on porous support with moveable binding reagents identical region, or its upstream or downstream.Scavenger binding reagents Can with bound analyte with moveable mark binding reagents identical calmodulin binding domain CaM, or with mark combine examination in analyte The different region of agent.Any or both analyses can use scavenger binding reagents, and scavenger binding reagents are at them Concentration, compatibility or the two on can be with different from each other.

For purposes of this application, term scavenger binding reagents are the other binding reagents for referring to bound analyte, The use of term " scavenger " is used for the purpose of making a distinction binding reagents with other binding reagents present in device.Remove Agent binding reagents are usually unlabelled.

According to embodiment, analytical equipment includes the first analysis, and it contains the first porous carrier, containing being provided with inspection The moveable mark binding reagents of area upstream, and second of analysis are surveyed, it, which contains, is provided with the removable of detection zone upstream Mark binding reagents, and same scavenger binding reagents provided in the detection zone upstream of second of analysis.The first point Analysis can be high analyte thing sensitivity analysis, and second of analysis can be harmonic analysis thing sensitivity analysis.

Scavenger reagent may be provided in moveable form.

Scavenger reagent can have the analytes different from the moveable mark binding reagents of second of analysis affine Property.In the embodiment of example, scavenger binding reagents with second analysis moveable binding reagents compared with, with compared with High analyte compatibility.The amount of scavenger binding reagents can change, to change spirit of second of analysis to analyte concentration Sensitivity.The amount of increase scavenger binding reagents reduces sensitivity for analysis, and this is due to that scavenger binding reagents can be tied More analyte is closed, the event of ratio for the mark binding reagents that can be attached to detection zone is significantly reduced.At the first It can change with the amount of mark binding reagents in second of analysis.The measurer of increase mark binding reagents has reduction hook effect Tendency, marks the amount of binding reagents, is particularly in the analysis compared with muting sensitivity, can be become according to the scope of analyte Change.

Scavenger binding reagents be able to may be combined with the identical or different region of analyte.In exemplary In, scavenger binding reagents being capable of calmodulin binding domain CaMs combinations different from analyte.Specifically, when measured analyte is During hCG, scavenger binding reagents can be combined with β subunits, and movably mark binding reagents to be combined with α subunits.

According to exemplary embodiment, analytical equipment includes the first analysis, it include containing for analyte can Mobile particle marks the glass fibre porous carrier materials of binding reagents, and provides in glass fibre porous carrier materials downstream Nitrocellulose porous carrier materials, nitrocellulose porous carrier materials have a detection zone, and detection zone is included for analysis The immobilization binding reagents of thing, and second of analysis, it includes glass fibre porous carrier materials, and the material contains for dividing Analyse thing first calmodulin binding domain CaM removable particle marker binding reagents and for analyte second calmodulin binding domain CaM can Mobile scavenger binding reagents, and the nitrocellulose porous carrier material in glass fibre porous carrier materials downstream is provided Material, nitrocellulose porous carrier materials have a detection zone, and second calmodulin binding domain CaM that detection zone contains for analyte is consolidated Surely binding reagents are changed.

It should be appreciated that the method for the sensitivity for analysis of above-mentioned change analysis is not exhaustive, and it can also combine Use.Analytical equipment can be comprising one or more features described above with impact analysis sensitivity.The selection of concrete analysis structure takes Certainly in analyte and its concentration range.

In order to avoid query, it is expressly recited herein, is described herein as " preferably ", " suitably ", " favorably " etc. Any feature, can have an independent existence in the present invention, or with any other feature for so describing with any combinations Mode exist, unless it is not so that context, which is clearly stated,.

With reference to following figure, the aspect to the present invention is further illustrated：

Fig. 1 is shown determines the type signal observed response and the ratio of typical competitive analysis for typical analysis Compared with.

Fig. 2 shows mapping of the signal intensity to hCG concentration in embodiment 1 and comparative example 1.

Fig. 3 shows mapping of the signal intensity to hCG concentration in the analytical equipment of embodiment 2.

