CN101663583A - Assay device - Google Patents

Abstract

Disclosed is an assay device for the determination of the presence and/or extent if an analyte in a liquid sample over an extended concentration range comprising a first assay and a second assay, wherein the first assay for an analyte comprises a first flow-path having a sole detection zone capable of immobilising a labelled binding reagent and the second assay for said analyte comprises a secondflow- path having a sole detection zone capable of immobilising a labelled binding reagent, wherein the presence of labelled binding reagent at the detection zones provides an indication of the presence and/or extent of analyte in said liquid sample.

Description

Analytical equipment

Technical field

The present invention relates to be used for the existence of determination and analysis thing in the concentration range that enlarges or analytical equipment, kit and the method for degree.

Background of invention

For the analyte in the test fluid sample, developed with commercialization simple sidestream immune analytical equipment, referring to for example EP291194.Such device generally comprises the carrier of porous, this carrier contain the drying that can combine with target analytes, movably, the mark binding reagents, immobilized binding reagents also can be combined in the analyte that provides in the detection zone in mark binding reagents downstream.Detection is immobilized at detection zone, the combination of mark, for the existence of analyte in the sample provides indication.

Perhaps, when target analytes was haptens, the immunoassay device can utilize competitive reaction, immobilized binding reagents in the analyte competition detection zone that wherein exists in the analyte of mark or analyte analog and the sample.Perhaps, analytical equipment can utilize inhibitory reaction, wherein provides immobilized analyte or analyte analog at detection zone, and analytical equipment contains the movably mark binding reagents at analyte.

When the check and analysis thing, sandwich immunoassays is the analysis of selecting usually.But sandwich assay is always unfeasible, and for example under for example haptenic situation of micromolecule, they are big inadequately, can not be thereon simultaneously in conjunction with two kinds of different binding partners.Use utilizes the dose response curve of the typical lateral flow devices preparation of sandwich assay method to demonstrate, and the level of signal increases along with the increase of analyte, and curve tends to be steady when the point of higher analyte level.When higher analyte level, owing to preferably catch the analyte that does not also combine with the reagent of mark at detection zone, signal begins to reduce.This phenomenon is called as hook effect (hook effect).Therefore and since observed semaphore or intensity under the higher analyte level may with under than harmonic analysis thing level observed identical or even lower, sandwich immunoassays has shown limited analyst coverage.

Competition or inhibition analysis method generally provide high signal when zero or low-level analyte.When analyte level raises, depend on the ratio of the amount of the amount of mark bond class of existence and analyte, signal level still may be high.When analyte level continues to increase, signal begins to reduce, because unconjugated analyte or compete immobilized binding reagents with the analyte or the analyte analog of mark perhaps combines with the mark binding reagents, reduced of the combination of mark binding reagents at detection zone.

Therefore, use sandwich assay to measure the interior analyte of expanded scope, may run into the problem of relevant hook effect.High analyte concentration begins to produce the reduction of analytic signal.Competition or inhibition analysis cause the loss of analytic signal under high analyte concentration, thereby analyte can measured scope be restricted.

Therefore, above-mentioned analytical approach is not suitable for Measurement and analysis thing level in the analyte ranges that enlarges.

US2005/0112780 discloses analytical equipment and has been used for the method for the dynamic detection range of extensive diagnostic device, comprises detection zone and the flowing through property porous carrier that is provided at the compensating basin in detection zone downstream.Detection relates to first kind of binding reagents, this reagent combines with detector probe, produces the detection signal that intensity is directly proportional with the amount of analyte, and the compensating basin contains second kind of capture agent, this reagent combines with detector probe, the signal that the intensity of generation and detection signal is inversely proportional to.Analysis can further comprise the 3rd calibration areas that produces signal.First kind of binding reagents can be selected from antigen, haptens or streptavidin.First kind and second kind of binding reagents can be selected from many thing classes, comprise antigen, haptens or streptavidin.

US2004/0197820 discloses the flowing through property porous carrier analytical equipment that is used to reduce hook effect that contains detection zone, and wherein said device can comprise the calibration areas in downstream.

US2006/0019404 discloses the analytical equipment of the dynamic range with expansion, contains lateral flow test strip, and this test-strips contains a plurality of detection zones, and they have the sensitivity that reduces gradually to analyte concentration.Analytical equipment can contain two kinds of carriers that respectively have a plurality of detection zones.Detect the amount of the label/signal that exists in a plurality of detection zones, to determine the concentration of analyte.

EP462376 discloses and has contained the analytical equipment of catching site and bond recovery site, wherein reception of bond recovery site and combination migration are by described described bond or bond compound of catching the site, and wherein be positioned at the immobilized bond that bond reclaims the site and catches the site, determine the amount of target analytes by detection.

The inventor shows, for a plurality of analytical equipments that are used for the detection zone of check and analysis thing wherein are provided on same porous carrier, the combination of upstream test section can change downstream test section in conjunction with feature, and any variation of upstream test section combination can cause that the interlock of downstream test section combination changes.Especially true under higher analyte concentration level, and may cause the poor accuracy analyzed.In addition, have been found that in the operational process of test, may take place to join between the corresponding binding reagents that exists in the detection zone, the manufacturing of installing and with them under the drying regime between storage life, also observed the intersection combination.This demonstrates accuracy and the sensitivity level analyzed influential.In the prior art, as if these problems also were not realized in the past.

Target of the present invention provides improved analytical equipment, kit and method, to enlarge the analyte ranges of described analysis.

Summary of the invention

First aspect, the invention provides and be used in the concentration range that enlarges, measuring the existence of fluid sample analyte and/or the analytical equipment of degree, comprise first kind of analysis and second kind of analysis, wherein first of analyte kind of analysis comprises first flow path, have can the fixation mark binding reagents first unique analyzing and testing district, second kind of analysis of described analyte comprises second flow path, have can the fixation mark binding reagents second unique analyzing and testing district, wherein there is the mark binding reagents in the detection zone, for the existence and/or the degree of analyte in the described fluid sample provides indication.

First kind of analysis can provide indication for the analyte level in first kind of concentration range, and second kind of analysis can provide indication for the analyte level in second kind of concentration range.

First kind and second kind of concentration range differ from one another.First kind and second kind of concentration range can overlap, so that continuous concentration range is provided.

Analytical equipment can provide indication for analyte level with respect to one or more threshold values.Analytical equipment can provide indication for the analyte level that is below or above a plurality of threshold values.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 of each storage is all corresponding to the level of analyte.

First kind and second kind of analysis can independently or lump together to the analyte level in certain scope indication is provided.

First kind of analysis can provide indication for the analyte level that is less than or equal to first threshold value, and second kind of analysis can provide indication for the analyte level that is higher than the 3rd threshold value.First and second analysis together can be for more than or equal to first threshold values but provide indication less than the analyte level of the 3rd threshold value.

Binding reagents at analyte or analyte analog can be provided in the detection zone with immobilized form.Binding reagents can be selected from binding reagents, analyte or analyte analog at target analytes, and this depends on that analyzing is the analysis of sandwich-type or the analysis of type of competition.Similarly, the mark binding reagents can comprise the mark binding reagents at target analytes, the analyte of mark or the analyte analog of mark.

