CN101078724B - Detection device and method for detecting analyte - Google Patents
Detection device and method for detecting analyte Download PDFInfo
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- CN101078724B CN101078724B CN2007100690702A CN200710069070A CN101078724B CN 101078724 B CN101078724 B CN 101078724B CN 2007100690702 A CN2007100690702 A CN 2007100690702A CN 200710069070 A CN200710069070 A CN 200710069070A CN 101078724 B CN101078724 B CN 101078724B
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Abstract
The invention discloses a measuring device for measuring analyzed matter and the method. The device comprises movable region, intercepting region and measuring region. The movable region comprises special combination analyzed matter molecule with marking material, which can flow as liquid flows. One of special combination molecules not related to analyzed matter is connected on the molecule. A matter like analyzed matter is fixed on the intercepting region and one of special combination molecules not related to analyzed matter is fixed on the measuring region. The invention can display measuring result direction and can accomplish reaction by only one operation. At the same time it can improve measuring sensitivity and accuracy.
Description
Technical field
The present invention relates to a kind of pick-up unit and detection method in the detection range; More specifically; Relate to a kind of method that whether has the device of analyte and use this device to detect in the sample that detects, particularly detect the apparatus and method of the haptens small-molecule substance in the sample.
Background technology
This technology of utilizing immune association reaction principle to detect whether to have analyte in the sample extensively is used in every field.Can come monitoring of diseases and human beings'health situation (early pregnancy, tumour, infectious disease, drugs or the like) with the analyte that it detects various biological specimens (saliva, blood, urine, serum, sweat or the like).The cardinal principle of this detection technique is to be based upon the performance that has specific bond between the immune molecule, for example antibody and antigen, haptens/antibody, biotin and antibiotin or the like.In addition, a lot of such detections can be accomplished on solid dielectric, cross flow reagent strip for example commonly used, and in glass or the plastics porous disc, device for immunochromatography or the like.Usually, on immune specific bond molecule, can combine some solid particles or chemical substance again, like this can be next qualitative through naked eyes or other instrument and equipments, the quantitative or semiquantitative testing result that draws.This solid particle can be colored colloidal solid (latex or a gold grain), and this chemical substance can be the material that has chromophoric group, and these materials can send specific wavelength and show testing result under other appropriate condition.
Utilize the detectable bar or the device of these principles can find in the prior art, the reagent strip that for example the following patent is described or contain the device of reagent strip: US4857453; US5073484; US5119831; US5185127; US5275785; US5416000; US5504013; US5602040; US5622871; US5654162; US5656503; US5686315; US5766961; US5770460; US5916815; US5976895; US6248598; US6140136; US6187269; US6187598; US6228660; US6235241; US6306642; US6352862; US6372515; US6379620; And US6403383.
Immune detection generally includes two kinds of principles, and Sanming City therapy and competition law are wherein common with the haptens small-molecule substance that competing method detects in the sample.Utilize method that competition law detects and reagent and pick-up unit in U.S. Pat 4235601; US4442204, US5208535 has detailed description in the US5229073.These devices all are to be described on the surveyed area or the result reads when not having change color on the zone or not having the color lines to occur; Testing result is judged to the positive; Expression detects sample possibly exist analyte, on the contrary, and when surveyed area or result read when occurring change color on the zone or having the color lines to occur; Testing result is judged to feminine gender, and expression detects in the sample possibly not exist analyte.
United States Patent (USP) 5028535,5089391,5627526; 5939272,5143852,5480792; A kind of detection method and device have been disclosed in the patents such as 5985579; When utilizing this device to detect haptens small-molecule substance,, change color on surveyed area, occurs or lines occur representing positive findings when the concentration that has small-molecule substance or small-molecule substance in the sample time greater than the value of a design in advance; Opposite negative result representes not exist in the sample and is analyzed haptens micromolecule or micromolecular concentration less than the value of design in advance, promptly intuitively shows the detection method or the device of positive findings.
Though utilize this apparatus and method can a plurality of analytes of one-time detection; But utilize this apparatus and method will on a reacting hole, react 10-15 minute detecting sample and reaction reagent earlier; Be added drop-wise to the reaction mixture body on the nitrocellulose filter of anticipating antibody then, also will exist the entire reaction time long like this with liquid wash for several times at last; And complex operation step, can not a step accomplish defectives such as whole detection reaction.
United States Patent (USP) 5798273 has disclosed a kind of reagent strip; Reagent strip comprises and is fixed with capture region and the surveyed area of being analyzed similar substance, the analyte similar substance (mark substance-analyte analog) that the material on surveyed area can the underlined material of combined belt.Utilize this reagent strip also can reach the testing goal of demonstration positive findings directly perceived, still this detection must be earlier sample, and antibody and mark substance form mixed solution and then be added to this mixed solution on the sample region of acceptance of reagent strip.
United States Patent (USP) 6699722; Application discloses 2006/0141639 and has disclosed a kind of reagent strip; This reagent strip comprises the sample region of acceptance successively along liquid flow direction, moving area, first capture region and second capture region; Wherein on moving area, comprising can be with the analyte analog that has mark substance (analog of mark substance-analyte) of liquid flow; On first capture region, be fixed with first capture antibody, on second capture region, be fixed with second capture antibody, the quality of analyte analog that has mark substance is greater than the analyte in the sample.When having analyte in the sample; Analyte flows forward with the analyte analog that has mark substance together; Because both quality are different; The speed that in liquid, flows is also different; Analyte arrives first capture region prior to the analyte analog that has mark substance and is caught fixingly by first capture antibody, and the analyte analog that has mark substance that arrives of back arrives on second capture region through first capture region like this, thereby Show Color is represented positive findings on second capture region.This reagent strip is difficult to utilization in reality, and testing result is also inaccurate.
Summary of the invention
The purpose of this invention is to provide a kind of new pick-up unit and the method that are used to detect analyte, to overcome the above-mentioned defective that prior art exists.
The said pick-up unit of the present invention; Comprise moving area; Stop zone and surveyed area; Wherein comprising on the moving area can be with the molecule of the specific bond analyte that has mark substance of liquid flow, also be connected with on this molecule with the incoherent specific bond molecule of analyte to it a kind of molecule; Stop the zone and go up the fixing a kind of and similar material of analyte; On the surveyed area fixing and the incoherent specific bond molecule of analyte to another kind of molecule.