Fig. 4 is shown for the analytical equipment in embodiment 2, changes the influence of the amount of scavenger antibody.

Comparative example 1 --- the preparation of the analytical equipment comprising single porous carrier, the carrier contains for sandwich First upstream test section of analysis and second downstream test section analyzed for inhibition

It is following to prepare analytical test strip, the test-strips contain for sandwich assay first upstream test section and be used for Second downstream test section of inhibition analysis, and the moveable mark binding reagents swum over the region are provided：

The preparation of downstream test section

By the anti-β-hCG antibody of 1.5mg/ml mouse in PBSA buffer solutions (self-control clone 3468) and in PBSA/ ovalbumins Middle 7.2KIU/ml hCG (Scipac) solution is mixed 1 hour, to provide anti-beta hCG-hCG conjugates.Obtained conjugate It is that (Whatman, pore-size is 8 microns to the wide nitrocellulose bands of the long x 40mm of 350mm, and thickness is micro- in 90-100 in size Rice between, on the back sheet for being laminated on 175 microns) on be deposited as line., will above using Biodot xyz3050 distribution platforms The conjugate of generation is with 1 μ l/cm speed to be made into about 1.2mm apart from nitrocellulose end of tape 16mm punishment wide and about The line of 300mm length.This results in second analyzed for inhibition downstream test section.

The preparation of upstream test section

For first detection zone (upstream test section) of sandwich assay, by being 3mg/ml by concentration in PBSA buffer solutions Anti- β-hCG antibody (self-control clone 3468) the same of anti-beta hCG-hCG conjugates is being applied with 1 μ l/cm speed Line is applied on nitrocellulose band to prepare.Anti- β-hCG antibody has been applied using Biodot xyz3050 distribution platforms in distance At the same end 10mm of the nitrocellulose band of anti-beta hCG-hCG conjugates, about 1.2mm is applied to wide and about The line of 300mm length.

NC bands are dried using series number #17494 Hedinair drying ovens, it is that 5 (single leads to be set to 55 DEG C and speed Cross).

Then NC is blocked using buffer solution is blocked, blocks buffer solution to contain 5% ethanol (BDH Analar 104766P) Plus 150mM sodium chloride (BDH Analar 10241AP) plus 50mM tromethamines (trizma base, Sigma T1503) add XX Tween 20 (Sigma P1379) and 1% (w/v) polyvinyl alcohol (PVA, Sigma 360627) mixture.

By the near-end for blocking buffer solution to be applied to band with 1.75 μ l/mm speed.Once after in blocking solution suction film, The molten of 2% (w/v) sucrose (Sigma S8501, in deionized water) is applied with 1.6 μ l/mm speed using same instrument Liquid, and it is drawn into nitrocellulose filter about 5 minutes).

Then NC bands are dried using series number #17494 Hedinair drying ovens, 75 DEG C are set to, speed is 5 (single It is secondary to pass through).

Mark the preparation of binding reagents

Mark binding reagents are prepared according to following scheme：

Latex particle is coated with anti-α hCG

1. with 100mM pH 8.5 disodium tetraborate buffer solution (BDH AnalaR 102676G) (DTB), it will come from Duke Scientific blue latex particles (a diameter of 400nm, DBl 040CB, 10% solid content (w/v)) are diluted to 2% Solid content (w/v).

2. by by the dilution latex of certain volume (2mls) in two Eppendorf centrifuge tubes in Heraeus Centrifuged 10 minutes with 17000rpm (25,848rcf) on Biofuge 17RS centrifuges, clean the latex of dilution.Take out and abandon Supernatant, precipitation is resuspended in 100mM DTB, obtains cumulative volume 1ml 4% solid content (w/v).

3. prepare mixture (95% ethanol (BDH AnalaR 104766P and the 5%w/v sodium acetates of ethanol and sodium acetate Sigma S-2889)。

4. 100 μ ls Ethanol-Acetic Acids sodium solutions are added in the latex cleaned of step 2, and (this is latex volume 10%).