Perhaps, reagent can be provided in the detection zone of compound that can incorporation of markings binding reagents-analyte-second kind of binding reagents with immobilized form.For example, second kind of binding reagents may be provided in movably form, with bond class biological example plain in conjunction with or otherwise be connected, the reagent that is fixed on detection zone is complementary binding partners, for example streptavidin or anti-Avidin antibody make to form immobilized mark binding reagents-analyte-second kind of binding reagents compound-biotin-streptavidin compound at detection zone.

Second kind of binding reagents may be provided in movably form, it can be in detection zone fixation mark binding reagents-analyte complex.For example, second kind of binding reagents can for example agarose or white latex combine with particle, detection zone can contain pore-size less than particle but the filter of the granularity of greater than flag binding reagents, make filter can intercept and capture the compound of any mark binding reagents-analyte-second kind of binding reagents of existence, and anyly can not pass through filter with the compound mark binding reagents of capture agent.

First kind and/or second kind of analysis can comprise the mark binding reagents, and it was provided at the upstream in first kind and/or second kind analyzing and testing district of drying regime with removable form before operative installations.

It is immobilized unlabelled in conjunction with the upstream that first kind and second kind of analysis provide on can each comfortable each detection zone, provides movably mark binding reagents.

Analytical equipment can contain two or more analyses, every kind of analyte that can both detect certain concentration range or be higher or lower than one or more threshold values.

First kind and second kind of analysis can be alone or altogether, provide indication for whether the analyte of specified level or analyte are higher or lower than certain threshold value.

Analytical equipment can have the common sample that is communicated with a plurality of flow path fluids and apply the zone.Therefore, the common sample that the is applied to device fluid sample that applies the zone can be transported to corresponding detection zone along the flow path of corresponding analysis.

As the replacement scheme that first kind and second kind analysis is provided in single analytical equipment, analyze and may be provided as analytical equipment separately, lumping together from the result of each device can provide indication for the level of analyte or measure.

Therefore, second aspect, the invention provides the existence that is used for determination and analysis thing in the concentration range that enlarges and/or the assay kit of degree, contain first analytical equipment and second analytical equipment, wherein first and second analytical equipment contain first kind and second kind of analysis of first aspect present invention respectively.

The third aspect the invention provides the existence and/or the degree methods that are used for determination and analysis thing in the analyte ranges that enlarges, comprises the following steps:

A) fluid sample is joined be provided at first unique upstream, analyzing and testing district, contain first kind of mark binding reagents movably and analyze and be provided at unique upstream, second analyzing and testing district, contain in second kind of analysis of mark binding reagents movably, described detection zone can the fixation mark binding reagents, wherein at the reagent of detection zone certification mark, for the degree and/or the existence of analyte in the fluid sample provides indication.

B) read analysis result.

Under the situation of level as the function of time of known analyte, corpus luteum hormone hCG for example, analytical equipment can provide time-based indication for the user, is the degree of the gestation of unit with sky or week for example.

For purposes of the present invention, term " flow path " is meant the matrix that liquid can be transported to second position from first position, can be for example capillary channel, microfluidic pathways, or porous carrier effluent porous carrier for example.Porous carrier can comprise one or more porous carrier materials, and they overlap in the mode of linearity or stacked arrangement, or the fluid connection.Porous carrier materials can be identical or different.First and second flow path can be provided on the matrix separately, and perhaps they can be provided on the common matrix, make the liquid of carrying along the flow path of first kind of analysis can not pass the flow path of second kind of analysis.For example, first kind and second kind of analysis can be provided on the same porous carrier, make first and second flow path be isolated from each other.Thereby this can make its atresia that becomes that first and second flow path are separated realization by for example part of cut porous carrier.As another kind of alternative, first and second detection zone can be arranged side by side basically, are provided on the same flow path, make any one not be provided at another downstream.

Specifically, flow path can comprise the effluent porous carrier.The mark binding reagents of each analysis can be provided at respectively on the different carrier materials with detection zone.Can comprise cellulose nitrate, acetate fiber, cellulose or cellulose derivative, polyester, polyolefin or glass fibre with the material that is fit to that detection zone is provided as porous carrier.Porous carrier can comprise cellulose nitrate.The advantage that it has is that binding reagents does not need prior chemical treatment just can be firmly fixed.If the porous solid phase material comprises for example paper, the fixedly needs of antibody in second zone use for example CKBr, N,N'-carbonyldiimidazole or tresyl chloride, connect by chemistry and carry out.

Analytical approach can provide with the form of analytical test strip, and wherein fluid sample flows along this test-strips.

Term " binding reagents " is meant in conjunction with right member, i.e. two kinds of different molecules, and wherein a kind of molecule combines with second kind of molecular specificity by chemistry or physics mode.These two kinds of molecules, being bonded to each other at them is to make them their binding partners can be distinguished on the next meaning with other analysis bank branch with similar quality, is correlated with.Specificity in conjunction with right member be called as part and acceptor (counter ligand), in conjunction with to the member with in conjunction with to gametophyte, or the like.Molecule also can be that the combination of molecular aggregate is to the member; For example, at the immune complex of second kind of antibody and the antibody and the corresponding antigen thereof that produce can be considered to the combination of immune complex to the member.The antibody fragment that binding reagents can comprise antibody or can combine with antigen.

Except antigen and antibodies to the member, other combination is to comprising such as but not limited to biotin and Avidin, carbohydrates and agglutinin, complementary nucleotide sequence, complementary peptide sequences, effector and acceptor molecule, the co-factor of enzyme and enzyme, the inhibitor of enzyme and enzyme, peptide sequence and specificity be at the antibody of this sequence or intact proteins, the bronsted lowry acids and bases bronsted lowry of polymerization, dyestuff and protein conjugates, peptide and specific proteins bond (for example ribonuclease, S-peptide and ribonuclease S-albumen), or the like.In addition, specificity is in conjunction with the member to the analog that can comprise the original specific binding members.

" label " in being used in mark binding reagents environment the time, is meant any material that can produce the signal that can detect by vision or instrument means.Be suitable for various label of the present invention and comprise the label that produces signal by chemistry or physics mode, for example can be by optical detection.Such label comprises enzyme and substrate, chromophore, catalyzer, fluorescent chemicals, chemiluminescence compound, electric active matter class, dye molecule, radioactivity label and particulate labels.Analyte itself can produce detectable signal inherently.Label can with the binding reagents covalent bond.

Label can comprise for example for example selenium or tellurium of gold, silver, colloid non-metallic particle of particle, and dyeing or painted particle for example is mixed with the polymer beads of dyestuff or dye sols.Dyestuff can have any suitable color, and is for example blue.Dyestuff can be a fluorescence.Dye sols can be from for example Foron Blue SRP (Sandoz) and Resolin BlueBBLS (Bayer) preparation of commercially available hydrophobic dye.The polymeric marker thing that is fit to can be selected from the category of synthetic polymer, for example polystyrene, polyvinyl toluene, polystyrene-acrylic acid and polyacrolein.The monomer that uses is normally water-fast, and emulsification in aqueous tenside comes induced polymerization by add initiating agent in emulsifying agent then so that form the monomer micelle.Produce spherical basically polymer beads.According to exemplary embodiment, label is the blue polymer particle.

