CN1894585B - Determing device for detecting interesting analyte in a sample and determing method - Google Patents
Determing device for detecting interesting analyte in a sample and determing method Download PDFInfo
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- CN1894585B CN1894585B CN2004800370304A CN200480037030A CN1894585B CN 1894585 B CN1894585 B CN 1894585B CN 2004800370304 A CN2004800370304 A CN 2004800370304A CN 200480037030 A CN200480037030 A CN 200480037030A CN 1894585 B CN1894585 B CN 1894585B
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Abstract
The present invention provides an assay kit for detecting an analyte of interest in a sample. The kit comprises a) a reporter species; b) a labeled species having first and second binding regions, wherein the first binding region is capable of binding to the analyte of interest and the second binding region is capable of binding to the reporter species; c) an immobilized species capable of binding to the first binding region of the labeled species; and d) immobilized capture reagent capable of binding to the reporter species. The arrangement is such that the sample is contacted with the labeled species, is then contacted with the immobilized species and is subsequently contacted with the immobilized capture reagent, the reporter species being added prior to exposure of the sample to the immobilized capture reagent. If no analyte is present in the sample, the labeled species becomes bound to the immobilized species and is therefore unable to bind to the immobilized capture reagent. If analyte is present in the sample, the analyte binds to the labeled species such that the labeled species is unable to bind to the immobilized species but can bind to the immobilized capture reagent via the reporter species, the presence of the analyte thus being determined by the presence of labeled species bound to the immobilized capture reagent via the reporter species.
Description
Technical field
The present invention relates to determinator (assay device), particularly and not exclusively, relate to lateral flow assay devices and whether they exist the application of analyte in test sample, described analyte is a haptens particularly and not exclusively.
Background technology
The current immunity detecting device that is used for haptenic existence of test sample and/or content is known, and is for example open by EP291194.Because haptens material volume is little, wherein to be combined in the association reaction of the sandwich trapping agent type on two different epi-positions of analyte be impossible to trapping agent (capture reagent) specificity.Replace the competitive reaction that employing wherein makes the trapping agent that analyte and another kind of material (the being generally identical analyte) competition in the sample is labeled.Usually, the effluent carrier comprises: contain the reagent area that particle is labeled reagent; With the detection zone that comprises fixed compound that is positioned at the reagent area downstream.When not having analyte, be labeled reagent in the detection zone combination, produce detectable signal.In the presence of the analyte in sample that usually is added in the upstream of reagent area, being labeled reagent can preferentially combine with analyte, thereby can not be in the detection zone combination.Therefore, the signal that forms at detection zone reduces along with the increase of analyte concentration.For semiquantitative is-the visual reading mensuration of non-to provide the possibility of result to cause obscuring of user or uncertainty by this way.For fear of this uncertainty, the manufacturer of this immunoassays prints positive on each device and negative findings looks like what kind of picture, to help user's sentence read result.Usually, the user of this mensuration for example is used to detect the some drugs abuse, will be laboratory technicians.Yet the increase of drug abuse makes this mensuration be used by untrained individuality more and more.Therefore, the easy degree of sentence read result seems extremely important.
The haptens that United States Patent (USP) 5451504 and 5874216 discloses the signal that wherein forms at detection zone to be increased along with the increase of analyte concentration detects uses immunoassay.
Still need to be used for the method that substitutes/improve of the existence of test sample analyte, wherein the signal of Chan Shenging increases along with the increase of analyte concentration.
Summary of the invention
According to first aspect, the invention provides the mensuration kit that is used for the test sample analyte of interest, it comprises:
A) reporter;
B) have the thing that is labeled of first and second lands, wherein first land can combine with analyte of interest, and second land can combine with reporter;
C) fixed compound that can combine with first land that is labeled thing; With
D) the immobilization trapping agent that can combine with reporter,
Be arranged so that the sample contact is labeled thing, contact fixed compound then, contact the immobilization trapping agent subsequently, before sample is exposed to the immobilization trapping agent, add reporter,
Wherein, if there is not analyte in the sample, then is labeled thing and combines with fixed compound also so can not combine with the immobilization trapping agent; And wherein, if analyte is arranged in the sample, then analyte be labeled thing and combine, make that being labeled thing can not combine with fixed compound, but can combine with the immobilization trapping agent by means of reporter, thereby measure the existence of analyte by the existence that is labeled thing that combines with the immobilization trapping agent by means of reporter.Fixed compound can be provided in holding back the district, and the immobilization trapping agent can independently be provided in the detection zone, and sample moves to detection zone from holding back the district.