5. antibody stoste (own (in-house) clone 3299) is diluted in DTB, the anti-of about 1200 μ g/ml is provided Body.

6. volume is heated about 2 points for the 1ml dilution antibody from step 5 in 41.5 DEG C of water-bath is set to Clock.Also Ethanol-Acetic Acid sodium is added to be heated 2 minutes in same water-bath the latex cleaned from step 4.

7. adding the antibody of dilution in latex adds Ethanol-Acetic Acid salt, it is well mixed, and in the water-bath for being set to 41.5 DEG C It is middle to incubate 1 hour, while being mixed using magnetic stirring apparatus and the magnet rotor (magnetic flea) placed in the mixture Close.

8. prepare 40mg/ml bovine serum albumin(BSA) (BSA) solution (Intergen W22903, in deionized water).It is logical Cross and isometric 40mg/ml BSA are added in the mixture of latex/antibody/Ethanol-Acetic Acid salt, and in 41.5 DEG C of water-bath Continue to stir and incubate 30 minutes, latex is blocked.

9. according to step 2, volume (is divided into 1ml in 10 minutes by mixture with 17000rpm centrifugations in Eppendorf pipes Batch).Supernatant is removed and abandoned, precipitation is resuspended in 100mM DTB.According to step 2 repeated centrifugation, remove simultaneously Supernatant is abandoned, precipitation is resuspended in (20% (w/v) sucrose Sigma in spraying (Air Brushing) buffer solution S8501,10%BSA (w/v), in 100mM tromethamine Sigma T1503,9) pH is adjusted to.Add spraying buffering Liquid, to provide 4% solid content (w/v) latex.

With reference to colloidal sol be sprayed at glass fibers in the mixture of BSA and sucrose, and with 50g/hr and 110mm/s speed Tie up on porous carrier (F529-09, Whatman), be dried using series number #17494 Hedinair conveyor ovens, if 65 DEG C are set to, speed is 5 (once-throughs).

Using the laminated film (Ferrisgate, 38mm are wide) for scribbling clear binder, by the glass of the latex with spraying Glass fibrous material is sticked on nitrocellulose filter, and it arranges the latex for causing to spray topmost, and glass fibre and nitric acid Length (350mm) overlapping about 2mm of the surface of cellulose along nitrate membrane strip.Glass fibre is adhered into nitric acid fine The end of element is tieed up, the upstream of first detection zone in upstream is located at.

Then the plate of lamination is cut into the wide test test-strips of 6mm.

Embodiment 1

Analytical equipment is prepared according to the mode similar to comparative example 1, simply first and second detection zone are divided Indescribably supply on first and second test-strips, wherein first and second detection zone are provided on nitrocellulose, Each test-strips contain the glass fibers being provided with nitrocellulose upstream, the α-hCG antibody for being coated with moveable latex mark Dimension.First and second detection zone are respectively provided in nitrocellulose test-strips at 16mm position.

Testing results bar

Embodiment 1 and comparative example 1 are tested using the own reader of the hCG buffer standards product with calibration Test-strips, the concentration of standard items is 0,25,50,100,250,500,1000,2500,5000,10000,15000,20000, 25000th, 50000,150000,200000 and 250000mIU/ml hCG.

Suppress the signal intensity that measures of detection zone as embodiment 1 (use -- ◆ -- represent) and comparative example 1 The function of the hCG concentration of the analysis of (use -- ■ -- represent), display is the signal of arbitrary unit to mIU/ml hCG in fig. 2.

Just as may be seen from the figure in see, the detection zone that suppresses of comparative example 1 is 0-100mIU/ in hCG scope Starting platform is shown under ml level, then intensity is reduced as expected under higher hCG levels.But, in higher water Under flat, the increase of signal intensity is still observed.By contrast, the signal intensity of embodiment 1 declines in higher hCG levels It is low, and then signal intensity does not increase under higher hCG levels.As seen, the analysis built according to comparative example 1 The scope that the inhibition zone of device can measure hCG is more limited.