Preferable, the affinity between the similar material of the molecule of above-mentioned specific bond analyte and analyte is greater than the molecule of specific bond analyte and the affinity between the analyte.
When not having analyte in the sample; Can along with the specific bond analyte that has mark substance of liquid flow molecule be in free state; Arrive when stopping the zone; The molecule and the analyte similar substance that have the specific bond analyte of mark substance form compound, and are fixed on the obstruction zone, thereby on surveyed area, just can not capture the negative result of molecule visual representation who has mark substance like this.When having analyte in the sample, the molecular specific that has mark substance combines analyte to form compound.This compound arrives when stopping the zone; Stop can not combine with this compound on the zone with the similar material of analyte; Thereby on surveyed area with the incoherent molecule of analyte to another kind of molecular specific combine, on surveyed area, capture the tape label material and the positive result of visual representation.
A preferred scheme; The molecule of the specific bond analyte on the moving area can be with liquid flow, and in practical operation, sample contacts with the molecule of specific bond analyte earlier and reacts; Handle the zone have with the similar material of analyte together flowing to then; Can let the analyte (if having) in the sample react more fully like this, and then and react with the analyte similar substance, thereby improve the sensitivity that detects; Can not cause false negative, thereby avoid omission.Better; The quantity of the underlined material molecule of accommodation zone; When the concentration of analyte in the sample greater than preset value the time, thereby the molecule of the underlined material of detection molecules combined belt on the surveyed area result who detects that shows directly perceived representes that analyte in the sample is greater than predefined value.
Preferably; Said and the incoherent specific bond molecule of analyte are to comprising; But be not limited only to this, biotin/affinity plain (biotin/avidin), biotin/Streptavidin (biotin/streptavidin); Antibody/antigen (antibody/antigen) (antibody and the analyte itself that do not comprise anti-analyte); The antibody of rhodamine/rhodamine (rhodamine/anti-rhodamine), the antibody (Mouse IgG/anti-mouseIgG) of mouse IgG/ mouse IgG, protein staphylococcus/animal immune Lysozyme or the like.
Preferably; On the moving area with the incoherent specific bond molecule of analyte be biotin to it a kind of molecule, on surveyed area fixing and the incoherent specific bond molecule of analyte to another kind of molecule be selected from Avidin or Streptavidin.
Preferably, be selected from corresponding antigen of analyte or antigen fragment with the similar material of analyte.
Preferably, the molecule of specific bond analyte is selected from analyte corresponding antibody or antibody fragment, comprises monoclonal antibody and polyclonal antibody.
Preferably, mark substance is selected from latex particle or colloid gold particle.
Preferably, comprise on the moving area can with liquid flow have latex particle the antibody of specific bond analyte, also connect biotin on this molecule; Stop the zone and go up the fixedly antigen of analyte; Fixedly Avidin or Streptavidin on the surveyed area.
Preferably, this device comprises reagent strip, and this reagent strip comprises the sample region of acceptance in order, and moving area stops the zone, and surveyed area detects control area and suction zone.Can visual display detecting result on the surveyed area, change color is arranged or have lines the expression positive findings to occur, no change color or do not have lines expression to occur to represent negative findings.
On the other hand, the present invention provides a kind of method that whether has analyte in the sample that detects, and comprises that sample passes through moving area in order, stops zone and surveyed area, realizes through following steps:
Let sample contact with moving area; Wherein comprise can be with the molecule of the specific bond analyte that has mark substance of liquid flow for moving area, also is connected with molecule with the incoherent specific bond of analyte on this molecule to it a kind of molecule; Let the sample of process moving area contact, wherein stop the zone and fix a kind of and similar material of analyte with the obstruction zone; Let contact with surveyed area through the sample that stops the zone, wherein comprise on the surveyed area with the incoherent specific bond molecule of analyte to another kind of molecule.
Preferable, the affinity of the molecule of specific bond analyte and the similar material of analyte is greater than the molecule of specific bond analyte and the affinity between the analyte.
Preferable, with the incoherent specific bond molecule of analyte to being selected from biotin/avidin, biotin/Streptavidin, the antibody of rhodamine/rhodamine, protein staphylococcus/animal immune Lysozyme etc.
Preferably, on the moving area with the incoherent specific bond molecule of analyte be biotin to it a kind of molecule; On the surveyed area with the incoherent specific bond molecule of analyte to another kind of molecule be selected from Avidin or streptomysin.
Preferable, the similar material of analyte is selected from analyte corresponding antigen or antigen fragment.
Preferable, the molecule of specific bond analyte is selected from analyte corresponding antibody or antibody fragment.
Preferable, mark substance is selected from latex particle or gold mark particle.
Definition:
Only if in addition definition has identical implication with scientific terminology with the employed term of those skilled in the art of the affiliated technical field of this invention in all technology of this use.
" in order " expression is along the tactic position of liquid flow direction; Such as; The pick-up unit that is used to detect analyte comprises moving area in order, stops zone and surveyed area; What show is this three's position relation, is appreciated that to moving area position in front, stops the zone between moving area and surveyed area.
" detection " expression chemical examination or test a kind of material or whether material exists; Such as; But be not limited to this, the metabolin of chemical substance, organic compound, mineral compound, metabolism product, medicine or drug metabolite, organic organization or organic organization, nucleic acid, protein or polymkeric substance.In addition, detect the quantity of expression test substances or material.Furtherly, immune detection, chemical detection, enzyme detection etc. are also represented in chemical examination.
The result that " intuitively detect " expression detects directly can reflect whether there is analyte in the sample or has what of analyte.For example; When the concentration that does not have analyte or analyte in the sample time less than the value of preestablishing; Change color on surveyed area, do not occur or the color lines occur representing negative findings; On the contrary, change color on surveyed area, occurs or the color lines occur, can the direct representation positive findings.This testing result can also can read through instrument directly through being observed visually.What result makes proofer (for example doctor) directly draw the amount that whether has analyte the sample or have analyte from testing result like this is.