Fluid sample can come from any source, for example industry, environment, agricultural or biogenetic derivation.Sample can come from or physiology source or be made of it, comprises blood, serum, blood plasma, interstitial fluid, saliva, sputum, crystalline body fluid, sweat, urine, milk, mucus, synovial membrane liquid, peritoneal fluid, through skin transudate, pharyngeal transudate, bronchoalveolar lavage fluid, endotracheal suction fluid, cerebrospinal fluid, seminal fluid, cervical mucus, vagina or urethra juice and amniotic fluid.Specifically, the source is human, and specifically, sample is a urine.

Analyte include but not limited to toxin, organic compound, albumen, peptide, microorganism, bacterium, virus, amino acid, nucleic acid, carbohydrates, hormone, steroids, vitamin, medicine (comprise since therapeutic purposes use and because the medicine that the purpose of violating a ban is used), the metabolin or the antibody of pollutant, pesticide and any above-mentioned substance.Term analyte has also comprised any antigenicity substance, haptens, antibody, big molecule, and combination.

Specifically, analyte is HCG (hCG).Analyte can have unique land or epi-position, perhaps can have more than one land.For example, analyte hCG contains and luteinizing principle (LH), α subunit that follicular stimulating hormone (FSH) is identical with thyrotropic hormone (TSH), and the exclusive β subunit of hCG.Antibody at α and β subunit can be used in conjunction with hCG in the sandwich immunoassays form.

Analytical equipment of the present invention is used in degree or the existence of the interior measuring h CG of concentration range of expansion.Scope can change between the 000mIU at about 10mIU to about 250.

According to embodiment, device can be measured the amount of hCG in the fluid sample, and according to the degree of the reference value that stores with the time-based indication user of unit pregnancy.Whether device can also be higher than by the level of determining hCG or be lower than basic threshold value, come denoted object conceived.Reference value and threshold value generally are stored in the device part as algorithm.The basis threshold value generally can be in the scope of 10-25mIU/ml.

According to embodiment, first kind of analysis can be higher than or being lower than basic threshold value comes whether denoted object is conceived respectively according to detected hCG level, if it is and/or conceived, the hCG level of indication in being less than or equal to first scope of first threshold value, second kind of analysis is for providing indication more than or equal to the hCG level in second scope of second threshold value, wherein first kind and second kind of analysis lump together, for greater than first threshold value but provide indication less than the hCG level in the 3rd scope of second threshold value.

First kind and/or second kind of analysis can further comprise the check plot, and it is gratifying being used to indicate the operation of analytical test, and promptly reagent is present in the proving installation, and they become in the process of operation test and move and transport along flow path.Immunochemistry can take place and interact in the reagent of check plot in also can indicating device, has confirmed the chemical integrity of device.Considering device when storing in certain temperature range under the drying condition and transporting, this is very important.The check plot generally is positioned at the downstream of detection zone, for example can comprise the secure bond reagent at the mark binding reagents.The mark binding reagents can be present in the upstream of check plot and detection zone with form movably.The mark binding reagents with can be identical or different at the mark binding reagents of analyte.

Analytical equipment can comprise the porous sample receptacle that is communicated with and is positioned at their upstreams with first and second flow path fluid.Porous sample receptacle can be shared for two kinds are analyzed.Therefore, the common sample that the is applied to device fluid sample that applies the zone can be transported to along the flow path of corresponding analysis in the relevant detection district.Porous sample receptacle can be provided in the inside of casing, or can stretch out described casing to small part, can be used for for example collecting urine stream.Porous sample receptacle can be used as fluid reservoir.Porous sample reception element can be made by any bibulous porous or fibrous material that can fast Absorption liquid.The porosity of material can be unidirectional (being the axle that the trend of hole or fiber is parallel to element fully or mainly), or multidirectional (omnidirectional, as to make element have unbodied spongy structure).Can use porous plastic materials, for example polypropylene, tygon (preferably having very high molecular weight), Kynoar, ethene vinyl acetate, vinyl cyanide and teflon.Other material that is fit to comprises glass fibre.

If desired, can provide absorption " groove " at the far-end of carrier material.Absorption cell can be made of for example Whatman 3MM chromatographic paper, and enough adsorptive poweies should be provided, and comes out from the detection zone wash-out to allow any unconjugated mark binding reagents.As the replacement scheme of such groove, the length of porous solid phase material extends to outside the detection zone just enough.

After binding reagents is applied to detection zone, can handle remaining porous solid phase material, to block any remaining binding site.Blocking-up can realize by for example using albumen (for example bovine serum albumin(BSA) or lactoprotein) or handling with polyvinyl alcohol (PVA) or monoethanolamine or its combination.In order to help the mark binding reagents to move freely when porous carrier is wetting by sample, porous carrier can also contain sugar for example sucrose or lactose, and/or other material, for example polyvinyl alcohol (PVA) (PVA) or polyvinylpyrrolidone (PVP).Such material can for example be deposited in the zone that will apply the mark binding reagents as aqueous solution.Such material can be used as first kind and applies thing and be applied on the porous carrier, applies label then; Perhaps, such material also can mix with label and be applied on the porous carrier, and perhaps the two is combined.Such material can be deposited on mark binding reagents place or its upstream.

Perhaps, porous carrier can not be blocked during fabrication; But the kit that will block porous carrier is contained in the material of porous carrier upstream.When test-strips was wetted, the instrument of blocking-up porous carrier was moved, and the blocking-up instrument flows into and by porous carrier, blocks when carrying out mobile.The blocking-up instrument comprises albumen for example BSA and casein, and polymkeric substance for example PVP, PVA, and carbohydrate and detergent Triton-X100 for example.The blocking-up instrument may reside in the macropore carrier material.

The cellulose nitrate porous carrier can have about at least 1 micron pore-size, for example greater than about 5 microns, and for example about 8-12 micron.

The cellulose nitrate porous carrier can be lined with for example plastic sheet, to increase its manipulation strength.This at a slice back lining materials for example passes through

Last formation skim cellulose nitrate can easily be made.

Dry binding reagents can be provided on the porous carrier materials that the porous carrier materials upstream of containing detection zone provides.The porous carrier materials of upstream can be a macropore.The carrier material of macropore is should protein combination low or do not have, perhaps should be easily by reagent for example BSA or PVA block, to minimize non-specific binding and to be convenient to macropore main body moving freely by the reagent of the wetting back of fluid sample mark.If desired, the macropore carrier material can be with surfactant or solvent pre-treatment, to give its more water wettability and to promote the quick absorption of fluid sample.The macropore carrier material that is fit to comprises plastic material for example tygon and polypropylene, or other materials such as paper or glass fibre.Under the situation of mark binding reagents with detectable particle mark, the macropore main body can have the pore-size than big at least 10 times of the maximum particle size of particulate labels.Bigger pore-size is the reagent of release mark better.As the replacement scheme of macropore carrier, the mark binding reagents can be provided on the nonporous matrix that the detection zone upstream provides, and described nonporous matrix has formed the part of flow path.

First kind and/or second kind of analysis can comprise the glass fibre macropore carrier that the upstream that is provided at the cellulose nitrate porous carrier and far-end and cellulose nitrate porous carrier overlap.

