CN103983773A - Fast immunochromatographic detection of oral fluid - Google Patents
Fast immunochromatographic detection of oral fluid Download PDFInfo
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Abstract
The invention relates to fast immunochromatographic detection of an oral fluid and also relates to an oral fluid collection swab separated from a side flow immunochromatographic test strip for detecting substances to be analyzed in the oral fluid. The side flow immunochromatographic test strip mainly comprises a sample pad, a conjugate pad, and detection zone and contrast zone pads. The pads comprise at least one base material. The conjugate pad is located in the downstream of the sample pad and is provided with conjugate stripes. The detection zone and contrast zone pads are located in the downstream of the conjugate pad. A specific binding reagent specifically binding with a target analyte is fixed to a detection zone. A second capture reagent is fixed to a contrast zone in the downstream of the detection zone. The invention also relates to a production method of the side flow immunochromatographic test strip, a side flow immunochromatographic method for detecting the analyte in the oral fluid by the side flow immunochromatographic test strip, and a kit containing the side flow immunochromatographic test strip.
Description
The application is dividing an application of application number is 200580049809.2, denomination of invention is " detections of mouth cavity fluid fast immune chromatographic " Chinese patent application, and this mother's case application is the application that the pct international patent application PCT/CN2005/000701 of submission on May 20th, 2005 enters the China national stage.
Technical field
The present invention relates to mouth cavity fluid fast immune chromatographic detects.More specifically, the present invention relates to the lateral flow immunochromatography inspection test-strips for detection of analyte in mouth cavity fluid, collect the method for mouth cavity fluid sample, produce the method for described examination bar, by using this examination bar to detect the lateral flow immune chromatography method of analyte in mouth cavity fluid and containing the kit that this tries bar.
Background technology
People have developed the analytical approach of various analytes in many qualitative or quantitative detection of biological somas and fluid.It is to adopt blood, urine, ight soil, biopsy or mouth cavity fluid that current most diagnosis detects.Compare with other material, collect mouth cavity fluid saliva and/or oral mucosa exudate for detection of bringing relatively less privacy to invade, it is comparatively safe, and can relatively easily complete fast.
So far, for exploitation is for the test-strips of collection, transmission and the sample preparation of mouth cavity fluid and for developing analysis, the especially analysis to various antibody and metabolin based on mouth cavity fluid, people have paid a lot of effort.
For example, WO88/07680 discloses qualitative by complicated radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) etc. or has quantitatively detected specific IgM, IgG and the existence of IgA or the method for its amount in human or animal's body fluid (being selected from saliva, tears, seminal fluid, urine and cerebrospinal fluid), described method is difficult to carry out relatively, time-consuming and cost many.
U.S. Patent No. 5,103,836 disclose a kind of from oral collection material for detection of method, it comprises the following steps: the absorption pad that soaks into hypertonic salt solution is inserted to oral cavity, and wherein the concentration of the salt of hypertonic solution in pad can reclaim the described material of high concentration effectively from oral cavity; From oral cavity, remove described pad; Preserve this pad to take out collected material for ELISA subsequently from pad.In disclosure file, disclosed absorption pad need to be with hypertonic solution pre-service so that the analyte of high concentration to be provided, and reuse complicated ELISA and detect analyte.
U.S. Patent No. 6,303,081 disclose for from oral collection and transhipment aqueous fluids to for detection of the inspection test-strips of lateral chromatography examination bar.Lateral chromatography examination bar is placed in outer cover (housing) and stretch in the chamber limiting along outer cover.To described lateral chromatography examination bar, provide at least one to inspect (inspection) position and inspect test result to realize selected position on lateral chromatography examination bar.Multi-hole center bar (wick) material at one end reaches the collection position of outer cover outside from described outer cover, at the other end, communicate with described lateral chromatography examination bar.Described multi-hole center strip material has particulate configurations, and described particulate absorption water-based mouth cavity fluid is to deliver to this fluid described lateral chromatography examination bar and to there is no absorption from oral instructions.This multi-hole center strip material is easily discharged into mouth cavity fluid lateral chromatography examination bar.Due to the roundabout stream of this multi-hole center strip material, naturally prevent the reversed flow from lateral chromatography examination bar to oral cavity.Biteplate (bite plate) is connected with described outer cover and can inserts between patient's tooth multi-hole center bar to be positioned in oral cavity, collect mouth cavity fluid.Described biteplate is maintained at appropriate location so that multi-hole center bar is positioned in oral cavity space by dental articulation power (occlusal force) conventionally.When inspection test-strips is in mouth, by observing lateral chromatography examination bar, obtain rapidly testing result.In U.S. Patent application No.2002/0192839, U.S. Patent application No.2002/0155029, all disclose for a step and collected mouth cavity fluid to detect and/or quantitatively improved inspection test-strips and the method for mouth cavity fluid analyte, the content of these patents or application all with its integral body by reference to being incorporated to herein.