Embodiment 2- contains first test-strips containing the first sandwich assay and also contained in addition to sandwich assay There is the preparation of the analytical equipment of second test-strips of scavenger reagent

The preparation of first analytical test strip

First analytical test strip is prepared according to the first (sandwich) analytical test strip in embodiment 1.

The preparation of second of analysis (scavenger) test-strips

Using the prepared product of the first analytical test strip according to embodiment 2, detection zone is prepared on nitrocellulose.

To be combined with the blue polystyrene latexs of 400nm (Duke Scientific) mouse anti human α-hCG mAb (gram The grand scavenger antibody mAb mouse anti human β-hCG antibody (own clone 3468) 3299) with 3mg/ml is mixed, and obtains indigo plant The final % of color latex is 3%, and 3468 final concentration are 0.075mg/ml, and the concentration of free anti-β-hCG antibody is 0.06mg/ml.Obtained mixture is sprayed on Whatman glass fibres (F529,25mm wide volume), BIODOT is used XYZS (series number 1673) is sprayed on F529-09 glass fibres under 90g/hr, with 2.02 μ g/cm.

Glass fibre is dried using series number #17494 Hedinair conveyor ovens, and it is 5 to be set to 65 DEG C and speed (once-through)., by repeat it is above-mentioned, but apart from spraying original position skew about 0.8mm (glass fibre it is more lower Trip), latex second by when deposition on the glass fibers.Glass fibre is dried as described above.

Comparative example 2

The analytical equipment that two of which detection zone is all provided on same porous carrier is constructed, it can not expanding Measurement analyte concentration in big analyte degree.

Using own detection zone optical reader, and scope is in the calibration of 0-250000mIU/ml hCG 12 concentration HCG buffer standards product, test the analytical equipment of embodiment 2.10 repeat samples to each concentration level are carried out Measurement, gives the tested analytical equipment that sum is 120.

The signal intensity of second of the analysis built according to embodiment 2 is shown in figure 3 to hCG concentration.

The first analytical test strip of embodiment 2, can determine before analysis curve flattens and be up to about 400mIU/ml HCG amounts.Second of analytical test strip of embodiment 2 can detect the hCG levels more than about l000mIU/ml. The measurement of signal can be determined between about 400mIU/ml and 1000mIU/ml on a kind of and second of analytical test strip HCG levels.

Change the influence for the amount for removing antibody

Second of analytical test strip of embodiment 2 is prepared for, depositing for antibody is simply removed in the preparation process of test-strips It is change in amount, gives 3468 final concentration of 0.12,0.16,0.2 and 0.24mg/ml.

Such as can Fig. 4 findings, increase remove antibody amount reduce detection zone capture analyte amount.

Claims (19)

1. for determining the presence of analyte and/or the analytical equipment of degree in fluid sample, bag in the concentration range of expansion The first analysis and second of analysis are included, wherein the first analysis bag of analyte is containing first flow path, and the flow path has only First of one analysis detection zone, can fixation mark binding reagents, and second of analysis bag of the analyte contains second Flow path, the flow path have it is unique second analysis detection zone, can fixation mark binding reagents, wherein exist in detection zone Mark binding reagents, provide instruction for the presence and/or degree of analyte in the fluid sample.

2. the device of claim 1, wherein first and/or second flow path include porous carrier.

3. the device of claim 2, wherein porous carrier are lateral flow porous carriers.

4. the device of any one of preceding claims, wherein the first and/or second of analysis bag containing provide it is described the first And/or second analysis detection zone upstream, the moveable mark binding reagents for analyte.

5. the device of any one of preceding claims, wherein the first and/or second of detection zone analyzed contain for analysis The immobilization binding reagents of thing.

6. the device of any one of preceding claims, wherein the first analysis are sandwich assays, and second of analysis is competitive Or inhibition analysis.