Analytical equipment or kit also further contain one or more degree that are used for definite label class that exists and/or the instrument of amount.For example, optical tooling comprises optical inspection tool, photodetector and one or more light source LED for example for example, and the position of light source makes can carry out illumination optical to detection zone, with the degree and/or the amount of the label class determining to exist.Analytical equipment can also comprise one or more power supplys, computational tool, signal conduction instrument, algorithm, show tools, storage tool and data input/output end port.Analytical equipment can contain casing, is used to hold first kind and second kind of other parts of analyzing and installing.Device can contain the threshold value of storage.

Analytical equipment generally contains the casing that comprises analysis.Casing can be fluid-tight, and with the plastic material that is fit to for example ABS make up.Analysis can also comprise the sample receiving element that is used to receive fluid sample.The sample reception element can extend from casing.

Casing can be used fluid-tight material construction.The also suitable isolated light on every side of casing.If be less than 10%, preferably be less than 5%, most preferably be less than the inside that 1% the visible light that incides the device outside is penetrated into device, then casing or shell are considered to isolated ambient light basically.The light tight synthetic plastics material that contains the disconnected pigment of suitable photoresistance is polycarbonate, ABS, polystyrene, general purpose polystyrene (polystyrol), high density polyethylene or polypropylene for example, is the suitable selection that is used to make casing.Outside at casing can provide eyelet, is communicated with analysis in being provided at the casing inner space.Perhaps, eyelet can be used for allowing the porous sample receiver to extend to the position of casing outside from casing.

First kind and second kind of analysis can provide for example to be arranged side by side, and perhaps being set at the another kind of face-to-face arrangement of analyzing the top with a kind of analysis wherein provides.Device can contain single photodetector, is used to detect the detection zone of the two.

Except the detection zone of measuring corresponding analysis and exist the check plot under the situation, optical tooling also can be measured reference region, does not promptly contain the part of the binding reagents of drying regime in the flow path.

The purpose of reference region provides signal value, and the signal value that obtains at detection zone can compare with it.The measurement of reference region can be measured from the reflection of flow path or the background level of transmitted light.Background level can be because for example for example existence of mark binding reagents of component of light reflection, fluid sample or the analysis of porous carrier.Therefore, can proofread and correct the amount of the mark binding reagents that exists with the signal that afforded redress indication detection zone with respect to the level of bias light in the light level that detection zone measures.Measurement in reference region also can compensate at any deviation between the fluid sample that is applied on the analytical equipment, and for example urine sample possibility difference on color is very big.

Be used for measuring the analytical equipment of fluid sample analyte level, can comprise the optical inspection tool of installation, be used to measure detection zone, check plot and reference region intensity of light reflected from analytical equipment.Optical tooling can comprise one or more light sources for example LED and one or more photodetector.

Analyzing usually can be through generation after a while, and during this period, the mark binding reagents is accumulated on test and the check plot.The typical time that is used for measuring the analytical test of urine hCG is 3 minutes.The analytical test time can begin automatically, for example when being measured to fluid sample by optical element and having arrived flow path or porous carrier a part of.

The light source that is fit to is LED.The color of LED is by the color decision of mark binding reagents.For the blue markings thing, the color of the LED that is fit to is red.LED can shine with specific frequency, so that the specific region of sub irradiation analysis apparatus.Light is from described regional reflex or be sent on the photodetector, and photodetector is noted electric signal.The quantity of the electric signal of record will depend on the running frequency of LED, therefore can write down one or more signals in time.Signal generally is expressed as % absorbance (%A).Can be behind the All Time of analytical test measured signal, or can more early measure, for example after having surpassed the signal specific threshold value.

Each measurement zone is generally by single LED illumination.Photodetector can detect the light from an above measured zone, therefore can detect the reflected light from an above LED.This can carry out irradiation process by order and realize, makes device can know from the light of which regional reflex and has arrived photodetector.During analyzing, the irradiation process of order can be carried out repetition with frequency fixing or that change, makes and can monitor signal level level in time in each zone.Analyzing bar can place side by side with arranging, and photodetector and light source are positioned at the top on bar plane, makes detection contrast and reference region towards light source and fluorescence detector.

Device can comprise detection adds liquid stream in analytical equipment instrument.For example, can monitor, determine whether and when fluid sample is applied on the device from the variation of the detected light level in one or more zones.The timing of analytical test can begin automatically, for example when fluid sample reaches the specific region.

Device can comprise the FLOW CONTROL instrument, wherein the variation from the detected light level in one or more zones can be used for determining whether and when fluid sample is applied on the device, and determines the flow velocity of fluid sample along device by measuring flowing between one or more measured zones.The definite of flow velocity can be used as another quality control inspection, if for example flow velocity is greater than or less than the setting level, analysis can be cancelled.Counting circuit can respond to signal, and Fluid Computation compares flow velocity and the upper and lower bound that calculates along the flow velocity of carrier flow, if the flow velocity that calculates outside upper and lower bound, then analysis result cancels.

Typical optical detection system will comprise at least one light source and at least one photodetector (for example photodiode).Optimized light source is light emitting diode or LEDs.Can measure reflected light and/or transmitted light by photodetector.For the purpose of present disclosure, reflected light is meant that the light from light source reflexes on the photodetector from porous carrier or other liquid transport agent.In this case, detecting device is arranged on usually on a side identical with light source of carrier.Transmitted light is meant the light that passes carrier, and general detecting device is arranged on the side opposite with light source of carrier.In order to carry out reflection measurement, can provide backing to carrier, for example white reflectivity Plastic layer.Therefore, will drop on the carrier from the light of light source, some will be from its surface reflection, and some will be penetrated in the carrier and in any degree of depth reflection, up to and comprise the degree of depth that provides the reflection horizon.Therefore, in fact the measurement of reflection type has comprised the segment thickness at least that porous carrier is crossed in transmittance.

Analytical equipment generally comprises one or more holes or window, and light can be radiated at from one or more irradiation sources by them and analyze or analyze on the specific region of bar.Window is used to limit the area that drops on the light on the specific region, and determines which part of analysis or test-strips is illuminated.Each irradiated zone can have corresponding window.Therefore, the device with 4 measured zone will have 4 windows.Collected by one or more photodetectors from the light of window reflection.For the analytical equipment that contains flow path, can measure fluid sample and between the zone, move the required time with a plurality of zones.

Can be regularly (for example roughly per second twice) measure from the light of each window reflection, can use lowpass digital filter to get rid of noise and make data smoothing.Cross the value that filters and can be used for detecting mobile and definite analysis result.

For each window, by the measured value (" calibration value ") when dry, before any fluid sample arrives described zone with the particular measurement in flow path zone, measured value in the time of may beginning to manifest divided by and line wetted when measured zone can calculate ratio.After this ratio equals to cause the reflectivity properties of flow path to change as the result that fluid sample passes through along flow path, the ratio of the light of reflection.For example, when flow path comprises porous carrier for example during cellulose nitrate, the variation of reflectivity properties may be quite remarkable.

For each window, at the window ratio of reference, contrast and test window, equal the measured value of porous carrier t=0 (before adding sample) when dry, divided by the measured value that adds the t time behind the sample:

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

Cross the calculating of the %A value that filters

For each time point t, can the use test line and these ratios of control line, use the reference ratio as contingent background baseline in the window that does not manifest outlet at all, calculate %A.