Although the analytic approach by above-mentioned inspection test-strips has advantages of directly, needs complex steps fast and not, for detected object, it is inconvenient in whole testing process, all wanting interlock inspection test-strips.In addition, for the analysis with engineering noise indication, when null result need to detect again, or detect for the diagnosis that need to confirm making a collection of specimens such as the such routine of HIV to detect, object will have to persist in into detection interlock inspection test-strips.Yet, should be understood that, after first collection, reach one hour, the collection of oral mucosa exudate reduces the exudate of the available IgG of being rich in of oral tissue surfaces conventionally, it is convenient not that this will make to collect subsequently to use " one-stop sample divider/detection combination " again to detect, because detected object must be waited until exudate higher level, otherwise come detection risk may have lower sensitivity (R.Mink, not Publishing Data) with sample.
Summary of the invention
The present invention keeps in mind the above-mentioned shortcoming existing in prior art and makes, an object of the present invention is to provide a kind of simple, convenient and cost-efficient mouth cavity fluid collection method with side direction immunity-chromatography test strip, it is suitable for sensitive and detects rapidly analyte in mouth cavity fluid.
Another object of the present invention is to provide a kind of method of producing described lateral flow immunochromatography inspection test-strips.
Another object of the present invention is to provide and is a kind ofly suitable for sensitive and detects rapidly the sensitive method of analyte in mouth cavity fluid.
Another object of the present invention is to provide for sensitive and detect rapidly the kit of mouth cavity fluid analyte.
Therefore, aspect first, the invention provides a kind of inspection of the lateral flow immunochromatography for detection of analyte in mouth cavity fluid test-strips, it is substantially by sample pad, conjugate pad, detection zone and check plot pad form, described pad is made by least one host material, wherein said conjugate pad is positioned on the downstream of sample pad and its draws striped with conjugate, described detection zone and check plot pad are positioned at downstream and inclusion test district and the check plot of this conjugate pad, wherein detection zone is fixed with specificity combinating reagent, described specificity combinating reagent is combined with target analyte specifically, and check plot is fixed with the second capture agent.
In an embodiment in this regard, analyte to be detected is selected from the antibody of anti-following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
In another embodiment, described host material is selected from inorganic powder, such as silicon dioxide and aluminium oxide; Glass fiber filter paper; Natural polymerization material is especially based on cellulosic material, chromatographic paper; The natural polymer of synthetic or modification is such as cellulose nitrate, cellulose acetate, Polyvinylchloride, polyacrylamide, cross-link dextran, agarose; With and combination.
In another embodiment, the host material of sample pad is glass fiber filter paper; The host material of conjugate pad is polyester material; The host material of detecting pad and contrast pad is nitrocellulose filter.
In another embodiment, conjugate comprises the label with the first capture agent coupling, and described the first capture agent is caught endogenous antibody in mouth cavity fluid.Described label is selected from colloid gold particle; Element or metal-sol particle, comprise selenium, silver, iron or carbon; Other pearl particle, comprises coloured latex, liposome and dye granule.Preferably, described label is colloid gold particle.
In another embodiment, described the first capture agent is selected from: the antibody of anti-IgG, IgM or IgA, albumin A, Protein G and concanavalin A.Preferably, described the first capture agent is albumin A.
In another embodiment, described specificity combinating reagent is selected from following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
In another embodiment, the antigen of communicable disease is restructuring or the synthetic peptide that represents HIV protein immunization determining area, and described HIV albumen is especially selected from the HIV envelope protein of the gpl20 of HIV-1 and the gp36 of gp41 and HIV-2.
In another embodiment, the second capture agent is selected from: the antibody of anti-IgG, IgM or IgA, albumin A, Protein G and concanavalin A, the antibody of wherein said anti-IgG is mountain goat anti-human igg antibody.
Aspect second, the invention provides the method for the defined lateral flow immunochromatography inspection of any one test-strips in production claim 1-17, it comprises: a) conjugate is drawn on conjugate pad to striped; B) described specificity combinating reagent is fixed on the detection zone of detection zone and check plot pad; C) the second capture agent is fixed on the check plot of detection zone and check plot pad; D) with described each pad of sealer sealing; And e) alignment gained pad is communicated with fluid each other.