Be labeled thing can be arranged in hold back the district upstream reagent area be provided.After reagent area applies sample,, and further can move to and hold back the district and move to detection zone subsequently if be labeled that thing can move and can combine in sample with the analyte of interest that exists in the sample.Reporter, be labeled thing, fixed compound and immobilization trapping agent and can be provided in substrate and/or in the substrate, it can be the effluent carrier.
Reporter can be movably, and can be present in the substrate of detection zone upstream.In one embodiment, reporter is provided at reagent area or its upstream.In another embodiment, reporter hold back the district be provided.
Specificity can be distinguished in conjunction with analyte of interest and reporter in first and second lands that are labeled thing, fixed compound can specificity in conjunction with first land that is labeled thing, the immobilization trapping agent can specificity in conjunction with reporter.
According to second aspect, the invention provides effluent carrier immunoassay apparatus, it comprises:
A) have the reagent area that mobility is labeled thing, mobility is labeled thing and has first and second lands, and wherein first land is that analyte of interest is specific, and second land is that reporter is specific;
B) be positioned at the district that holds back in reagent area or its downstream, hold back the district and comprise immobilized analyte of interest to be detected;
C) be positioned at the detection zone of holding back the downstream, district, detection zone comprises the specific immobilization trapping agent of reporter; And d) is positioned at the mobility reporter of detection zone upstream.
According to the third aspect, the invention provides the method for the existence that is used for the test sample analyte of interest, this method comprises:
Make the sample contact have the thing that is labeled of first and second lands, wherein first land can combine with analyte of interest, and second land can combine with reporter;
The fixed compound that the sample contact can be combined with first land that is labeled thing;
The immobilization trapping agent that the sample contact can be combined with reporter;
Wherein before sample contact immobilization trapping agent, add reporter, and if wherein do not have analyte in the sample, then be labeled thing and combine with fixed compound also so can not combine with the immobilization trapping agent by means of reporter; And if wherein in the sample analyte is arranged, then analyte be labeled thing and combine, make that being labeled thing can not combine with fixed compound, but can combine with the immobilization trapping agent by means of reporter, thereby measure the existence of analyte by the existence that is labeled thing that combines with the immobilization trapping agent by means of reporter.
Sample can be exposed to before being exposed to detection zone to be held back under the district, and described detection zone is present in the substrate with immobilization trapping agent, and the described district that holds back is present in the substrate with fixed compound.
The method of third aspect present invention can comprise makes sample be exposed to reagent area, and described reagent area is present in and has in the substrate that is labeled thing.When sample contact reagent area, being labeled thing can move in sample, and that moves then is labeled thing and can combines with analyte of interest in the sample (if existence).
The method of third aspect present invention can comprise the sample that contains analyte of interest under a cloud joined and comprises that mobility reporter, mobility are labeled the one step in the substrate of thing, fixed compound and immobilization trapping agent.Perhaps, reporter and/or be labeled thing and before putting on substrate, be introduced in the sample.
According to other aspect, the invention provides the method that is used for haptenic positive detection, wherein fluid sample is joined in the effluent carrier, so, if there is analyte, then form with mobility be labeled that thing combines first in conjunction with compound.Mobility is labeled thing also specificity association reaction of experience and reporter before or after forming the analyte compound, and described reporter is penetrated in the detection zone subsequently, so this report thing thing specificity immobilization trapping agent that is in the news is caught.Can measure and be present in being labeled reagent or making its video picture in the detection zone, with the indication that provides analyte to exist.
When not having analyte, mobility is labeled thing and combines compound with reporter formation.This compound is trapped the district subsequently and catches and therefore can not be penetrated in the detection zone.
According to another aspect, the invention provides mensuration kit of the present invention or device and be used for application at the test sample analyte.
The invention provides the mensuration kit or the device that are used for the test sample analyte, wherein the signal of Chan Shenging increases along with the increase of analyte in sample concentration proportionally.Sample can be fluid, as liquid, for example takes from experimenter's body fluid, or for deriving from the liquid of solid.This indentifying substance or other reagent can be used for obtaining fluid sample from solid.