Analytical equipment can contain the contrast threshold value (CLT) of storage, if the signal value＜CLT that arrives for blank determination wherein, because control line does not fully manifest, the result will be rejected, and if described value＞CLT will determine that contrast is gratifying.

According to embodiment, analytical equipment can contain two analytical test strips, each all contains porous carrier, one of them analysis is high sensitivity (HS) analysis, it is sensitive promptly analyzing the analyte level under the harmonic analysis substrate concentration, another is muting sensitivity (LS) analysis, and it is sensitive promptly analyzing for the analyte under the higher analyte concentration.Specifically, analyte is hCG.

Analytical equipment can comprise two tests (detection) district, and each analytical test strip all contains test section, reference region and check plot.The signal in HS and LS district can be measured, and can be defined as follows:

The %A value that filters of crossing in HS and LS district can be defined as follows:

By with the difference of the window ratio of reference (ref.) window ratio and (contrast or test window) considered divided by reference window ratio and multiply by 100%, provided normalized relative attenuation percent (%A).

The %A value will be the value that obtains in complete analysis developing time (FDT) usually.

Flow detection and affirmation

Flow detection

The fluid that the window ratio of each window can be used for detecting through window flows.When ratio had reduced flow detection threshold percentage (FDT%), flowing to be classified as arrived window.This value that was equivalent to filter is higher than the same ratio of its corrected value.

For time t,

Or

For each window, the time of record when the standard first fit, be used for mobile really with.

Flow and confirm

Can be stored in the device with mobile corresponding various parameters, and be used for fluid sample is classified along the mobile of porous carrier of analytical equipment.Device can show any mobile error that causes owing to operative installations.

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

Device can comprise the threshold value of many storages, for example the control line threshold value.It is effective contrast that the value that is greater than or equal to this threshold value can be confirmed as, and the value that is lower than this threshold value can be confirmed as be invalid contrast, i.e. test will be rejected.

The measured value that analytical equipment can also comprise one or more storages overflows parameter, if wherein any measured value greater than or much smaller than desired value, the result will be rejected.This can make analytical equipment repel for example hardware fault, and for example interruption of wiring board or short circuit, dead battery, optical window are kept off, LED fault or the like.

Analytical equipment can contain other threshold value, and for example early stage decision threshold (EDT) if wherein signal exceeds this threshold value any time in test process, then provides earlier results, i.e. the time rating of spending early than test run (complete signal presentation time).Under the situation of carrying out the hCG measurement, will provide conceived indication by show tools.Analytical equipment can also contain the storage values corresponding to minimum presentation time (MDT), and wherein when in a single day MDT is exceeded, analytical equipment will only provide earlier results.

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

According to embodiment, the analytical equipment that detects the hCG analyte in urine is provided, wherein said device comprises:

Be used for shining and detecting the illumination optical and the testing tool of the mark binding reagents of detection zone; Be used to calculate the hCG level or corresponding to the computational tool of the value of hCG level; The show tools that is used for the display analysis test result; The basic threshold value that stores, wherein the hCG level does not have pregnancy corresponding to the value indication of the basic threshold value that is lower than storage, and wherein the hCG level is conceived corresponding to the value indication of the basic threshold value that is equal to or higher than storage; Two other first and the threshold value of second storage, wherein the hCG level is corresponding to being higher than basic threshold value but the value of being less than or equal to first threshold value indicates conceived level to be in first scope, the hCG level indicates conceived level to be in the 3rd scope corresponding to the value that is higher than basic threshold value and is higher than second threshold value, the hCG level is corresponding to being higher than basic threshold value and being greater than or equal to first threshold value but the value indication hCG level that is lower than second threshold value is in the 3rd scope, and wherein show tools can be indicated not conceived state or conceived state and conceived degree.

First kind of analysis is different with second kind of analysis, makes corresponding analysis can measure the analyte of varying level.

For example, first kind can be used different analytical structures with second kind of analysis, for example analyzes and uses sandwich association reaction for first kind, analyzes for second kind and uses competition or inhibitory reaction.First kind of analysis can comprise the movably mark binding reagents at analyte that is provided at the detection zone upstream, described detection zone contains the unlabelled immobilization binding reagents at analyte, second kind of analysis can comprise the movably binding reagents at analyte, is provided at the upstream at the unmarked binding reagents of immobilization of removable binding reagents.Perhaps, second kind of analysis can comprise movably labelled analyte or analyte analog reagent, is provided at the upstream at the unmarked binding reagents of immobilization of analyte or analyte analog.For example, sandwich assay can be a high-sensitivity analysis, and promptly it can measure the analyte that is in the low concentration scope, and inhibition or competition analysis can be the muting sensitivity analyses, and promptly it can measure the analyte of higher concentration degree.

Analytical equipment can, for example, contain first kind and second kind of analysis, wherein the unmarked binding reagents of first kind of analysis is different with the unmarked binding reagents of second kind of analysis, and/or the mark binding reagents of first kind of analysis is different with the mark binding reagents of second kind of analysis.For example, this can be the concentration difference, or to the compatibility difference of analyte, analyte analog or binding reagents.The high-affinity binding reagents will relatively hang down the analyte sensitivity height of the binding reagents of compatibility.Similarly, the low concentration binding reagents will be lower than the analyte sensitivity of high concentration binding reagents.First kind of analysis and second kind of analysis can change in this way, make them can measure the analyte of variable concentrations degree.

Therefore, analytical equipment can contain first kind of analysis of high analyte thing sensitivity, it contains the removable marking binding reagents that is provided at the detection zone upstream, has finite concentration or compatibility, and second kind of analysis of harmonic analysis thing sensitivity, it contains the movably mark binding reagents that is provided at the detection zone upstream, has low concentration or compatibility.For choosing or additionally, first kind of analysis contains the immobilization binding reagents of finite concentration or compatibility at detection zone, and second kind of analysis can contain the immobilization binding reagents with low concentration or compatibility at detection zone.

Sensitivity for analysis can be controlled by the ratio that changes binding reagents and label.The thing if the use particle serves as a mark can change the amount that is applied to the binding reagents on the label so.Another lever of controlling sensitivity for analysis is the amount that changes the label that uses in assay determination.For example, can reduce sensitivity for analysis by reducing binding reagents and the ratio that is used for the label class of mark binding reagents.Therefore, analytical equipment can comprise the analysis of first kind of high analyte thing sensitivity and the analysis of second kind of harmonic analysis thing sensitivity, wherein first kind of analysis comprised the removable particle mark binding reagents that is provided at the detection zone upstream, they have the ratio of binding reagents and particulate labels, and wherein second kind of analysis comprises the removable particle mark binding reagents that is provided at the detection zone upstream, and they have the binding reagents that is lower than first kind of analysis and the ratio of particulate labels.

The another kind of means of controlling sensitivity for analysis are the optical density that change label.By making the low label of optical density, sensitivity for analysis can reduce.This can be by for example providing polymer beads label with low concentration dyestuff or by using the lower coloured label of fluorescence detector sensitivity being realized.Therefore, analytical equipment can contain the analysis of first kind of high analyte thing sensitivity and the analysis of second kind of harmonic analysis thing sensitivity, wherein first kind of analysis comprises the removable particle mark binding reagents that is provided at the detection zone upstream, described label has optical density, and wherein second kind of analysis comprises the removable particle mark binding reagents that is provided at the detection zone upstream, and wherein said label has than first kind analyzes low optical density.