In one embodiment, before drawing striped, conjugate is stabilized in simple or glycoconjugate solution, and wherein said sugar juice comprises sucrose, trehalose, potassium stannate and carbamide peroxide.
In another embodiment, use biotin/streptavidin connexon that described specificity combinating reagent is fixed on detection zone and check plot.
In another embodiment, use biotin/streptavidin connexon that described specificity combinating reagent is fixed on check plot.
In another embodiment, the detergent that described sealer comprises nonionic, cationic, negative ion and amphoteric ion type; Sugar, comprises sucrose, fructose; Or protein, comprise that bovine serum albumin(BSA), animal's whole blood are clear, casein and skimmed milk power.
In another embodiment, animal's whole blood is clearly hyclone.In another embodiment, animal's whole blood is clearly for being selected from the birds serum of goose serum, turkey serum and chicken serum.
Aspect the 3rd, the invention provides the lateral flow immune chromatography method for detection of analyte in mouth cavity fluid, it comprises: (a) use the gatherer separating with the defined lateral flow immune chromatograph testing examination of any one in claim 1-17 bar to collect mouth cavity fluid; (b) gatherer dropped in the sample buffer of certain volume to mouth cavity fluid is discharged in damping fluid and obtain potpourri; (c) the defined lateral flow immune chromatograph testing examination of any one in aforementioned claim bar is put into described potpourri; (d) existence by observing check plot signal to be to determine the validity of detection, and by observing the existence of signal in detection zone in 15-60 after detection starts minute, determines the existence of analyte.
In another embodiment, the gatherer in step (a) is untreated polyester swab (swab), for example Texwipe Large Alpha Swab TX714A.
In another embodiment, sample buffer is the buffer solution of potassium phosphate of pH7.2+/-0.2, and it also comprises 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween80,0.2%Tetronic T-904 and 0.0.285% (active component) ProClin950.
In another embodiment, the volume of sample buffer is 1000 μ l.
In another embodiment, described method is also included in the step of taking out potpourri sample between step (b) and step (c), and the volume of wherein said sample is 200 μ l.
In another embodiment, described signal is for there being colo(u)r streak, as blush (reddish) line.
In another embodiment, in 20-45 minute, observe described signal.
Aspect the 4th, the invention provides the kit for detection of analyte in mouth cavity fluid, it comprises at least one above-mentioned lateral flow immunochromatography inspection test-strips.Described inspection test-strips is closed in drying receptacle.Each kit optionally comprises sample buffer, mouth cavity fluid gatherer, the package insert of appended examination bar instruction is provided, containing being useful on, inspection test-strips is carried out the positive of quality testing and the tubule of negative control (vial), be can be used for determining that the present invention measures timer and/or the bio-hazard thing container handling when completing.
In a preferred embodiment of this aspect, sample buffer is the buffer solution of potassium phosphate of pH7.2+/-0.2, and it also comprises 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin950.
In another embodiment, gatherer is untreated polyester swab, for example Texwipe Large Alpha Swab TX714A.
Therefore, an advantage of the invention is the simplicity of design and the low cost of material, this is owing to lacking outer cover, such as this is found in other mouth cavity fluid fast detecting (for example, referring to United States Patent (USP) 6303081, U.S. Patent application 20020155029 and 20020192386) and other quick lateral flow detects (United States Patent (USP) 6,303,081,5,935,864,6,485,982,6,534,320,6,352,862,6,187,598 and 6,027,943) in.This cost that not only makes product manufacture is lower, but also the more manufacturing process complicacy of low-complexity is provided, and this has further reduced cost.
Another advantage of the present invention be this analysis self be independent of sample divider (for example United States Patent (USP) 6303081, U.S. Patent application 20020155029 and 20020192839 and United States Patent (USP) 5935864 in), this makes user to sample, carry out repeated detection in the situation that not needing to collect in addition.This is especially favourable in the lateral flow immunochromatographic measurement with engineering noise indication, wherein invalid result need to detect again, or need to confirm that it is also especially favourable that the diagnosis detecting detects to collected sample for the such routine of for example HIV, because reach one hour after first collection, oral mucosa exudate collect conventionally reduce oral tissue surfaces can with the effluent that is rich in IgG, it is convenient not to make to collect subsequently again to detect with " one-stop sample divider/detection is combined ".
The further advantage of the present invention is that (for example United States Patent (USP) 6 with other mouth cavity fluid detection, 303,081) compare, described mouth cavity fluid fast immune chromatographic examination bar very compact (for example 5mm * 1mm * 75mm), this reduces transportation and storage cost.