Measure the single continuous strip that kit or device can comprise material, on this material, suitably be furnished with cut section, make that the flow direction of sample is to holding back the district, up to detection zone from reagent area (if existence).Perhaps, determinator can comprise: the appointed function that is used for carrying out reagent area, holds back district and each district of detection zone is appointed as in independent chamber, these independent chambers; With the device of the product of each chamber being transferred in the next chamber.Described each district is meant the independent section (section) of identical or different material.These sections can overlap each other partially or completely, make sample to lead to another section from a section.On one or more sections of material, can provide mobility reagent (promptly with good release characteristic, reagent can be resuspended in the sample and can be along material or by the material infiltration), and can on one or more sections with good combination feature, provide immobilized reagent.Described section can be through handling, to strengthen or to reduce their combination and/or release characteristic before using.Examples of material with good release characteristic is a glass fibre, and the examples of material with good combination feature is a nitrocellulose.This processing and material are described in EP291194 and EP383619.
The effluent carrier of certain embodiments of the invention can be selected from suitable porous carrier, as nitrocellulose.Perhaps, can use the permission sample to move through any suitable material of holding back district and detection zone.In addition, the effluent carrier can be by strengthening the support of solid backing, to increase the manipulation strength of device.Carrier can comprise also that above a kind of material these material arrangements are fluid contact each other.
Perhaps, the effluent carrier can be microfluid (microfluidic) carrier with one or more capillary channels.
Perhaps, the substrate of mensuration kit of the present invention can be one or a series of layers or the post that comprises immobilization and optional mobility reagent.In one embodiment, fixed compound and immobilization trapping agent are arranged so that sample and are labeled thing and contact fixed compound, contact the immobilization trapping agent then, wherein add reporter before contact immobilization trapping agent.
Mensuration kit of the present invention also can comprise as United States Patent (USP) 6352862 disclosed fluids and immerses part and/or sample reception part, and the check plot that is positioned at the detection zone downstream.Therefore, in another embodiment of mensuration kit of the present invention, measure kit and comprise the contrast marker (it can be movably) that is positioned at immobilization analyte upstream; With the check plot that is positioned at immobilization trapping agent downstream, the check plot comprises the immobilization trapping agent that can combine with the contrast marker.The contrast marker can permeate by holding back with sample to be distinguished and detection zone, and arrives the check plot, and there, the contrast marker is immobilized the trapping agent combination.The detection of the contrast marker of check plot confirm sample successfully infiltration by measuring kit, thereby and provide mensuration successful fully positive signal for the user.Be labeled the effect that thing also can play the contrast marker.Can produce signal in the check plot by any suitable method.In one embodiment, the check plot can comprise the immobilization acceptor that is used for mobility contrast marker, and it can be present in any position of upstream, check plot in the determinator, as long as the detection of contrast marker can differentiate with the detection of analyte.Hold back district and detection zone along with sample moves through along determinator, sample has carried mobility contrast marker.Perhaps, the check plot can comprise the immobilization anhydrous substances, and it is induced variable color when fluid sample arrives the check plot.Provide the another kind of alternative method of positive signal can be included in the determinator of the present invention in the check plot.
Can in addition mensuration kit of the present invention or device be combined in the shell, this shell can be the non-permeability of fluid, as the disclosed shell of EP291194.
In one embodiment, provide the current immunity detecting device, the end in contact sample that this current immunity detecting device is installing usually.Sample flow at first arrives reagent area (if existence) by poriness fluid carrier material, arrives then and holds back the district, further arrives detection zone by capillarity.Perhaps, can pass through other power,, promote sample each continuum by determinator of the present invention as gravity, centrifugal force, stirring etc.
The reagent area of certain embodiments of the present invention comprises that mobility is labeled thing, is labeled thing and has first and second lands, and wherein first land is that analyte of interest is specific, and second land is that reporter is specific.Mobility is labeled thing and can be deposited in the substrate of reagent area by many methods well known by persons skilled in the art, makes mobility be labeled thing and remained fixed in reagent area before adding sample.After adding sample, mobility is labeled thing and becomes movably, is carried remainder by substrate by sample.The thing that is labeled that moves moves freely in sample, and can interact with reporter and analyte (if being present in the sample) and combine with the two.
The mobility reporter can be present in the reagent area.Can be applied in the substrate of the present invention by the reporter of method known to those skilled in the art concentration known.In certain embodiments, therefore reporter was present in before applying sample in the substrate or in the substrate, and became movable by adding sample in basad.It can not be material in the Already in interested sample that reporter can be selected from.In one embodiment, select reporter make its can not hinder analyte of interest in conjunction with behavior.Similarly, analyte of interest can not hinder detection zone in conjunction with behavior.In one embodiment of the invention, reporter is ovalbumin-FITC bond.