7. any one of claim 1-5 device, wherein the first analysis bag are containing offer in the first analysis detection zone upstream, pin To the moveable mark binding reagents of analyte, wherein the detection zone includes the combination examination for the immobilization of analyte Agent；

Wherein second analysis bag analyzes detection zone upstream, first calmodulin binding domain CaM for analyte containing offer at second Moveable mark binding reagents and the scavenger binding reagents for analyte, wherein it is described second analysis detection zone energy Enough binding marker binding reagents.

8. the device of claim 7, wherein second of analysis detection zone contains the binding reagents of the immobilization for analyte.

9. the device of claim 7 or 8, wherein for second is analyzed, the mark of scavenger binding reagents and immobilization Binding reagents are compared, and have higher binding affinity to analyte.

10. any one of claim 7-9 device, wherein the moveable mark binding reagents and scavenger knot of second of analysis Close reagent to be combined with first of analyte and second calmodulin binding domain CaM respectively, second of analysis detection zone contains for analyte Second calmodulin binding domain CaM immobilization binding reagents.

11. any one of claim 7-10 device, wherein the moveable mark binding reagents and scavenger of second of analysis Binding reagents are provided in same area.

12. any one of claim 7-11 device, wherein scavenger binding reagents are provided in the form of immobilization.

13. the device of any one of preceding claims, wherein mark binding reagents are marked with optically detectable particle.

14. the device of any one of preceding claims, contains common porous sample receiver.

15. the analytical equipment for determining the presence of analyte and/or degree in fluid sample, includes the first analysis and second Analysis is planted, wherein the first described analysis bag contains the porous carrier with detection zone, detection zone contains the fixation for analyte The binding reagents of change, and it is provided with the upstream of detection zone the moveable mark binding reagents for the analyte；And its Described in second analysis bag contain detection zone, the detection zone contains the binding reagents of the immobilization for analyte, and in inspection The upstream in survey area is provided with moveable mark binding reagents and the scavenger reagent for the analyte for analyte, The detection of binding reagents is marked wherein present in detection zone, is that the presence of analyte in the fluid sample and/or degree are carried For indicating.

16. the analytical equipment of any one of preceding claims, wherein the first analysis is highly sensitive analyte analyzation, and the Two kinds of analyses are the analyte analyzations of muting sensitivity.

17. the device of any one of preceding claims, containing casing, wherein the first and second of analysis is provided at the machine In shell.

18. one or more of device of preceding claims, for detecting the hCG analytes in urine, the wherein device bag Contain：

A) it is used to irradiating and detecting illumination and the detection instrument for marking optical bond reagent in detection zone；

B) it is used for the calculating instrument for calculating hCG levels or the value corresponding to hCG levels；

C) it is used for the show tools for showing analysis test result；

D) the value instruction that the basic threshold of storage, wherein hCG levels correspond to less than the basic threshold of storage is not pregnant, wherein HCG levels correspond to or indicate pregnancy higher than the value of the basic threshold of storage；

E) threshold value of two first and second other storages, wherein corresponding to the value less than or equal to first threshold value HCG levels indicate that pregnancy level is within the scope of first, and hCG levels correspond to the value instruction pregnancy water higher than second threshold value Flat to be within the scope of the 3rd, hCG levels correspond to greater than or equal to first threshold value but indicated less than the value of second threshold value HCG levels are within the scope of second, and wherein show tools can indicate the degree of non-Pregnancy status or Pregnancy status and pregnancy.

19. in fluid sample determine analyte presence and/or degree assay kit, comprising the first analysis and Second of analysis, wherein the first described analysis bag contains the porous carrier with detection zone, the detection zone contains for analysis The binding reagents of the immobilization of thing, and the moveable mark for the analyte is provided with reference to examination in the upstream of detection zone Agent；Wherein described second of analysis bag contains detection zone, and the detection zone contains the binding reagents of the immobilization for analyte, And be provided with the upstream of detection zone for the moveable mark binding reagents of analyte and the removing for the analyte The detection of binding reagents is marked present in agent reagent, wherein detection zone, is provided for the presence and/or degree of analyte in sample Indicate.