Another advantage of the present invention is that described mouth cavity fluid collection method provides the volume enough detecting such as authenticity for other detection judgement.
The further advantage of the present invention is that (for example United States Patent (USP) 6 than other mouth cavity fluid detection, 303,081) reduced complexity of producing, because needn't configure outer cover around at lateral flow chromatography examination bar, this causes material cost and process time to be all minimized.
Accompanying drawing explanation
Fig. 1 illustrates the program detecting according to the mouth cavity fluid fast immune chromatographic of a preferred embodiment of the present invention, and wherein Fig. 1-1 illustrates the step of detection to 1-15.
Fig. 2 illustrates according to a preferred embodiment of the present invention, three kinds of possible outcomes that mouth cavity fluid fast immune chromatographic detects, and wherein Fig. 2 A, 2B and 2C represent respectively feminine gender, the positive and invalid result.
Embodiment
By to following detailed description of the preferred embodiments also by reference to the accompanying drawings, can understand better the present invention.
Aspect first, the invention provides a kind of inspection of the lateral flow immunochromatography for detection of analyte in mouth cavity fluid test-strips, it is comprised of sample pad, conjugate pad, detection zone and check plot pad substantially, described pad is made by least one host material, wherein conjugate pad is positioned at the downstream of sample area pad, and draw striped thereon with conjugate, described detection zone and check plot pad are positioned at the downstream of this conjugate pad, and in detection zone, be fixed with the specificity combinating reagent with target analyte specific binding, in check plot, be fixed with the second capture agent.
Described sample pad receives oral cavity fluid sample.Sample pad consists of the material that shows low target antibody reservation conventionally.In some embodiments, sample pad also can play the effect of mechanical filter, and it can catch any unwanted particle such as dust, fragment, precipitation, mucus or blood.
Described conjugate pad is positioned at the downstream of sample pad and contains conjugate, and described conjugate comprises directly or indirectly and the label of the first capture agent coupling.
Described detection zone is positioned at the downstream of conjugate pad and contains specificity combinating reagent, and described specificity combinating reagent is combined with target antibody specifically, and whereby, the conjugate of mark can be fixed in matrix.
Described check plot is arranged in the downstream of stream detection zone.The second capture agent is contained in check plot, and described the second capture agent is caught the antibody of being caught by the first capture agent, and is preferably fixed in check plot to form control line, and described control line is concentrated any by the labeled antibody conjugate of the second capture reagent bind.
As used herein, term " mouth cavity fluid " refers to be found in alone or in combination intraoral one or more fluids.It includes but not limited to saliva and oral mucosa exudate.Will be appreciated that, mouth cavity fluid for example can comprise, from multiple source (the attached body of gland parotid gland, salivary gland, sublingual gland, gums mucous membrane and oral mucosa) the combination of fluid, and term mouth cavity fluid comprises the fluid from each these source alone or in combination.Term saliva refers to the combination of mouth cavity fluid, for example, conventionally see oral cavity, especially after chewing.As used herein, term " oral mucosa exudate " refer to by serum component from oral mucosa space to oral cavity the fluid that produces of passive diffusion.Oral mucosa exudate usually forms a kind of component of saliva.
As used herein, term " analyte " is used in reference to part to be detected in particular analysis.Conventionally the analyte detecting in the present invention analyzes includes but not limited to the antibody of anti-following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.Particularly preferred analyte comprises the antibody of the antigen of infectivity resistant disease, and described communicable disease is such as HIV, hepatitis etc.Antigen can be but be not limited to such as hepatitis B antigen, antibody can be but be not limited to the antibody of HIV, the antibody of the antibody of HTLV, helicobacter pylori, the antibody of the antibody of the antibody of hepatitis, measles, parotitic antibody and rubella.The metabolic product of medicine and drug abuse or drug abuse can be but be not limited to tetrahydrocannabinol, nicotine, ethanol, bitter edible plant alkali, phenytoinum naticum, paracetamol, lithium, stable, nortriptyline (nortryptyline), quinalbarbitone, phenobarbital.Hormone can be but be not limited to testosterone, estradiol, 17-hydroxyprogesterone, progesterone, thyroxine, thyrotropic hormone, follicle-stimulating hormone (FSH) and luteinizing principle.
As used herein, " specificity combinating reagent " is the reagent with target analyte specific binding, as mentioned above, described target analyte is selected from following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
As used herein, " antigen " is a kind of material that stimulates antibody to generate when it is introduced in mammal or birds.The preferred antigen of the present invention comprises human immunodeficiency virus (HIV) protein, the particularly gp36 of the gp120 of virus envelope protein HIV-1 and gp41 and HIV-2.