It is specific and reporter is specific that first and second lands that are labeled thing can be respectively analyte of interest, and other analyte is had few specificity or do not have specificity (as not detecting) above background.But the land can comprise any molecule that can combine with expection target molecule specificity and can not combine with any other molecule with detection level.When reporter is when having the material in single epi-position site such as haptens, reporter can combine with carrier such as BSA or ovalbumin, and self does not hinder mensuration these carriers.Can use standard associated methods well known by persons skilled in the art that reporter is combined with the appropriate carriers molecule.
When reporter be bigger, can association reaction take place in different epi-position sites the time, this epi-position site can be identical or different, can directly use reporter and unnecessary it is combined with carrier.
Each land that is labeled thing can be and can combine with interested material specificity to form specificity in conjunction with right specificity land.Specificity comprises antibody and antigen in conjunction with right example, wherein antigen can be peptide sequence, complementary nucleotide or peptide sequence, high molecular bronsted lowry acids and bases bronsted lowry, dyestuff and casco, peptide and specific proteins bond, enzyme and accessory factor, effector and acceptor molecule, wherein the term acceptor is meant any compound or the composition of the specific or polarity orientation (that is, epi-position or decisive site) that can discern molecule.
The fragment that antibody includes but not limited to polyclonal, monoclonal, bispecific, humanized and chimeric antibody, single-chain antibody, Fab fragment and F (ab ') 2 fragments, produced by the Fab expression library, antiidiotype (anti-Id) antibody and above-mentioned any epi-position binding fragment.The part of antibody comprises Fv and Fv ' part.Can partly modify Fv, be called as the synthetic structure of strand Fv (scFv) molecule with generation.This comprises and is covalently bonded in V
hAnd V
lThe peptide connector in district, it helps the stability of molecule.Antibody can be naturally occurring antibody, as the monoclonal antibody that obtains by Koehler and Milstein method with by for example antigen being expelled to the polyclonal antibody that obtains in the animal.Antibody also can be partially or completely humanized.Spendable other synthetic structure comprises complementary determining region (CDR) peptide.These peptides are to comprise the synthetic peptide of antigen in conjunction with determinant.Also can use the peptide simulation.These molecules are normally simulated the structure of CDR ring and are comprised the limited organic ring of conformation of AI side chain.
The thing that is labeled that can combine with analyte of interest and reporter can be the direct particulate labels of for example using the first specific bond reagent and the common sensitization of the second specific bond reagent, the described first specific bond reagent is that analyte or analyte are specific like thing, and the described second specific bond reagent is that reporter is specific.The examples of substances of common sensitization is open in EP833157.Perhaps, mobility is labeled thing and can be the bispecific catches that is labeled, and it has analyte specificity and specific two lands of reporter.The example of bispecific trapping agent provides in EP962771.
Mark can be any suitable entity, and can be direct particle mark, as metal-sol such as aurosol, or for example has been mounted with organic granular such as the polyurethane or the latex of suitable dyestuff (comprising fluorescent dye).Mark can be can be detected and quantitative any suitable mark.The other example of suitable mark comprises particle such as carbon black; Can react to produce enzyme such as the horseradish peroxidase and the alkaline phosphatase of color response product; Can produce the molecule that can detect light such as bioluminescence, chemiluminescence, phosphorescence and fluorescence; Or any other mark well known by persons skilled in the art.
In comprising those embodiments of reagent area, the district that holds back of the present invention can be with respect to sample flow to the downstream that is positioned at reagent area.Hold back the district and comprise and can fixed form be connected in the suprabasil fixed compound of determinator, and this fixed compound combines with any thing that is labeled, described first land that is labeled thing does not combine with analyte of interest in the sample as yet.Like this, holding back the district stops do not combine with analyte of interest in the sample any to be labeled the thing arrival detection zone that advances, thereby false positive results (when in fact not having analyte of interest in fluid sample, producing signal at detection zone) appears in prevention.Any thing (and this be labeled thing also combine with reporter) that is labeled that combines with analyte of interest in the sample will be by holding back the district and entering detection zone.Therefore, at analyte and be used to hold back the district being labeled of binding site do not have competitive reaction between the thing, any analyte that exists in the sample can directly cause being labeled thing and enter detection zone.
Fixed compound can be analyte of interest or its analog.The analog of analyte of interest comprise the related substances that derives from animal similar molecule (when this animal dis the time) or synthesize the molecule that structurally imitates analyte or particularly simulate the concrete land of analyte.
Fixed compound can be connected in the substrate of holding back in the district by any method known to those skilled in the art.Fixed compound can be directly connected in to be held back in the district, perhaps can be connected in self to be fixed on the support molecule of holding back in the district.Perhaps, fixed compound can be fixed to that to be retained as with the fluid carrier material be on the fluid propping material such as solid plastic film that contact.Perhaps, fixed compound can be connected on the surface of the mensuration material of contact sample when sample passes through device.For example, when immunoassay apparatus was the microfluid device, fixed compound can be connected in and carry on the inside surface of sample by the capillary channel of device.