" sample " comprises body fluid (for example, urine and other body fluid, and clinical sample).Liquid sample possibly be derived from solid or semisolid sample, comprises excrement, biological tissue and food sample.These solids can be transformed into liquid sample through any suitable method with semisolid sample, for example in a kind of suitable liquid, mix, stamp broken; Macerate, hatch, dissolving or enzymolysis solid sample are (for example; Water, phosphate buffer or other damping fluid)." biological specimen " comprises the sample that is derived from animal alive, plant and food; Also comprise urine, saliva, blood and blood constituent, cerebrospinal fluid, vaginal swab; The culture of seminal fluid, ight soil, sweat, secretion, tissue, organ, tumour, tissue and organ; Condition medium cell culture and there is no matter be the people's or animal.Food sample comprises finished COF and last product, meat, cheese, wine, milk and potable water.Plant sample comprises the sample of the condition medium that is derived from any plant, plant tissue, plant cell cultures and there." environmental samples " is those samples that are derived from environment (for example, the sample of lake water sample or other water body, sewage sample, soil sample, underground water sample, seawater sample, the samples of runoff water).Sewage also can be included in the environmental samples with relevant refuse.
Molecule of " specific bond " expression is through physics or another molecule of chemical mode specific bond, and mutually combining between these two molecules can combine difference mutually with other.Specific bond between this two molecules is except the antibody that comprises antigen and antigen; Another antibody that also comprises antibody and anti-this antibody; The albumen of biotin and antibiotin, between the polypeptide fragment, DNA and DNA; RNA and RNA, and pass through specific bond pairing molecule of recombinant technique acquisition or the like.This combination can be direct combination, can also be to close through the intermolecular access node of its special pairing.These specific bond are that those skilled in the art combine the present invention to expect easily.Be meant a kind of material can only with corresponding single-minded combination of another kind of material.In some concrete schemes; Specific binding molecule possibly be a kind of antibody or a kind of antibody fragment; A kind of antigen; A kind of acceptor of binding partner or the fragment of acceptor, it is right that perhaps biotin-Avidin or biotin-streptomysin Avidin combines the combination of a right composition or other type.
" analyte " comprises some haptens materials in apparatus and method of the present invention, these haptens comprise drugs (like drug abuse)." drug abuse " (DOA) is meant that the non-medical destination uses medicine (playing the paralysis nerve usually).Abuse these medicines and can cause body & mind to suffer damage, produce dependence, habit-forming and/or dead.The example of drug abuse comprises cocaine; Amphetamine (for example, black beauty, white amphetamine tablet, dextroamphetamine, dexie, Beans); Crystal methamphetamine (crank, meth, crystal, speed); Barbiturate is (like
; Roche Pharmaceuticals; Nutley, New Jersey); Sedative (paramedicines of promptly sleeping); Lysergic acid diethylamide (LSD); Suppressant (downers, goofballs, barbs, blue devils, yellow jackets, methaqualone); The anti-antidepressant of tricyclic antidepressants (TCA, i.e. imipramine, amitriptyline and doxepin); Hog (PCP); Tetrahydrocannabinol (THC, pot, dope, hash, weed, etc.); Opiate (being morphine, opium, codeine, heroin, the hydroxyl dihydrocodeinone); Anxiolytic and hypnotic sedative agent; Anxiolytic is one type and is mainly used in anxiety reduction, anxiety, fear; Set the mind at rest; Have the medicine of hypnosis sedation concurrently; Comprise Benzodiazepines (benzodiazepines, BZ), part class, open loop BZ class, diphenylmethane derivatives, piperazine carboxylic acid salt, piperidine carboxylic acid salt, Kui azoles quinoline ketone, thiazine and the thiazole of atypia BZ class, the phenodiazine NB23C class that merges, tall and erect type of benzene nitrogen, BZ acceptor, other heterocyclic, imidazole type calmness/anodyne, propanediol derivative one carbamates, fatty compound, anthracene derivative etc.Use this device also can be used to detect and belong to medical usage but the detection of overdose easily, like tricyclic antidepressant (imipramine or analog) and Paracetamol etc.Can resolve into different small-molecule substances after these medicines are absorbed by the body, these small-molecule substances are present in the body fluid such as blood, urine, saliva, sweat or there is above-mentioned small-molecule substance in part body fluid.
Analyte can also be human chorionic gonadotrophin (hCG), lutropin (LH), ovarian stimulation plain (FSH), hepatitis C virus (HCV), hepatitis B (HBV), hepatitis B surface antigen, the medicine of AIDS virus and any abuse.Analyte can or liquefy at any liquid and detect in the sample, urine for example, saliva, saliva, blood, blood plasma, perhaps serum.The example of other analyte also has the acid of flesh ammonia acid anhydride, cholerythrin, nitrite, protein (nonspecific), blood; Leucocyte, blood sugar, heavy metal and toxin, bacterium composition (for example, special protein and the sugar of the bacterium of particular type; Colon bacillus 0157: H7 for example, staphylococcus aureus, salmonella, C.perfringens; Campylobacter, listeria monocytogenes, enteritis vibrios, perhaps cured shape bacillus).Any other analyte of suitable lateral flow assay form can detect with this device.
" the similar material of analyte " comprises; But be not limited to this; Go up connecting or coupling is associated with the antigenic substance that protein molecular can cause immune response at above-mentioned analyte (haptens), can also be the material of analyte other chemical structures of deriving and the antigenic substance that is connected with immunogen protein, can also be the growth of analyte; Isomeride, autoploid or similar antigenic substance on structure or function.These haptens materials itself can not cause that immune response produces antibody, and immunogenic substances could let animal body produce antibody to have only connection or coupling to join upward.These immunogenic substances include, but are not limited to this, albumen; Naturally or synthetic polypeptide or some carbohydrates, for example hemocyanin (Keyhole limpet hemocyanin, KLH), BHb (Bovine gammaglobulin; BGG), bovine serum albumin (Bovine Serum Albumin; BSA), bovine thyroid albumen (Bovine Thyroglobulin, BTG), ovalbumin (Ovalbumin, OVA), sperm whale myoglobin (SpermWhale Myoglobin; SWM), tetanus toxoid (Tetanus Toxoid; TT), methylated bovine serum albumin (Methylated Bovine Serum Albumin, mBSA), human immunoglobulin(HIg) IgG or IgA (Human immunoglobulins IgG, IgA) or the immunogen protein of other prior aries.