As used herein, " antibody " refers to the protein being comprised of one or more polypeptide of substantially being encoded by immunoglobulin gene or immunoglobulin gene fragment.Generally acknowledged immunoglobulin gene comprises κ, λ, α, γ, δ, ε and μ constant region gene, and the immune globulin variable region gene of huge amount.The light chain of antibody is divided into κ or λ.Heavy chain is divided into γ, μ, α, δ or ε, itself so that determine that the type of immunoglobulin (Ig) is respectively IgG, IgM, IgA, IgD and IgE.
The basic structural unit of known antibodies (also claiming immunoglobulin (Ig)) comprises the tetramer.Each tetramer is comprised of two pairs of identical polypeptied chains, and every pair has a light chain (about 25kD) and a heavy chain (about 50-70kD).Main approximately 100 to 110 or more amino acid whose variable region of being responsible for antigen recognizing of N end definition of each chain.Term variable region light chain (V
l) and variable region heavy chain (V
h) refer to respectively these light chains and heavy chain.
Antibody can be used as complete immunoglobulin (Ig) or exists as the fragment by a large amount of well-characterized of producing with various peptide enzymic digestions.Therefore, for example pepsin region produces F (ab ')
2, F (ab ')
2be the dimer of Fab, Fab is from as by disulfide bond and V
h-V
h1 connected light chain.F (ab ')
2thereby the disulfide bond that can be reduced to destroy hinge region under temperate condition connects by F (ab ')
2dimer is converted into Fab ' monomer.Fab ' monomer is the Fab with a hinge area part (about the more detailed description of other antibody fragment referring to Fundamental Immunology, W.E.Paul, Ed., Raven Press, N.Y., 1993) substantially.Although various antibody fragments are to define according to the digestion of complete antibody, technician will know such Fab ' fragment can be by chemistry or by utilizing recombinant DNA method from the beginning synthetic.Therefore, term antibody also comprises antibody fragment as used herein, and described antibody fragment produces or uses recombinant DNA method from the beginning synthetic by modifying complete antibody.
As used herein, term " matrix " refers to the insoluble material that can support that fluid flows.Host material can be source natural origin and/or synthetic, (bibulous) of water suction or do not absorb water, fibrous or granular.Matrix of the present invention can form the continuous strip of same material or different materials potpourri, and described different materials is along common band continuous distribution, or discontinuously arranged district's band to be formed on band zones of different with different physics or chemical property.As an alternative, can be formed by identical or different host material the pad of series of discrete, and the reagent for measuring is added to each pad.Then pad can be placed to fluid communication with each other to form continuous stream.For the material that builds matrix of the present invention can be inertia or can with one or more reagent reactings of the present invention, it is insoluble that prerequisite is that described material is still in implementing invention described herein.Host material can be selected from inorganic powder, such as silicon dioxide and aluminium oxide; Glass fiber filter paper; Natural polymerization material particularly based on cellulosic material such as filter paper, chromatographic paper; The natural polymer of synthetic or modification is such as cellulose nitrate, cellulose acetate, Polyvinylchloride, polyacrylamide, cross-link dextran, agarose; With and combination.In a preferred embodiment, described matrix is nitrocellulose filter.
In another preferred embodiment of the present invention, glass fiber filter paper is as the matrix of sample pad, and polyester material is as the matrix of conjugate pad, and nitrocellulose filter is as the matrix of detection zone and check plot pad.
" water suction " (bibulous) refers to that some absorbent material supports the ability of the different solute migration speed of otherness during fluid is flowed through this material.Therefore the absorbent material that, has water suction character can carry out chromatography by solute based on physics and/or chemical property.
As used herein, term " label " refers to directly or indirectly by detectable atom or the molecular structure of analyte specific binding companion and analyte specific binding.Label of the present invention can be by physics or chemical detection.Preferred label be when in conjunction with time visible to bore hole, to indicate positive test symbol.Label of the present invention is selected from colloid gold particle; Element or metal-sol particle, comprise selenium, silver, iron or carbon; Other pearl particle, comprises coloured latex, liposome and dye granule; In preferred embodiment of the present invention, described label is colloid gold particle.These labels are well known in the art, and are described in for example G.Frens Nature, in 241,20-22 (1973) and U.S. Patent No. 4,313,734, its disclosure with its integral body by reference in being incorporated herein.
" collaurum " used in this invention refers to the colloidal sol of fine gold grain, and it can ad infinitum be held in aqueous suspension.