In fact, the capture rate of holding back the district is lower than 100%, therefore, even when not having analyte in the fluid sample, be not that all reporter-labelled reagent must be trapped district institute in conjunction with compound and catches, so they can be penetrated in the detection zone.Yet for fear of false positive results occurring, the amount of reporter can be chosen as and make and can not observe labelled reagent in detection zone when adding the sample that does not have analyte in device in the determinator.How the addition of reporter can and be handled and be decided according to the type of effluent carrier.Best addition is the most easily determined by normal experiment.Detection zone can be positioned at the downstream of holding back the district with respect to the sample flow direction, and can comprise the immobilization trapping agent that can combine with reporter.The immobilization trapping agent can combine with reporter, just fixedly is labeled thing when being labeled thing and combining thereby have only when reporter.Therefore, have only when the analyte that exists in the sample be labeled thing (be labeled thing and combine reporter) when combining and reporter in detection zone, when combined, just can produce positive signal again at detection zone.When not having analyte in the sample, the reporter arrival detection zone that still advances, in detection zone, reporter combines with the immobilization trapping agent, but owing to be not labeled thing in the detection zone, can not produce signal.
In one embodiment of the invention, determinator comprise hold back the district and detection zone.Before sample is joined device, sample can be mixed with the suitable buffer solution that comprises the reporter that is labeled thing and chooses wantonly, the described thing that is labeled has first and second lands, wherein first land can combine with analyte of interest, and second land can combine with reporter.Solution can suitably cushion, with prevent to may be at analyte, be labeled the binding interactions that takes place between thing and the reporter and cause any obstruction.Can be with sample and buffer solution mixing reasonable time, if with the analyte that allows to exist in the sample, be labeled any association reaction of generation between thing and the reporter.After mixing, sample can be joined in the determinator in the position of holding back the upstream, district.Sample passes through detection zone with any way as mentioned above then at first by holding back the district then.Therefore, analyte, be labeled binding interactions initial between thing and the reporter and can before sample is joined determinator, take place.
Analyte of interest is optional from any material that can combine behavior with first land that is labeled thing.Determinator of the present invention is suitable for detection and comprises micromolecule such as haptenic analyte.Haptens may be defined as has only the little antigenic determinant that can cause immune response when combining with carrier.Haptens can be incorporated into antibody, but himself can not cause antibody response separately, for example by haptens is expelled in the animal health.Haptenic non-limitative example comprises that amphetamine is drugs such as MDA (3,4-methylene-dioxy amphetamine), MDMA (is also referred to as " Ecstasy ", 3,4-methylene-dioxy-N-meth), MDEA (3,4-methylene-dioxy-N-ethyl amphetamine), BDB (3,4-methylenedioxyphenyl-2-butylamine), MBDB (3,4-methylenedioxyphenyl-N-methylbutylamine) and MDPA (3,4-methylene-dioxy-N-propyl group amphetamine); Opiate such as morphine and codeine and their synthetic analogues comprise heroin, Hydromorphone, hydrocodone, Oxycodone and Oxymorphone; Lysergic acid diethylamide (LSD) and metabolin thereof; Tetrahydrocannabinol and cocaine; Toxic drugs diazepam, nicotine, paracetamol, caffeine, Risperidone, phenobarbital; Steroids such as progesterone, estradiol and metabolin thereof, testosterone; Pesticide; Dyestuff such as fluorescein isothiocynate, texas Red etc.
In the present invention, term " body fluid " comprises all liq that can derive from mammalian body, comprises for example blood, blood plasma, urine, lymph liquid, gastric juice, bile, serum, saliva, sweat, tissue fluid and cerebrospinal fluid.Decide according to analyte of interest, can consider other fluid and fluid sample, as derive from industry, environment or agricultural source those.In addition, body fluid can be processed (for example serum) or process.If needed, can be with sample pretreatment obtaining and/or to discharge analyte of interest, disposal route can relate to handles solid sample to obtain comprising the fluid sample of analyte of interest.The method that obtains humoral sample from the experimenter is known in those skilled in the art.
The change that the preferred characteristics in each aspect of the present invention are in addition necessary is applicable to each others.The prior art file of mentioning herein is with the allowed by law this paper that at utmost is merged in.