Antibody or antibody fragment
" antibody " is meant immunoglobulin (Ig), no matter is natural or some or all of synthetic.This term also comprises the derivant of the antibody that wherein keeps binding ability, also comprise any contain with the binding domain homologue of immunoglobulin (Ig) or the protein that combines the territory of homology to a great extent.These protein possibly be to be derived from natural materials, also possibly be some or all of synthetic.A kind of antibody possibly be monoclonal or polyclonal.A kind of antibody possibly be a member in any immunoglobulin class, comprises any mankind's immunoglobulin class: IgG, IgM, IgA, IgD, IgG and IgE." antibody fragment " is a part less than total length of the derivant or the antibody of antibody.Antibody fragment can remain to the remarkable site of the binding ability of a few full length antibody.
Antibody fragment can be generated by any way.For example, antibody fragment can generate through enzymolysis or complete antibody of chemical cracking, perhaps also can be through the genetic recombination from the coded portion antibody sequence.In other words, the antibody fragment generation of can partly or wholly recombinating.Antibody fragment can be a single chain antibody fragments arbitrarily.In other words, antibody fragment can comprise many peptide chains that interconnect, for example, and through disulfide linkages.Antibody fragment also can be a kind of arbitrarily polymolecular compound.One has the antibody fragment of function to comprise about at least 50 amino acid usually, and more antibody fragment comprises about at least 200 amino acid usually.
Describe in detail
Moving area
In pick-up unit, comprise on the moving area with the incoherent specific bond molecule of analyte to it a kind of molecule.When on surveyed area, add with the incoherent specific bond molecule of analyte to it in a kind of molecule, on moving area with the incoherent specific bond molecule of analyte to it a kind of molecule can be connected to the specific bond molecule to another kind of molecule.The incoherent specific bond molecule of analyte is to comprising; But be not limited only to this; Biotin/affinity plain (biotin/avidin); Biotin/Streptavidin (biotin/streptavidin), antibody/antigen (antibody/antigen) (antibody and the analyte itself that do not comprise anti-analyte), the antibody of rhodamine/rhodamine (rhodamine/anti-rhodamine); The antibody of mouse IgG/ mouse IgG (Mouse IgG/anti-mouse IgG), protein staphylococcus/animal immune Lysozyme or the like.More specifically; When on the molecule of the specific bond analyte that has mark substance on the moving area, connecting; Mark; Or coupling is when join going up with the incoherent specific bond molecule of analyte it a kind of molecule, on surveyed area the just fixing and incoherent specific bond molecule of analyte to another kind of molecule.
Preferable, select for use biotin/avidin this to right with the molecule of the incoherent specific bond of analyte.Biotin is one of water soluble ingredient of vitamin B complex, can be used as the coenzyme (being called biotin or biotin again) of carboxylase, participates in various carboxylation reactions.Two ring texturees are arranged in the biotin molecule, and wherein the I ring is the imidazolone ring, is and the plain main position that combines of affinity; II ring is thiazole ring, on a valeric acid side chain is arranged, its terminal carboxyl group is the macromolecular unique structure of binding antibody and other biological.Biotin molecule is little, and is simple in structure, very easily with biomacromolecules such as antibody, nucleic acid, polysaccharide and plurality of enzymes with the covalent bond stable bond, the primeval life activity to bond has no adverse effects simultaneously.Avidin is also claimed avidin or avidin, is a kind of alkaline glycoprotein that from egg protein, extracts.The alkaline glycoprotein that natural Avidin is made up of 4 subunits, isoelectric point are 10.5.Avidin is high to the affinity of biotin, is 1,000,000 times of antigen-antibody reaction, tool high degree of specificity and stability.Avidin character is very stable, and per molecule can combine 4 biotin derivatives, and it plays amplification as the bridge between the biotinylated molecule.Simultaneously, Avidin is again a glycoprotein, can direct and various biomolecule such as couplings such as IgG and theophylline, protein join.
Preferable, select for use biotin/Streptavidin this to right with the molecule of the incoherent specific bond of analyte.(Strepavidin SA) is a kind of protein that streptomycete secretes to Streptavidin in incubation.Streptavidin PI is 6.0, is a kind of and affine a kind of protein that have similar characteristic, and a Streptavidin molecule also can combine 4 biotin molecules.Because its non-specific binding is low more than Avidin, the non-special absorption of p-poly-phenyl ethene, nitrocellulose filter, DNA is lower, in SABC, stronger background colour can not occur, is that a kind of molecule of more satisfactory specific bond is right.There is the least possible material that contains the analyte acceptor can pass through (free form and composite form) this zone when making liquid move to stationary phase, reduced false-positive possibility through such effect of amplifying step by step.
In pick-up unit, moving area also comprises can be with the molecule of the specific bond analyte of liquid flow, and this material can be along with liquid flow and through stopping zone, surveyed area.Here said analyte comprises any interested material in the sample, comprises but does not limit to antibody, antibody fragment, haptens material or the like.
The molecule of specific bond analyte can be the antibody or the antibody fragment of analyte, or the like, the perhaps material be familiar with of other persons skilled in the art.The molecule of specific bond analyte is labeled the material mark usually, and the mark substance here can be an enzyme, water-insoluble particles; Metal colloid particles (colloid gold particle) for example; Latex particle or the like, perhaps some water-soluble mark substances can also the fluorescence labeling material.Preferable selection is to select the latex particle material that serves as a mark for use; This be because; Latex molecule relatively other mark substances is bigger, and the molecule of specific bond analyte that has latex particle is when stopping the zone, and oarse-grained material speed is slower than finely ground particle substance; Can let the molecule of the specific bond analyte that has mark substance do on the zone longly slightly to stop and stop molecule and fully react stopping like this, help reducing " false positive " of testing result.
In addition; Can also comprise that on moving area some have the molecule of mark substance; This molecule be fixed on the molecule that detects on the control area and combine to play the checking testing result whether effectively or the effect of contrast, be used for representing that whether effectively or the completion of expression chemical examination etc. testing result.