As used herein, " capture agent " is any molecule that is attached to specifically target antibody.Capture agent of the present invention is preferably fixed on matrix with deterministic model, typically is the line vertical with stream.Preferred capture agent is the antibody of anti-IgG, IgM or IgA; Albumin A, Protein G or concanavalin A.
As used herein, " the first capture agent " of the present invention is the capture agent with label coupling.Preferably, the antibody that described the first capture agent is anti-IgG, IgM or IgA; Albumin A, Protein G or concanavalin A.In a preferred embodiment, albumin A or Protein G and label coupling, form conjugate.
" albumin A " used in this invention refers to the high stability surface receptor being produced by staphylococcus aureus (Staphylococcus aureus), its can with the immunoglobulin (Ig) of a large amount of species especially Fc of the IgG (Boyle that partly combines, M.D.P.and K.J.Reis.Bacterial Fc Receptors.Biotechnology5:697-703,1987).At least 2 IgG molecules of an albumin A molecular energy while combination (
j., Meloun, B.and Hjelm, H.Protein A is isolated from Staphylococcus aureus after digestion with lysostaphin.Eur J Biochem29:572-578,1972).
" Protein G " used in this invention refers to the cell surface associated protein of being combined with IgG with high-affinity from hammer mattress.It has the IgG binding structural domain (referring to Lian, et al., 1992.Journal of Mol.Biol.228:1219-1234 and Derrick and Wigley.1994.Journal of Mol.Biol.243:906-918) of three height homologies.
As used herein, " the second capture agent " is to be fixed on check plot to catch the capture agent of the antibody of being caught by the first capture agent.Suitable the present invention's the second capture agent is antibody, albumin A, Protein G or the concanavalin A of anti-IgG, IgM or IgA.In a preferred embodiment, described the second capture agent comprises the anti-IgG antibody that provides the species of mouth cavity fluid from being different from.Even more preferably, this second capture agent is anti-human IgG antibody; Most preferably, this second capture agent is mountain goat anti-human igg antibody.
As used herein, " stream " refers to when the route of oral cavity fluid sample through matrix time institute warp.Described stream is preferably uniline, but can comprise several routes, and wherein every route can simultaneously, sequentially or independently be supported liquid flow for other route.
When " downstream " refers to that liquid passes through matrix away from the directed stream of liquid application point.
When " upstream " refers to that liquid passes through matrix towards the directed stream of liquid application point.
Aspect second, the invention provides the method for production lateral flow immunochromatography inspection test-strips, it comprises: a) conjugate is drawn on conjugate pad to striped; B) specificity combinating reagent is fixed to the detection zone of detection zone and check plot pad and the second capture agent is fixed to check plot; C) with sealer, seal each pad; And e) alignment gained pad is communicated with fluid each other.
conjugate is drawn on conjugate pad to striped
Conjugate is deposited in the matrix of conjugate pad in the following manner: when it contacts with mouth cavity fluid sample, it is easy to migration in fluid stream.For realizing this point, the matrix of conjugate pad is made by the polyester of netted bonding (spun-bonded).Conjugate is utilized together with drawing striped solution to for example contact tip or gasoloid tip stroke striped on pad.Before drawing striped, preferably by conjugate stabilization.For example, can be by conjugate stabilization in 20% sucrose, 5% trehalose and 0.1% carbamide peroxide.
When oral cavity fluid sample is flowed through conjugate pad, conjugate is dissolved and add the fluid by inspection test-strips to flow.
specificity combinating reagent is fixed to detection zone
Use the methods known to those skilled in the art, be applicable to specificity combinating reagent of the present invention and can comprise from any source natural, chemosynthesis or recombinant production and obtain.For example, the peptide moiety of preferred SEQ ID No.1 to 5 can be used the chemosynthesis of solid-phase peptide synthetic technology, or by operability, the nucleic acid of coding expectation peptide is connected to and in expression vector and in suitable host, expresses described nucleic acid and carry out recombinant production.Once separated, can use known technology by described peptide biotinylation.