Embodiment
Can understand characteristics of the present invention and advantage more completely with reference to following examples, but should be appreciated that following examples are specific embodiments of the present invention, the present invention should not regarded as the concrete characteristics that are subject to specific embodiments as described below.With reference to the following drawings, wherein:
Fig. 1 explanation is designed for a mensuration of the present invention that detects Risperidone, the measurement result diagram when it is included in check negative sample and positive.
Fig. 2 shows determinator of the present invention, and it comprises the Whatman glass fiber material section that is connected with the band of nitrocellulose membrane by lamination.
Fig. 3 explanation is measured with the standard Risperidone and is compared, and detects the measurement result of the existence of Risperidone in patient's sample.
Fig. 4 is illustrated in the of the present invention different determinators after use standard Risperidone is measured detection.
Embodiment has described the positive reading haptens of Antipsychotic Drug Risperidone and has measured. In this embodiment, reporter is the FITC (fluorescein-5-isothiocyanates, isomers 1) that is incorporated into the ovalbumin carrier. Carry out many experiments with guarantee to resist-FITC antibody is not combined with Risperidone, and guarantees that anti-Risperidone antibody significantly is not combined with FITC. The schematic diagram that Fig. 1 provides exemplary haptens to measure comprises the result when detecting positive and negative sample.
Anti--FITC antibody
By Fitzgerald Inc.Concord MA, USA provides (clone M25285, M25286, M49209).After preliminary screening, select clone M25286 to be used for these experiments.
The preparation of ovalbumin-FITC bond
(Molecular Probes USA) mixes with the dimethyl sulfoxide (DMSO) (DMSO) (Aldrich Chemicals) of 1ml with the FITC of 10mg.The ovalbumin (Sigma Chemical Co.) of preparation 5mg/ml in 50mM carbonate buffer solution (pH 9.3).The series reaction of preparation ovalbumin and FITC is than (from 1: 10 to maximum 1: 40).With reactant wrap in the paper tinsel at room temperature standing over night and second day morning by gel filtration (with the PD10 post of phosphate buffered saline (PBS) PBSA balance) purifying.All bonds diluted with 1: 100 in PBSA and characterize by the scanning of UV/ visible light.Produce following ovalbumin and FITC combine than: 1: 2.5,1: 3.9,1: 3.8 and 1: 2.6.Select in conjunction with being used for these experiments than the bond OBB02 that is 1: 3.9.
The preparation of ovalbumin-Risperidone bond
Anhydrous dimethyl formamide (DMF) (ROMIL) in the preparation Risperidone, N-hydroxy-succinamide (Sigma) and 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide HCl (Sigma) stoste.The solution of preparation ovalbumin (Sigma) in 0.1M sodium phosphate buffer (pH 7).
In vial (bijou) preparation Risperidone, N-hydroxy-succinamide and 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide HCl (with 1: 1: 1 ratio) activator mixture and at room temperature shake～2 hours.Above-mentioned activator mixture is added drop-wise in the solution of ovalbumin and at room temperature on magnetic stirring apparatus, stirs and spend the night.The amount of selecting mentioned reagent is to produce the reaction ratio of 25: 1 (Risperidone is to ovalbumin).Select this ratio to increase, reduce the possibility of precipitation in conjunction with usefulness.
In the second day morning, use PD10 post (Amersham Biosciences) that bond is passed through steric exclusion chromatography method purifying.After being applied to post, bond is eluted among phosphate buffered saline (PBS) (adding sodium azide) pH 7 (Sigma).Bond characterizes by HPLC, obtains 6.8: 1 combination than (Risperidone is to ovalbumin).
The preparation of effluent carrier
According to disclosed method among the EP291194, use the application of sample pen (pen) that is connected in volume pump and syringe that reagent is quantitatively joined nitrocellulose membrane (the 8 μ m Schleicher﹠amp that Mylar does backing; Schuell, code C10888-1) on.For 3 zones of film preparation ovalbumin-Risperidone bond (Risperidone 009,2.39mg.ml).The baseline of first area range determination band is 8.5mm, the 1.5mm of being separated by in the downstream, first area of zone subsequently.To resist-FITC-clone M25286-(2.5mg/ml) is precipitated as single district, apart from band baseline 16mm (observing the district).After deposition, film is added sealing in 3% (w/v) sucrose (BDH Analar K30139086236) 55 ℃ of dryings 5 minutes and at 1% (w/v) polyvinyl alcohol (PVA) (PVA) (Airvol9K 18034004).Make the lock solution chromatogram by film, up to film by saturated.After saturated, with film 75 ℃ of dryings 5 minutes.