The another kind of mode of measuring high analyte thing level is to use non-particulate mark binding reagents.When measuring by sandwich binding analysis, high-caliber analyte needs high-caliber binding reagents.Label is under the situation of particulate labels therein, at porous carrier inner or its on to provide high-caliber analyte to cause sterically hindered, cause sensitivity for analysis poor.On the contrary, under than harmonic analysis thing level, use non-particulate mark binding reagents may produce low signal owing to optical density is low.But under high analyte thing level, non-particulate label can exist with easy detected enough high levels.Therefore, analysis can comprise the sensitivity analysis of first kind of high analyte thing, but it contains the particle mark binding reagents that is provided at the optical detection of detection zone upstream, and the sensitivity analysis of second kind of harmonic analysis thing, but it contains the non-particulate mark binding reagents that is provided at the optical detection of detection zone upstream.But the example of the non-particulate label of optical detection can be a dyestuff.Dyestuff can be a fluorescence.

Sensitivity for analysis may be subjected to the influence of the flow velocity of porous carrier.The method that reduces sensitivity for analysis is to use the porous carrier (for example cellulose nitrate) with high flow velocities.Therefore, analytical equipment can contain the sensitivity analysis of first kind of high analyte thing, and it contains the porous carrier with certain flow rate, and the sensitivity analysis of second kind of harmonic analysis thing, and it contains the higher or porous carrier faster of first kind of analysis of velocity ratio.

For selecting or additionally, can controlling sensitivity for analysis from speed that its source discharges by changing the mark binding reagents.The another kind of method that reduces sensitivity for analysis be with process that fluid sample contacts in, the snap-out release of mark binding reagents from porous carrier is provided.The release of mark binding reagents can by in device, provide sugar, albumen or other polymeric material for example methylcellulose change.Such material can be provided near the of binding reagents or its upstream.

The use of scavenger reagent

The another kind of method that reduces analyte sensitivity provides the scavenger binding reagents that combines with analyte.The scavenger binding reagents can be provided in the upstream of detection zone, can be immobilized, movably or the two.The scavenger binding reagents can be provided on the porous carrier and the identical zone of binding reagents movably, or its upstream or downstream.The scavenger binding reagents can bound analyte on the identical calmodulin binding domain CaM of mark binding reagents movably, the perhaps zone different on the analyte with the mark binding reagents.Any or this two kinds of analyses can be used the scavenger binding reagents, the scavenger binding reagents they concentration, compatibility or the two on can differ from one another.

For purposes of this application, term scavenger binding reagents be meant can bound analyte other binding reagents, the use of term " scavenger " only be for will binding reagents with device in other binding reagents of existing make a distinction.The scavenger binding reagents generally is unlabelled.

According to embodiment, analytical equipment comprises first kind of analysis, it contains first kind of porous carrier, contain the movably mark binding reagents that is provided at the detection zone upstream, and second kind of analysis, it contains the movably mark binding reagents that is provided at the detection zone upstream, and the scavenger binding reagents that is provided at the detection zone upstream of second kind of analysis equally.First kind of analysis can be the sensitivity analysis of high analyte thing, and second kind of analysis can be the sensitivity analysis of harmonic analysis thing.

Scavenger reagent may be provided in movably form.

Scavenger reagent can have the analyte compatibility different with the movably mark binding reagents of second kind of analysis.In the embodiment of example, the scavenger binding reagents is compared with the movably binding reagents of second kind of analysis, has higher analyte compatibility.The amount of scavenger binding reagents can change, to change the sensitivity of second kind of analysis to analyte concentration.The amount that increases the scavenger binding reagents has reduced sensitivity for analysis, and this is because the scavenger binding reagents can reduce the event of ratio of the mark binding reagents that can be attached to detection zone effectively in conjunction with more analyte.The amount of mark binding reagents can change in first kind and second kind of analysis.The measurer that increases the mark binding reagents has the tendency that reduces hook effect, and the amount of mark binding reagents, particularly in the analysis than muting sensitivity can change according to the scope of analyte.

The scavenger binding reagents may can with identical or different regional combination of analyte.In exemplary, the scavenger binding reagents can with the different calmodulin binding domain CaM combinations of analyte.Specifically, when determined analyte was hCG, the scavenger binding reagents can combine with the β subunit, and movably the mark binding reagents can combine with the α subunit.

According to exemplary embodiment, analytical equipment comprises first kind of analysis, it comprises the glass fibre porous carrier materials that contains at the removable particle mark binding reagents of analyte, and the cellulose nitrate porous carrier materials that is provided at glass fibre porous carrier materials downstream, the cellulose nitrate porous carrier materials has detection zone, detection zone comprises the immobilization binding reagents at analyte, and second kind of analysis, it comprises the glass fibre porous carrier materials, this material contains at the removable particle mark binding reagents of first calmodulin binding domain CaM of analyte with at the movably scavenger binding reagents of second calmodulin binding domain CaM of analyte, and the cellulose nitrate porous carrier materials that is provided at glass fibre porous carrier materials downstream, the cellulose nitrate porous carrier materials has detection zone, and detection zone contains the immobilization binding reagents at second calmodulin binding domain CaM of analyte.

Should be realized that the method for the sensitivity for analysis that above-mentioned change is analyzed is not exhaustive, and can be used in combination.Analytical equipment can comprise one or more above-mentioned features with impact analysis sensitivity.Analyte and concentration range thereof are depended in the selection of concrete analysis structure.

For fear of query, clearly state at this, be described as any characteristics of " preferably ", " suiting ", " favourable " etc. in this article, can have an independent existence in the present invention, perhaps any other characteristics with such description exist in any combination way, are not like this unless context offers some clarification on.

Figure with reference to following further specifies aspect of the present invention:

Fig. 1 has shown for the comparison of the observed type signal response of typical assay determination with typical competitive analysis.

Fig. 2 has shown that signal intensity among embodiment 1 and the comparative example 1 is to the mapping of hCG concentration.

Fig. 3 has shown that signal intensity in the analytical equipment of embodiment 2 is to the mapping of hCG concentration.

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

The comparative example 1---comprise the preparation of the analytical equipment of single porous carrier, described carrier Under contain first upstream test section of being useful on sandwich assay and be used for that inhibition analyzes second The trip detection zone

Be prepared as follows analytical test strip, described test-strips contains first upstream test section that is useful on sandwich assay and is used for second downstream test section that inhibition is analyzed, and the movably mark binding reagents that is provided at described regional upstream:

The preparation of downstream test section

With 1.5mg/ml mouse anti β-hCG antibody in the PBSA damping fluid (self-control clone 3468) and in the PBSA/ ovalbumin solution of the hCG (Scipac) of 7.2KIU/ml mixed 1 hour, so that anti--β hCG-hCG bond to be provided.The bond that obtains is deposited as line being of a size of on the wide cellulose nitrate band of the long x40mm of 350mm (Whatman, pore-size are 8 microns, and thickness is laminated on 175 microns the back sheet) between the 90-100 micron.Use the Biodotxyz3050 distribution platform, the bond that produces above is made into the line of the wide and about 300mm length of about 1.2mm with the speed of 1 μ l/cm in distance cellulose nitrate end of tape 16mm punishment.This has formed and has been used for second downstream test section that inhibition is analyzed.