Can use any the methods known to those skilled in the art that suitable antigen is fixed to matrix, described method is not destroyed the specific binding of antigen to target antibody.Preferably, for recombinant protein antigen, antigen is fixed directly in matrix without further modifying.Preferably, use biotin/streptavidin connexon that antigenic synthetic peptide is fixed in matrix, most preferably, antigen is coupled to biotin compound with streptavidin, then streptavidin is coupled to matrix.For water absorptivity matrix, use the technology that well known to a person skilled in the art, conventionally before sealing, the streptavidin of compound is coupled to matrix.Preferably, in contrasting the solution of each biotin structure, the streptavidin binding site equivalent that contains at least 4: 1 ratio realizes coupling, although other ratio is such as in 0.5: 1,1: 1,2: 1,3: 1 and 5: 1 and all centres (mark) ratio be also included within, as a part of the present invention.For water absorptivity matrix, resulting composite can be applied to host material dry simply, subsequently with sealing with suitable sealer.The suitable antigen that has enough high molecular and can directly be combined with matrix is such as a lot of recombinant proteins needn't adhere to by the method for biotin/streptavidin connexon.The amino acid sequence that is applicable to exemplary antigenic peptides of the present invention sees SEQ ID No:1 to 5.
Antigen is fixed to matrix and preferably for the mode of concentrated labeled antibody conjugate, carries out, described antibody coupling matter is specifically in conjunction with immobilized antigen.By the antibody coupling matter of concentrated mark, the signal being produced by label is reinforced, thereby improve sensitivity, also reduces as far as possible to obtain the possibility of error result.
the second capture agent is fixed on check plot
The second capture agent is contained in check plot, and is preferably fixed in check plot to form the control line of concentrated label conjugate.Adopt known technology, comprise the above-mentioned technology for immobilized antigen, will be applicable to the second capture agent of the present invention and be fixed to matrix.Can use biotin/streptavidin connexon that the second capture agent is fixed to matrix, in the case, most preferably, as mentioned above, before streptavidin is coupled to matrix, the second capture agent be coupled to biotin and meet with it with streptavidin.Preferably, capture agent is such as mountain goat anti-human igg F (ab ')
2can not use biotin/streptavidin connexon and be fixed directly in matrix.
pad is sealed with sealer
Although can use intrinsic absorptive host material to make pad of the present invention, the fluid that the present invention that flows through examines test-strips flows preferably in itself for non-absorptive.
By application sealer, water-absorbing material can be converted into the material that shows non-water absorptivity flow characteristics.These sealers can be detergent, sugar or protein, and it can hinder the interaction force that produces water absorption character.Exemplary protein sealer comprises that bovine serum albumin(BSA) self or its methylate or succinylation form, animal's whole blood clear (for example horse or hyclone), and other blood protein.Preferred sealer is that birds serum is such as goose or turkey serum, most preferably chicken serum.Other example of protein blocking agent comprises casein and skimmed milk power.Sealer based on detergent is selected from nonionic, cationic, anionic and amphoteric ion type, and its selection is the character based on being closed matrix.Tween20 is the useful especially detergent for closing membrane.The exemplary sugar that can be used as sealer comprises sucrose and fructose.
Can to remove undesirable surface reaction, apply sealer by pad described in the sealer solution-treated by effective concentration.Generally speaking, this processes and adopts lock solution under about room temperature, to carry out several minutes to a few hours, and described lock solution is such as the protein solution of 1-20mg/ml protein.Gained coating material then by air be dried, freeze-drying or other drying means be for good and all adsorbed onto surface.
The matrix of using intrinsic water absorptivity but can changing non-water suction flow characteristics into is for fixedly specificity combinating reagent and the second capture agent are particularly useful.For example, can the second capture agent be applied to matrix before applying sealer and can fix in position.Once the second capture agent has been fixed to matrix, then can apply sealer.
For example, operation the present invention during, by sealer of the present invention to be enough to the stoping amount of target antibody and host material interphase interaction to be applied to sample pad.A kind of particularly advantageous sealer for sample pad is birds serum, more preferably chicken serum.In a preferred embodiment, sample pad is soaked into the solution that contains following composition: polyvinylpyrrolidone, bovine serum albumin(BSA), birds serum, borate and/or carbonate buffer solution (about 0.5M) and Triton X-100 or Tween-20 detergent.To the sample pad of being made by glass fiber material, most preferred sample sealing damping fluid is by forming below: 40% chicken serum (hot deactivation aseptic filtration), 0.25M saleratus, 0.05M dipotassium hydrogen phosphate, 0.1%Tween80,100mM potassium stannate and 0.2% carbamide peroxide, its pH is 8.2 to 8.5.Pad compression also will be padded 30 ℃ of dried overnight to remove excessive damping fluid.The wettable that an advantage of the method is sample pad and capillary action increase.