These can be processed at the reagent of liquid flow on the solid phase, and are for example on some water-absorbing materials, plain such as spun glass, filter paper, on the nitrocellulose filter or the like.The molecule of specific bond analyte and the similar substance of analyte are handled on above-mentioned carrier through conventional means; Certainly can also comprise on above-mentioned carrier that some other reagent improves or the conditioned reaction condition, for example handles some buffer solution solution reagents and comes pH value of conditioned reaction or the like.In these solid surface treatment compositions and methods and reagent configuration, selecting also is that one of ordinary skill in the art is through consulting prior art and combining technology of the present invention to accomplish easily.It is not emphasis of the present invention.
In a preferred mode, pick-up unit comprises reagent strip, on reagent strip; Moving area can be positioned at the upper reaches that stop the zone; Also can be positioned at the downstream of sample region of acceptance, certainly, moving area can be positioned on the same reagent strip with obstruction zone and surveyed area.But in pick-up unit, also can be positioned on the different reagent strips, as long as on liquid flow direction, liquid can pass through moving area successively, and it is just passable to stop zone and surveyed area.
The affinity size
" affinity " is meant the tightness degree that antibody combines with antigen, is another critical nature of monoclonal antibody.Affinity is strong more, with antigen combine firm more.The monoclonal antibody of different purposes has different requirement to affinity.The height of affinity is by the decision of the appropriate degree of spatial configuration between the binding site of the size of antigen molecule, antibody molecule and the antigenic determinant.Help to keep Coulomb force, Van der Waals force and the space repulsion that the stable intermolecular force of antigen antibody complex has hydrogen bond, hydrophobic bond, side chain opposite charges gene.Affinity often representes with affinity costant K that the unit of K is L/mol, and the scope of K also has nearly 1014/mol at 108～1010/mol usually.The purposes of antibody is confirmed in the screening of the mensuration antagonist of affinity of antibody, and the homogeneity of checking antibody etc. are all significant.
In current micromolecule detects; During particularly drugs or drug abuse detect; In order better to distinguish drug abuse or drug abuse patient and normal therapeutic property medication patient; At the commercial floor level that usually need improve detection, promptly when patient's body contains the test substance of certain higher concentration, make the result of detection still negative.So just can not let those be that to be mistaken as because of therapeutic medication person be the drug addict originally.In order to address this problem; In a preferred mode; The molecule of selection specific bond analyte and the affinity of the similar material of analyte are more effective greater than the molecule and the affinity between the analyte of specific bond analyte; When particularly requiring to improve the minimum (cut-off) that detects, adopt the method more effective.Detailed says; The molecule of specific bond analyte and the affinity between the analyte are represented with K1; Affinity between the molecule of specific bond analyte and the analyte similar substance is represented with K2; When the molecule of selecting such specific bond analyte, even K1 less than K2 the time, needs in the sample more analyte molecule to compete the site on the combination specific bond analyte molecule with the analyte similar substance; Can improve the level of the minimum (cut-off) of detection like this, promptly can also make testing result negative in the high concentration analyte relatively.
Concrete must saying can be selected conduct of analyte corresponding antigen and the similar material of analyte for use.When having analyte in the sample, at first on moving area, combine to form compound with the molecule of the specific bond analyte that has mark substance.When mixing sample arrive to stop the zone, the analyte antigen that stops on the zone closed and is analysed thing by part and can both combine with the molecule of the specific bond analyte that has mark substance, both thereby form competitive relation.Because the affinity of analyte antigen and the molecule of the specific bond analyte that has mark substance is greater than the affinity of molecule of analyte with the specific bond analyte that has mark substance itself, thereby the preferential selection of molecule that has a specific bond analyte of mark substance combines with analyte antigen.So; If the concentration ratio of analyte is lower in the sample; On surveyed area, then be shown as negative result; Have only when having q.s in the sample, just can read positive result, can reach the level that improves the minimum (cut-off) that detects through such method at surveyed area.
In order to satisfy the different detection requirement, the molecule that utilizes K1 to be less than or equal to the specific bond analyte of K2 still is feasible in technical scheme of the present invention.In addition, in persons skilled in the art other technologies scheme of combining disclosed technical scheme and combining prior art to produce also is included in.
Stop the zone
A preferred scheme; In pick-up unit, stop the downstream that the zone is positioned at moving area, stopping fixing a kind of and analyte similar substance on the zone; This material can combine with the molecule of specific bond analyte, but can not bind receptor-analyte compound.This molecule can be the analog of analyte, homologue, growth, the antigen of isomeride or analyte or antigen fragment.
These reagent that stop on the zone can be handled on solid phase material, filter paper for example, and cellulose membrane, nylon membrane, a preferred scheme is a nitrocellulose filter.Stopping method and the mode of molecule processing on film is existing known technology.In addition, obstruction can also comprise some other auxiliary reagent on the zone, and these reagent can improve the obstruction molecule and be fixed on effect on the film, makes the obstruction molecule more stable, more even distribution or the like.The configuration of these auxiliary reagents and to come out all be the prior art technique known, the those skilled in the art in this area combine prior art to accomplish.
In addition, with being connected of the similar material of analyte and specific bond analyte molecule, mode and method that coupling joins also are the prior art technique known, and the those skilled in the art in this area combine prior art to accomplish.
Surveyed area
In a preferred scheme, fixing molecule can directly be used for showing the result of detection on surveyed area.Molecule on the surveyed area comprise with the incoherent specific bond molecule of analyte to another kind of molecule.When for example being connected with on the corresponding moving area with the incoherent specific bond molecule of analyte to it a kind of molecule; On surveyed area, be provided with this molecule to another kind of molecule; When the moving area material that has mark when liquid flow direction arrives surveyed area; Can interosculate with incoherent two the specific bond molecules of analyte, thereby intuitively on surveyed area must read positive findings.The advantage of implementing this programme is, avoided by molecular substance and with analyte specific bond process in disturbed and " false negative " result of occurring by complex process, make testing result more accurate.