The preparation of upstream test section

First detection zone (upstream test section) that is used for sandwich assay is by being that anti-β-hCG antibody (self-control clone 3468) of 3mg/ml is coated with line with the speed of 1 μ l/cm and prepares with concentration in the PBSA damping fluid on the same cellulose nitrate band that has applied anti--β hCG-hCG bond.Anti-β-hCG antibody uses Biodot xyz3050 distribution platform at the same terminal 10mm place apart from the cellulose nitrate band that has applied anti--β hCG-hCG bond, is applied to the long line of the wide and about 300mm of about 1.2mm.

Use the Hedinair drying oven of series number #17494 to come dry NC band, being set to 55 ℃ is 5 (single passes through) with speed.

Use the blocking-up damping fluid to block NC then, the blocking-up damping fluid contains 5% ethanol (BDHAnalar 104766P) and adds 150mM sodium chloride (BDH Analar 10241AP) and add 50mM tromethamine (trizma base, Sigma T1503) adds the potpourri of XX Tween 20 (Sigma P1379) and 1% (w/v) polyvinyl alcohol (PVA) (PVA, Sigma 360627).

The blocking-up damping fluid is applied to the near-end of band with the speed of 1.75 μ l/mm.In case blocking solution uses same instrument to apply the solution of 2% (w/v) sucrose (Sigma S8501 is in deionized water) with the speed of 1.6 μ l/mm after sucking in the film, and make it be drawn in the nitrocellulose filter about 5 minutes).

Use the Hedinair drying oven of series number #17494 to come dry NC band then, be set to 75 ℃, speed is 5 (single passes through).

The preparation of mark binding reagents

The mark binding reagents prepares according to following scheme:

Wrap by latex particle with anti--α hCG

1. the disodium tetraborate damping fluid (BDH AnalaR 102676G) of usefulness 100mM pH 8.5 (DTB), will (diameter be 400nm from the blue latex particles of Duke Scientific, DB1 040CB, 10% solid content (w/v)) is diluted to 2% solid content (w/v).

By with the dilution latex of certain volume (2mls) in two Eppendorf centrifuge tubes on Heraeus Biofuge 17RS hydro-extractor with 17000rpm (25,848rcf) centrifugal 10 minutes, clean the latex of dilution.Taking-up also abandons supernatant, will precipitate to be suspended in again among the 100mMDTB, obtains 4% solid content (w/v) of cumulative volume 1ml.

3. potpourri (95% ethanol (BDH AnalaR 104766P and 5%w/v sodium acetate Sigma S-2889) for preparing ethanol and sodium acetate.

4. in the latex of the cleaning of step 2, add 100 μ ls ethanol-sodium acetate solutions (this be latex volume 10%).

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

6. with volume be 1ml from about 2 minutes of the heating in being set to 41.5 ℃ water-bath of the dilution antibody of step 5.Also will add ethanol-sodium acetate from the latex of the cleaning of step 4 heated 2 minutes in same water-bath.

7. add in ethanol-acetate the antibody that adds dilution at latex, mix, and in being set to 41.5 ℃ water-bath incubation 1 hour, use magnetic stirring apparatus simultaneously and the magnet rotor (magnetic flea) that is placed in the potpourri mixes.

8. bovine serum albumin(BSA) (BSA) solution (Intergen W22903 is in deionized water) for preparing 40mg/ml.By the isopyknic 40mg/ml BSA of adding in the potpourri of latex/antibody/ethanol-acetate, and in 41.5 ℃ water-bath, continue to stir incubation 30 minutes, latex is blocked.

9. according to step 2, with potpourri with centrifugal 10 minutes of 17000rpm (with volume in the Eppendorf pipe, be divided into 1ml batch).Remove and abandon supernatant, will precipitate and be suspended in again among the 100mM DTB.According to step 2 repeated centrifugation, remove and abandon supernatant, will precipitate and be suspended in (20% (w/v) sucrose Sigma S8501,10%BSA (w/v) in spraying (Air Brushing) damping fluid again, in 100mM tromethamine Sigma T1503, pH is adjusted to 9).Add the spraying damping fluid, to provide the latex of 4% solid content (w/v).

In conjunction with colloidal sol in the potpourri of BSA and sucrose, and be sprayed at the speed of 50g/hr and 110mm/s that (F529-09 Whatman), uses the Hedinair conveyor oven of series number #17494 to carry out drying on the glass fibre porous carrier, be set to 65 ℃, speed is 5 (single passes through).

Use scribbles the laminated film (Ferrisgate of clear binder, 38mm is wide), the glass fiber material that will have the latex of spraying sticks on the nitrocellulose filter, it is arranged and makes the latex that sprays topmost, and the surface of glass fibre and cellulose nitrate is along the overlapping about 2mm of length (350mm) of nitrocellulose filter band.Glass fibre is adhered to the end of cellulose nitrate, be located at the upstream of first detection zone of upstream.

Plate with lamination is cut into the wide test test-strips of 6mm then.

Embodiment 1

Prepare analytical equipment according to the mode similar to comparative example 1, just first and second detection zone are provided at respectively on first and second test-strips, wherein first and second detection zone are provided on the cellulose nitrate, and each test-strips contains and is provided at the cellulose nitrate upstream, is coated with the movably glass fibre of the α of latex mark-hCG antibody.First and second detection zone respectively are provided at the position of 16mm on the cellulose nitrate test-strips.

The operation test-strips

Use has the test-strips that the own reader of the hCG damping fluid standard items of calibration has been tested embodiment 1 and comparative example 1, and the concentration of standard items is 0,25,50,100,250,500,1000,2500,5000,10000,15000,20000,25000,50000,150000,200000 and 250000mIU/ml hCG.

Suppress signal intensity that detection zone measures as embodiment 1 (use--◆--expression) and the function of the hCG concentration of comparative example's 1 (using--■--to represent) analysis, be presented among Fig. 2, for the signal of arbitrary unit to mIU/ml hCG.

As what can see from this figure, comparative example 1 inhibition detection zone is to demonstrate initial platform under the level of 0-100mIU/ml in the scope of hCG, and intensity reduces as expection under higher hCG level then.But, under higher level, still observe the increase of signal intensity.By contrast, the signal intensity of embodiment 1 reduces under higher hCG level, and not increase of signal intensity under higher hCG level subsequently.As finding, the scope that the inhibition zone of the analytical equipment that makes up according to comparative example 1 can measuring h CG is more limited.

Embodiment 2-has comprised first test-strips of containing first kind of sandwich assay and except sandwich Also contain the preparation of analytical equipment of second test-strips of scavenger reagent outside the analysis

The preparation of first analytical test strip

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

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

Use has prepared detection zone according to the prepared product of first kind of analytical test strip of embodiment 2 on cellulose nitrate.