The preparation of the latex particle that in anti--Risperidone and anti--FITC, coats
Duke Scientific blue latex (CB 1615) (diameter 400nm) is coated in 0.5% (w/v) solid mixture that comprises anti--Risperidone (100ug/ml cloned 6352: 1) and anti--FITC (25ug/ml, clone M25286).After at room temperature cultivating 60 minutes, latex particle was at room temperature sealed 30 minutes in 20mg/ml BSA (bovine serum albumin, Intergen Type H7N21207).After preparation, particle is washed and exchanges in the drying buffer liquid (comprise the 100mM borate of 6.5%BSA and 20% sucrose (w/v), pH 8.5).
The deposition of reagent on the Whatman glass fiber material
The latex particle that as above coats is deposited on (in drying buffer liquid) becomes a line on the Whatman fiberglass carrier in anti--Risperidone and anti--FITC.Use the Hamilton syringe latex to be deposited on the carrier (0.23uls/mm) and become lines with the pump that is connected in X-Y plotter.In addition, preparation ovalbumin-FITC bond OBBO2 in drying buffer liquid 1: 20 dilution and be deposited on the lines that (0.23uls/mm) on the identical glass fiber material becomes separation by Hamilton pump and syringe.By lamination the Whatman glass fiber material is connected with the nitrocellulose membrane that comprises ovalbumin-Risperidone bond and anti--FITC detection zone, with the determinator shown in the production drawing 2.
The check of device
By in the negative urine of Risperidone pond, diluting, prepare the Risperidone standard items from 1.18mg/ml Risperidone stoste.The determinator that builds is placed plastic casing, make the Whatman glass fibre～3mm can be maintained in the pond of Risperidone standard items.Device is kept vertical in 300 μ l aliquot sample of the Risperidone standard items that as above prepare, at room temperature kept five minutes.
After five minutes, will install at room temperature dry.Use Persona
TMMonitor (being sold by Unipath Ltd) carries out the signal intensity in optics detection by quantitative district.
Measured several patients' sample, the result as shown in Figure 3.
Fig. 4 represents with the different determinator bands of urine Risperidone standard items after testing.These are measured bands and clearly illustrate in depletion when how the Risperidone level in urine raises of the signal on ovalbumin-Risperidone bond zone.On the contrary, the signal intensity of detection zone increases along with the increase of Risperidone concentration, produces positive response.
Claims (11)
1. be used for the mensuration kit of test sample analyte of interest, it comprises:
A) reporter;
B) have the thing that is labeled of first and second lands, wherein first land can combine with analyte of interest, and second land can combine with reporter;
C) fixed compound that can combine with first land that is labeled thing; With
D) the immobilization trapping agent that can combine with reporter,
Be arranged so that the sample contact is labeled thing, contact fixed compound then, contact the immobilization trapping agent subsequently, before sample is exposed to the immobilization trapping agent, add reporter,
Wherein, if there is not analyte in the sample, then is labeled thing and combines with fixed compound also so can not combine with the immobilization trapping agent; And wherein, if analyte is arranged in the sample, then analyte be labeled thing and combine, make that being labeled thing can not combine with fixed compound, but can combine with the immobilization trapping agent by means of reporter, thereby measure the existence of analyte by the existence that is labeled thing that combines with the immobilization trapping agent by means of reporter.
2. the mensuration kit of claim 1, wherein fixed compound is provided in holding back the district, and the immobilization trapping agent independently is being provided in the detection zone, and sample moves to detection zone from holding back the district.
3. the mensuration kit of claim 2, wherein be labeled thing be arranged in hold back the district upstream reagent area be provided, and wherein in use, when sample contact reagent area, if be labeled that thing moves and can combine in sample, and can further move to and hold back the district and move to detection zone subsequently with the analyte of interest that exists in the sample.
4. the mensuration kit of claim 2, wherein reporter is provided in the upstream of holding back the district or holding back the district.
5. be used for the effluent carrier immunoassay apparatus of test sample analyte of interest, it comprises:
A) mobility reporter;
B) have the reagent area that mobility is labeled thing, mobility is labeled thing and has first and second lands, and wherein first land is that analyte of interest is specific, and second land is that reporter is specific;
C) be positioned at the district that holds back in reagent area or reagent area downstream, hold back the district and comprise immobilized analyte of interest; With
D) be positioned at the detection zone of holding back the downstream, district, detection zone comprises the specific immobilization trapping agent of reporter;
Wherein the mobility reporter is positioned at the upstream of detection zone;
Wherein, if there is not analyte in the sample, then is labeled thing and combines with immobilized analyte also so can not combine with the immobilization trapping agent; And wherein, if analyte is arranged in the sample, then analyte be labeled thing and combine, make that being labeled thing can not combine with immobilized analyte, but can combine with the immobilization trapping agent by means of reporter, thereby measure the existence of analyte by the existence that is labeled thing that combines with the immobilization trapping agent by means of reporter.