In another preferred scheme, the molecule on the surveyed area except comprise with the incoherent specific bond molecule of analyte to another kind of molecule, also be connected with the antibody or the antibody fragment of specific bond analyte molecule, perhaps the antibody of separate sources.For example when the specific bond molecule was certain small-molecule substance, the molecule of this small-molecule substance of specific bond was the antibody of this material on the moving area, and the molecule that is fixed on the surveyed area is the antigen of this small-molecule substance; When the molecule of specific bond analyte is the Dan Kelong antibody of certain haptens material; The antibody (antiantibody) of the monoclonal antibody of this haptens material of specific bond on the moving area, the molecule that is fixed on the surveyed area is the monoclonal antibody of this haptens material.The advantage of this embodiment is, except and the incoherent specific bond molecule of analyte between coupling join, can also through with moving area on the further specific bond of molecule.
Better, the Avidin or the Streptavidin of fixing and biotin specific bond are connected with biotin molecule on the corresponding moving area on surveyed area.When the specific bond analyte molecule that has mark when liquid flow direction arrives surveyed area because the specific bond between biotin and Avidin or the Streptavidin, thereby on surveyed area, intuitively observe positive findings.
In another preferred scheme, fixedly specific bond has the molecule of mark substance on surveyed area, and this mark substance includes, but are not limited to this, enzyme, dyestuff, fluorescent material, chemiluminescent substance, colloid gold particle or latex particle.
Another preferred mode is that surveyed area is positioned on the nitrocellulose filter, and method and mode that detection molecules is fixed on the film are the prior art technique known.Certainly, on surveyed area, can also comprise other reagent, some buffer reagents for example, closed reagent or the like, the interpolation of these reagent can improve the environment that reacts on the surveyed area, makes detection reaction more accurate.
Pick-up unit
Pick-up unit comprises moving area, stops zone and surveyed area.
In a preferred scheme, pick-up unit comprises host material composition reagent strip.On reagent strip, comprise moving area, stop zone and surveyed area.Comprising on the moving area can be with the molecule of the specific bond analyte that has mark substance of liquid flow; Also be connected with on this molecule with the incoherent specific bond molecule of analyte to it a kind of molecule; Stop the zone and go up fixing and analyte similar substance, on the surveyed area fixing and the incoherent specific bond molecule of analyte to another kind of molecule.In a concrete scheme, the reagent rule comprises a kind of absorbent material, and the host material of supporting liquid flow is provided." host material " is meant a kind of material of supporting liquid flow and transportation.In a concrete scheme, host material is a kind of absorbent material.Flow of liquid is crossed this device and is realized by means of capillary motion effect.In different concrete schemes; Host material can be the bar that homogenous material constitutes; Also can be by multiple in liquid interactional absorbent material synthetic, the material of " suction " is meant the material that those can stably absorb moisture and moisture is transported through capillary motion effect therein.The example of absorbent material comprises nitrocellulose, filter paper, spun glass, polyester and other suitable material.At one specifically is in the mode, and pick-up unit comprises the passage of accepting sample and the transparent window of observing testing result can read the result of detection through window channel.This device that contains reagent strip and the reagent strip position relation in device is a prior art, for example following patent or apply for that disclosed device can be applied on the reagent strip of the present invention, and for example United States Patent (USP) 6303081; 6248598; 6998273,6406992,6514769 etc.
The molecule of specific bond analyte and with the incoherent specific bond molecule of analyte can be that the form of solid-state or liquid exists to the existence form of it a kind of molecule in pick-up unit; In the preferred scheme, the molecule of specific bond analyte and it a kind of molecule is present on the reagent strip with solid-state form with the incoherent specific bond molecule of analyte.Through the moving area time, these materials on the moving area can be dissolved in and form liquid phase in the liquid and move with liquid downstream as liquid.
Preferred, also wrap sample region of acceptance that is positioned at the moving area upper reaches and the suction zone that is positioned at the surveyed area downstream at pick-up unit, between surveyed area and suction zone, also comprise the testing result control area.
Detection method
The present invention provides a kind of method that whether has analyte in the sample that detects, and comprises that sample passes through moving area in order, stops zone and surveyed area, realizes through following steps:
Let sample contact with moving area; Wherein comprise can be with the molecule of the specific bond analyte that has mark substance of liquid flow for moving area, also is connected with molecule with the incoherent specific bond of analyte on this molecule to it a kind of molecule; Let the sample of process moving area contact, wherein stop the zone and comprise and the similar material of analyte with stopping the zone; Remove and stop the compound that has mark substance in the zone; Let contact with surveyed area through the sample that stops the zone, wherein comprise on the surveyed area with the incoherent specific bond molecule of analyte to another kind of molecule.
Preferably; Let sample and the antibody that has the specific bond analyte of mark substance react; Also be connected with biotin on this antibody; With being fixed on antibody on the solid dielectric and the specific bond analyte underlined material of the similar molecule combined belt of analyte, the molecule of the mark substance that has biotin with the Avidin or the Streptavidin specific bond that are fixed on the solid dielectric at last comes the quantity that shows whether the analyte in the sample exists or exist directly perceived.
Preferably, moving area comprises the monoclonal antibody of the specific bond analyte that has latex particle, also is connected with biotin on this monoclonal antibody, lets sample contact with this monoclonal antibody earlier; Again with the antigen of analyte stop free be connected with biotin and latex particle monoclonal antibody; Remove the compound that contains the latex particle mark in the mixed liquor; Whether detect directly perceived the demonstration with Avidin or Streptavidin again exists analyte in the sample or has what of quantity.
Preferably, pattern detection occurs on the reagent strip, and sample is in order through the sample region of acceptance, and moving area stops the zone, surveyed area, testing result control area and suction zone.
The beneficial effect that the present invention obtains: use this pick-up unit of the present invention and method, when perhaps haptenic concentration was greater than preset value when having hapten molecule in the sample, the testing result of demonstration was directly perceived positive; When perhaps haptenic concentration was less than preset value when not having hapten molecule in the sample, the testing result of demonstration was directly perceived negative.This shows; Method of the present invention, ocular and clear, reagent strip of the prior art all are on surveyed area, not have change color or do not occur representing negative findings in the color lines; Both represented not exist in the sample analyte; On the contrary, change color occurs or have lines the expression negative findings to occur, promptly represent to exist in the sample analyte.In addition, the device of the analyte in the visual inspection sample of the present invention and mode and these similar devices of the prior art and method relatively make settling at one go of detection, and sensitivity is higher, and is more accurate, operates easier.