Scavenger antibody mAb mouse anti human β-hCG antibody (own clone 3468) of mouse anti human α-hCG mAb (clone 3299) that will combine with the blue polystyrene latex of 400nm (Duke Scientific) and 3mg/ml mixes, the final % that obtains blue latex is 3%, 3468 final concentration are 0.075mg/ml, and the concentration of free anti-β-hCG antibody is 0.06mg/ml.The potpourri that obtains is sprayed at (F529, the volume that 25mm is wide) on the Whatman glass fibre, uses BIODOT XYZS (series number 1673) under 90g/hr, be sprayed on the F529-09 glass fibre with 2.02 μ g/cm.

Glass fibre uses the Hedinair conveyor oven drying of series number #17494, and being set to 65 ℃ is 5 (single passes through) with speed., above-mentioned by repeating, but the original position of distance spraying is offset about 0.8mm (the more downstream of glass fibre), latex for the second time by the time be deposited on the glass fibre.According to top description glass fibre is carried out drying.

The comparative example 2

Made up wherein two detection zones and all be provided at analytical equipment on the same porous carrier, it can not make Measurement and analysis substrate concentration in the analyte degree that enlarges.

Use own detection zone optical readings device and scope hCG damping fluid standard items, tested the analytical equipment of embodiment 2 in the calibration of 12 concentration of 0-250000mIU/ml hCG.10 repeat samples to each concentration level are measured, and have provided to add up to 120 tested analytical equipment.

The signal intensity of the second kind of analysis that makes up according to embodiment 2 is presented among Fig. 3 hCG concentration.

First kind of analytical test strip of embodiment 2 can be measured the hCG amount up to about 400mIU/ml before analytic curve flattens.Second kind of analytical test strip of embodiment 2 can detect the hCG level greater than about 1000mIU/ml.The measurement of signal can be determined at the hCG level between about 400mIU/ml and the 1000mIU/ml on first kind and second kind of analytical test strip.

Change the influence of the amount of removing antibody

The second kind of analytical test strip that has prepared embodiment 2, the amount of just removing antibody in the preparation process of test-strips changes, and provided 3468 final concentration and be 0.12,0.16,0.2 and 0.24mg/ml.

But, increase the amount that the amount of removing antibody has reduced the analyte of catching at detection zone as Fig. 4 finding.

Claims (19)

1. be used in the concentration range that enlarges, determining the existence of fluid sample analyte and/or the analytical equipment of degree, comprise first kind of analysis and second kind of analysis, wherein first of analyte kind of analysis comprises first flow path, described flow path has first unique analyzing and testing district, can the fixation mark binding reagents, and second kind of analysis of described analyte comprises second flow path, described flow path has second unique analyzing and testing district, can the fixation mark binding reagents, the mark binding reagents that wherein exists in the detection zone is for the existence and/or the degree of analyte in the described fluid sample provides indication.

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

3. the device of claim 2, wherein porous carrier is the effluent porous carrier.

4. each device of aforementioned claim, wherein first kind and/or second kind of analysis comprise be provided at described first kind and/or upstream, second kind of analyzing and testing district, at the movably mark binding reagents of analyte.

5. each device of aforementioned claim, wherein the detection zone of first kind and/or second kind analysis contains the immobilization binding reagents at analyte.

6. each device of aforementioned claim, wherein first kind of analysis is that sandwich assay and second kind of analysis are competitiveness or inhibition analysis.

7. each device of claim 1-5, wherein first kind of analysis comprise be provided at upstream, first kind of analyzing and testing district, at the movably mark binding reagents of analyte, wherein said detection zone comprises the immobilized binding reagents at analyte;

Wherein second kind of analysis comprise be provided at upstream, second kind of analyzing and testing district, at the movably mark binding reagents of first calmodulin binding domain CaM of analyte with at the scavenger binding reagents of analyte, wherein said second kind of analyzing and testing district can the incorporation of markings binding reagents.

8. the device of claim 7, wherein the immobilized binding reagents at analyte is contained in second kind of analyzing and testing district.

9. claim 7 or 8 device, wherein for second kind of analysis, the scavenger binding reagents is compared with immobilized mark binding reagents, and analyte is had higher binding affinity.

10. each device of claim 7-9, wherein the movably mark binding reagents of second kind of analysis and scavenger binding reagents combine with first and second calmodulin binding domain CaM of analyte respectively, and the immobilized binding reagents at second calmodulin binding domain CaM of analyte is contained in second kind of analyzing and testing district.

11. each device of claim 7-10, wherein the movably mark binding reagents of second kind of analysis and scavenger binding reagents are provided in the same area.

12. each device of claim 7-11, wherein the scavenger binding reagents provides with immobilized form.

13. each device of aforementioned claim, but wherein the mark binding reagents carries out mark with the particle of optical detection.

14. each device of aforementioned claim contains common porous sample receiver.

15. be used for determining the existence of fluid sample analyte and/or the analytical equipment of degree, comprise first kind of analysis and second kind of analysis, wherein said first kind of analysis comprises the porous carrier with detection zone, detection zone contains the immobilized binding reagents at analyte, and provides the movably mark binding reagents at described analyte in the upstream of detection zone; Comprise detection zone with wherein said second kind of analysis, described detection zone contains the immobilized binding reagents at analyte, and provide in the upstream of detection zone at the movably mark binding reagents of analyte with at the scavenger reagent of described analyte, the detection of the mark binding reagents that wherein exists in the detection zone is for the existence and/or the degree of analyte in the described fluid sample provides indication.

16. each analytical equipment of aforementioned claim, wherein first kind of analysis is the analysis of high-sensitivity analysis thing and second kind of analyte analyzation that analysis is a muting sensitivity.

17. each device of aforementioned claim contains casing, wherein first kind and second kind of analysis are provided in the described casing.

18. one or multinomial device of aforementioned claim are used for detecting the hCG analyte of urine, wherein this device comprises:

A) be used for shining and detecting the illumination and the testing tool of detection zone mark optical bond reagent;

B) be used to calculate hCG level or corresponding to the computational tool of the value of hCG level;

C) be used for the show tools of display analysis test result;

D) the basic threshold value of Chu Cuning, wherein the hCG level does not have pregnancy corresponding to the value indication of the basic threshold value that is lower than storage, and wherein the hCG level is conceived corresponding to the value indication of the basic threshold value that is equal to or higher than storage;

E) threshold value of two other first and second storage, wherein indicate conceived level to be in first scope corresponding to the hCG level of the value of being less than or equal to first threshold value, the hCG level indicates conceived level to be in the 3rd scope corresponding to the value that is higher than second threshold value, the hCG level corresponding to being greater than or equal to first threshold value but the value indication hCG level that is lower than second threshold value be in second scope, wherein show tools can be indicated not conceived state or conceived state and conceived degree.

19. be used for determining the existence of analyte and/or the assay kit of degree at fluid sample, comprise first kind of analysis and second kind of analysis, wherein said first kind of analysis comprises the porous carrier with detection zone, described detection zone contains the immobilized binding reagents at analyte, and provides the movably mark binding reagents at described analyte in the upstream of detection zone; Comprise detection zone with wherein said second kind of analysis, described detection zone contains the immobilized binding reagents at analyte, and provide in the upstream of detection zone at the movably mark binding reagents of analyte with at the scavenger reagent of described analyte, the detection of the mark binding reagents that wherein exists in the detection zone is for the existence and/or the degree of analyte in the sample provides indication.

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