6. each mensuration kit or device among the claim 2-5, it comprises in addition:
-be positioned at the mobility contrast marker of detection zone upstream; With
-being positioned at the check plot in detection zone downstream, the check plot comprises the immobilization trapping agent that can combine with the contrast marker.
7. each mensuration kit or device among the claim 2-5, wherein analyte is a haptens.
8. the method that is used for the existence of test sample analyte of interest, this method comprises:
Make the sample contact have the thing that is labeled of first and second lands, wherein first land can combine with analyte of interest, and second land can combine with reporter;
The fixed compound that the sample contact can be combined with first land that is labeled thing;
The immobilization trapping agent that the sample contact can be combined with reporter;
Wherein before sample contact immobilization trapping agent, add reporter, and if wherein do not have analyte in the sample, then be labeled thing and combine with fixed compound also so can not combine with the immobilization trapping agent by means of reporter; And if wherein in the sample analyte is arranged, then analyte be labeled thing and combine, make that being labeled thing can not combine with fixed compound, but can combine with the immobilization trapping agent by means of reporter, thereby measure the existence of analyte by the existence that is labeled thing that combines with the immobilization trapping agent by means of reporter.
9. the method for claim 8, wherein sample was exposed to before being exposed to detection zone and holds back under the district, and described detection zone is present in the substrate with immobilization trapping agent, and the described district that holds back is present in the substrate with immobilized analyte of interest.
10. the method for claim 9, wherein sample is exposed to reagent area, and described reagent area is present in and has in the substrate that is labeled thing, is labeled thing then and moves in sample.
11. each mensuration kit or device are used for the application of test sample analyte in the claim 1 to 7.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0328892A GB0328892D0 (en) | 2003-12-12 | 2003-12-12 | Positive signal assay for analytes |
| GB0328892.5 | 2003-12-12 | ||
| US58511804P | 2004-07-01 | 2004-07-01 | |
| US60/585,118 | 2004-07-01 | ||
| PCT/GB2004/005146 WO2005059547A2 (en) | 2003-12-12 | 2004-12-10 | Assay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1894585A CN1894585A (en) | 2007-01-10 |
| CN1894585B true CN1894585B (en) | 2011-06-15 |
Family
ID=30130153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2004800370304A Active CN1894585B (en) | 2003-12-12 | 2004-12-10 | Determing device for detecting interesting analyte in a sample and determing method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1894585B (en) |
| GB (1) | GB0328892D0 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0806771D0 (en) * | 2008-04-12 | 2008-05-14 | Spd Swiss Prec Diagnostics Gmb | Assay devices comprising bubble-forming means |
| US8088594B2 (en) * | 2010-03-16 | 2012-01-03 | Saladax Biomedical Inc. | Risperidone immunoassay |
| EP2888287A4 (en) * | 2012-08-21 | 2016-04-20 | Ortho Clinical Diagnostics Inc | Antibodies to paliperidone haptens and use thereof |
| CN104737017B (en) * | 2012-08-21 | 2017-03-08 | 詹森药业有限公司 | Antibody of Risperidone and application thereof |
| GB201905090D0 (en) * | 2019-04-10 | 2019-05-22 | Spd Swiss Prec Diagnostics Gmbh | Assay device |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5451504A (en) * | 1991-07-29 | 1995-09-19 | Serex, Inc. | Method and device for detecting the presence of analyte in a sample |
| US5874216A (en) * | 1996-02-23 | 1999-02-23 | Ensys Environmental Products, Inc. | Indirect label assay device for detecting small molecules and method of use thereof |
-
2003
- 2003-12-12 GB GB0328892A patent/GB0328892D0/en not_active Ceased
-
2004
- 2004-12-10 CN CN2004800370304A patent/CN1894585B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5451504A (en) * | 1991-07-29 | 1995-09-19 | Serex, Inc. | Method and device for detecting the presence of analyte in a sample |
| US5874216A (en) * | 1996-02-23 | 1999-02-23 | Ensys Environmental Products, Inc. | Indirect label assay device for detecting small molecules and method of use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| GB0328892D0 (en) | 2004-01-14 |
| CN1894585A (en) | 2007-01-10 |
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