Description of drawings
Fig. 1 reagent strip structural drawing.
1: the sample region of acceptance; 2: moving area; 3: stop the zone; 4: surveyed area; 5: the testing result control area; 6: nitrocellulose filter; 7: suction zone;
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used to explain the present invention, and unrestricted scope of the present invention.
Embodiment detects hemp in the urine (THC)
The making and the processing of each parts of reagent strip are described in conjunction with accompanying drawing 1:
Sample region of acceptance 1.The material of sample region of acceptance is a spun glass, with buffer solution (Borax0.05M, pH9.3; Surface-active agents: 2% S-17: (
ON-870)) handling the back dries in 37 ℃ baking oven.
Moving area 2 comprises the monoclonal antibody (THC-Ab) of THC, the common label (Biotin-THC-Ab-latex) that forms of mark substance latex particle (latex) and biotin (Biotin).
The making of label (Biotin-THC-Ab-latex):
1) THC-Ab of 2.5 mg/ml (buy from immune biotech company, Immunetic, Inc.) (PH=8.0) dialysis 4 hours in 1000 milliliters soda mint (NaHCO3) buffer solution forms solution 1.
2) earlier be mixed with 10mM to biotin with pure water, the biotin solution of getting 47 microlitres is then mixed with solution 1 also and is at room temperature stirred 1 hour time formation solution 2.
3) in 1000 milliliters phosphate buffer solution (PH is 7.4), dialyse solution 2 12 hours, form solution 3.
4) under 655 nanometers, measure the concentration of the Biotin-THC-Ab in the solution 3 with spectrophotometer, and be mixed with the solution 4 that ultimate density is 0.5 mg/ml.
5) in clean glass container, measure 3L latex particle latex solution of (35nm-40nm) in optimum range, and insert a stirrer.Container is placed on the stirrer, stir.In latex solution, adjust about latex solution pH to 6.8 behind the phosphate buffer solution of adding pH=7.0 simultaneously.The Biotin-THC-Ab that adds 25mg then continues stirring reaction 1h.
6) latex solution that sealing is finished under the speed of 11000rpm centrifugal 35 minutes is removed supernatant.
7) collecting precipitation, the concentration that makes the latex label solution with washing buffer adjustment in needed scope, the OD value of mensuration solution under 540 nano wave lengths.
8) the final concentration of aignment mark thing (Biotin-THC-Ab-latex) is 0.0125%.
The processing of moving area 2.Earlier the polyester film with buffer solution (PVA5g/L; Na
2HPO
47.1g/L; S-14 (Triton X-100) 0.1%; NaN
30.2g/L) handle the back and in 37 ℃ baking oven, dry, and then marks for treatment thing (Biotin-THC-Ab-latex) in the above, standard be 1 microlitre/centimetre, the mode of processing is for spraying with a spray kind microprocessor device automatically.Handling the back well dries in 37 ℃ baking oven.
Stop the processing in zone 3.On fenced areas 3, handle the antigen (BSA-THC-Ag) that has THC; BSA concentration 5g/L wherein; The concentration of Treatment Solution is 0.6 mg/ml, the standard of processing be 2.5 microlitres/centimetre, the mode of processing is automatically handled three lines with automatic little processing controls Membrane jetter.
The processing of surveyed area 4.On surveyed area 4, handle Streptavidin (from Fitzgerald, Inc company buy), the standard of processing be 1.0 microlitres/centimetre, the mode of processing is for automatic little processing controls Membrane jetter lines of processing automatically.
Nitrocellulose filter 6 comprises obstruction zone 3, surveyed area 4 and testing result control area 5.Be placed on the nitrocellulose filter of handling well in 37 ℃ the baking oven and dry.
The making of reagent strip.Be made into each parts of handling well wide 0.4 centimetre; Long 10 centimetres reagent strip; Let sample region of acceptance 1 and moving area 2, nitrocellulose filter 6 (comprise and stop zone 3, surveyed area 4 and testing result control area 5) and absorbent filter 7 be superimposed in order, these parts all are bonded on the non-water absorptivity card.
Detect the process of analyte THC:
If do not contain THC in the sample or contain a small amount of THC:
1) a certain amount of sample drop is added on the sample region of acceptance 1;
2) sample flow on the moving area 2 and with the mark substance on the moving area and is dissolved in the liquid sample, and mark substance can not combine.If have THC in the sample, then mark substance and THC combine to form compound;
3) through the mobile fenced areas territory 3 that arrives of the sample continuation of moving area 2, thereby the THC antigen on whole or a large amount of mark substances and the obstruction zone combines to be fixed on the obstruction zone.If contain a certain amount of THC in the sample, the remaining mark substance that is not combined by THC is fixed on and stops on the zone, and the compound that mark substance and THC combine to form can not be stopped.
4) continue the mobile surveyed area 4 that arrives through stopping regional sample, do not have or label mass-energy seldom and the combination of the Streptavidin on the surveyed area, thereby be presented as negative findings, do not have in the expression sample perhaps not reach and judge the standard that contains THC.
If contain a certain amount of THC in the sample:
1) a certain amount of sample drop is added on the A of sample region of acceptance;
2) sample flow on the moving area 2 and with the mark substance on the moving area and is dissolved in the liquid sample, and mark substance and THC combine to form compound;
3) sample through moving area 2 continues the mobile fenced areas territory 3 that arrives, and the remaining mark substance that is combined by THC is not fixed on and stops on the zone, and mark substance and THC combine the compound of formation not stopped.
4) continue the mobile surveyed area 4 that arrives through the sample that stops the zone; A large amount of combinations the mark substance that has biotin of THC on surveyed area with the Streptavidin specific bond; Owing to have latex particle; On surveyed area, can see peach line or section, the positive result of visual representation.According to the depth of line or section color, contain what of amount of THC in the judgement sample.
Present embodiment is pressed the proportioning of material variable concentrations and processing on moving area and the surveyed area, is provided with following several groups respectively and tests and choose best enforcement proportioning:
Streptavidin (Sterpavidin) concentration is made as respectively: 0.1mg/ml, 0.2mg/ml, 0.3mg/ml; BSA-THC-Ag:2.0mg/ml;
Concentration is that 0.0125% Biotin-THC-Ab-latex selects 2ul respectively for use, 4ul and 6ul,
Principle according to combination in twos is divided into 9 groups; Comprise the combination (A combination) of the Streptavidin of three kinds of variable concentrations under the 2ulBiotin-THC-Ab-latex; The combination of the Streptavidin of three kinds of variable concentrations under the 4ulBiotin-THC-Ab-latex (B combination), and the combination of the Streptavidin of three kinds of variable concentrations under the 6ulBiotin-THC-Ab-latex (C combination).Dripping different samples respectively through on the pick-up unit of different disposal, comprise negatives, standard items and three times of standard items (3X), after 10 minutes, carry out the rank statistics and obtain following result according to the surveyed area result displayed:
Table one: the A combine detection is the rank statistics as a result
Table two: the B combine detection is the rank statistics as a result
Table three: the C combine detection is the rank statistics as a result
In this experimental data, according to those skilled in the art's approval, the general estimation colour developing is positive visible more than 3 grades, and rank is high more, and it is obvious more to develop the color, and positive findings is stable more; And 1 grade, 2 grades and 3 grades be considered to negative findings, and rank is low more, and negative findings is more stable.Visible by above three table results, the result of each ratio proportioning is more satisfactory in this experiment, and wherein optimum each set of dispense under the THC-Ab-latex concentration 2ul/4mm compares testing result.
Claims (7)
1. pick-up unit that is used to detect analyte; Comprise moving area in order, stop zone and surveyed area; It is characterized in that: moving area comprises can be with the analyte that has mark substance of liquid flow corresponding antibody or antibody fragment, also be connected with on this antibody or the antibody fragment with the incoherent specific bond molecule of analyte to it a kind of molecule; Stop the zone and go up corresponding antigen or the antigen fragment of fixing a kind of analyte; On the surveyed area fixing and the incoherent specific bond molecule of analyte to another kind of molecule; The antigen that antibody that described analyte is corresponding or antibody fragment are corresponding with analyte or the affinity of antigen fragment are greater than corresponding antibody of analyte or the affinity between antibody fragment and the analyte.
2. the pick-up unit that is used to detect analyte according to claim 1 and 2; It is characterized in that described and antibody or the protein staphylococcus/animal immune Lysozyme of the incoherent specific bond molecule of analyte to being selected from biotin/avidin, rhodamine/rhodamine.
3. the pick-up unit that is used to detect analyte according to claim 1 and 2 is characterized in that, described and the incoherent specific bond molecule of analyte are to being selected from biotin/Streptavidin.
4. the pick-up unit that is used to detect analyte according to claim 1 and 2 is characterized in that, on described moving area with the incoherent specific bond molecule of analyte be biotin to it a kind of molecule; On described surveyed area fixing and the incoherent specific bond molecule of analyte to another kind of molecule be selected from Avidin.
5. the pick-up unit that is used to detect analyte according to claim 1 and 2 is characterized in that, on described moving area with the incoherent specific bond molecule of analyte be biotin to it a kind of molecule; On described surveyed area fixing and the incoherent specific bond molecule of analyte to another kind of molecule be selected from Streptavidin.
6. the pick-up unit that is used to detect analyte according to claim 1 and 2 is characterized in that, described mark substance is selected from: latex particle or colloid gold particle.
7. the pick-up unit that is used to detect analyte according to claim 1 is characterized in that, described analyte is a THC; Described moving area comprise concentration be 0.0125% can also be connected with biotin on this antibody with the THC antibody that has the latex mark of liquid flow; Stopping regional upward fixed concentration is the THC antigen of 2.0mg/ml; Fixed concentration is the Streptavidin of 0.1mg/ml on the surveyed area.
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| CN2007100690702A CN101078724B (en) | 2007-06-08 | 2007-06-08 | Detection device and method for detecting analyte |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100690702A CN101078724B (en) | 2007-06-08 | 2007-06-08 | Detection device and method for detecting analyte |
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| CN101078724B true CN101078724B (en) | 2012-01-04 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP3157356U (en) * | 2006-07-26 | 2010-02-18 | インバーネス・メデイカル・スウイツツアーランド・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Analytical equipment for biological samples |
| WO2009070812A1 (en) * | 2007-11-29 | 2009-06-04 | Inverness Medical Switzerland Gmbh | Assay |
| CN102169120B (en) * | 2010-02-25 | 2014-09-17 | 艾博生物医药(杭州)有限公司 | Detector |
| CN102928594B (en) * | 2011-12-27 | 2015-11-25 | 北京宝瑞源科技孵化有限公司 | phencyclidine detection kit and preparation method thereof |
| CN102565382B (en) * | 2012-01-06 | 2013-12-04 | 苏州浩欧博生物医药有限公司 | Immunochromatography method for detecting allergen-specific IgE antibodies in blood samples |
| CN102937647B (en) * | 2012-02-16 | 2015-11-25 | 北京宝瑞源科技孵化有限公司 | Lysergide detection kit and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002014869A2 (en) * | 2000-08-17 | 2002-02-21 | Lifepoint, Inc. | Assay method and device for detecting the presence and concentration of analytes |
| WO2006073500A2 (en) * | 2004-07-29 | 2006-07-13 | Relia Diagnostic Systems, Llc | Lateral flow system and assay |
| WO2006115866A1 (en) * | 2005-04-20 | 2006-11-02 | Becton, Dickinson And Company | Semi-quantitative immunochromatographic device |
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2007
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002014869A2 (en) * | 2000-08-17 | 2002-02-21 | Lifepoint, Inc. | Assay method and device for detecting the presence and concentration of analytes |
| WO2006073500A2 (en) * | 2004-07-29 | 2006-07-13 | Relia Diagnostic Systems, Llc | Lateral flow system and assay |
| WO2006115866A1 (en) * | 2005-04-20 | 2006-11-02 | Becton, Dickinson And Company | Semi-quantitative immunochromatographic device |
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