The composition of sealing damping fluid and pH change with the material type of pad.For example,, by the conjugate pad made by polyester material being immersed in the damping fluid that contains following composition and by its sealing: polyvinylpyrrolidone, chicken serum, bovine serum albumin(BSA), 0.1% carbamide peroxide, 100mM potassium stannate and sal tartari and/or borate buffer.Then by conjugate pad in dry 120 minutes of 50 ℃ and forced ventilation, then air-dry overnight under environment temperature.For the detection zone of being made by nitrocellulose filter and check plot pad, most preferred sample sealing damping fluid composition is: 0.15% bovine serum albumin(BSA), 0.075Tween20 in the potassium salt soln of pH7.8+/-0.1 buffering.
Aspect the 3rd, the invention provides the lateral flow immune chromatography method for detection of analyte in mouth cavity fluid, it comprises: (a) use the gatherer separating with lateral flow immunochromatography inspection test-strips to collect mouth cavity fluid; (b) gatherer dropped in the sample buffer of certain volume to mouth cavity fluid is discharged in damping fluid and obtain potpourri; (c) lateral flow immunochromatography inspection test-strips as above is placed in to the about 15-60 minute of potpourri; (d) by observing the existence of check plot signal, determine the validity detecting, and by observing the existence of detection zone signal, determine the existence of analyte.
The principle of said method sees following.Mouth cavity fluid is examined upwards migration of test-strips by matrix by wicks and along measuring.It is conjugate rehydration, and such as erythroid protein A-colloidal gold reagent on examination bar, and the IgG in sample is combined with conjugate and is formed IgG/ conjugate compound.Described IgG/ conjugate compound continues along the upwards migration of examination bar, and first the detection zone of containing specificity combinating reagent in test-strips is examined in arrival, wherein said specificity combinating reagent can be combined with target analyte specifically, and compound is fixed in detection zone and occurs signal, for example erythroid have a colo(u)r streak.This Indicator Reaction or positive findings.In detection zone, do not have signal designation sample not contain target analyte and form non-reacted or negative findings.IgG/ conjugate compound continues to make progress migration until it arrives check plot along measuring examination bar.The second capture agent is contained in check plot, for example, be fixed on the anti-human F of goat in the line of measuring inspection test-strips (ab ')
2igG fragment.Remaining IgG/ conjugate compound and immobilization F (ab ')
2fragment combination, and occur signal, for example erythroid have a colo(u)r streak.The appearance of this signal is to test the evidence of normally working and containing IgG.No matter sample is reactive or non-reacted for target analyte, is carrying out all having signal to occur in check plot between all effective detection periods.Sample continues to enter final absorption pad through check plot migration, and its help pulls IgG/ conjugate compound by examination bar and removes any background color.
In this regard, the sample divider that can be used for this test can be from oral collection oral mucosa exudate, and known described oral mucosa exudate has the required higher diagnostic IgG of other products (for example, referring to U.S. Patent No. 5,103,836).In fact, in a preferred embodiment, for the gatherer of this mensuration, be ready-made untreated polyester swab, i.e. Texwipe Large Alpha Swab, TX714A (Texwipe Inc., Upper Saddle River NJ).
In an embodiment in this regard, for diluting the sample buffer of mouth cavity fluid, be 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30ug/ml affinity element, 0.2%Tween80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin950 with pH7.2+/-0.2 potassium phosphate buffering.For diluting the volume of the sample buffer of mouth cavity fluid, be 1000 μ l.
In another preferred embodiment, the method is also included in the step of taking out potpourri sample (aliquot) between step (b) and step (c).In a specific embodiments, the volume of described sample is 200 μ l.
When proper operation, the second capture agent will continue in conjunction with markd antibody coupling matter, until unconjugated labeled antibody conjugate is consumed totally, or the second capture agent is by saturated.Because even contain endogenous IgG from the mammiferous mouth cavity fluid sample of health, and preferably surpass the molar weight of immobilized antigen with the molar weight of the labelled reagent of label coupling, the antibody coupling matter of mark should always can be used in conjunction with the second capture agent, at control line, produces signal.Therefore, the human IgG that can't detect the signal designation of control line or lack q.s is to produce object line, or inspection test-strips defectiveness, or examination bar misoperation.
Typically, after inserting mouth cavity fluid, described inspection test-strips can be observed label signal between 15 to 60 minutes, more preferably between 15 to 45 minutes, most preferably between 15 to 30 minutes.After detection starts early than 15 minutes or be later than and within 60 minutes, read testing result and may give the result make mistake.The signal producing by coloured label as above can directly detect from inspection test-strips without further processing conventionally.Fluorescent marker may need photofluorometer to detect.In method well-known to those skilled in the art, use silver salt solution can strengthen the signal being produced by metal-sol label.Similarly, when using enzyme, label must contact with the substrate of enzyme labeling thing, to produce, can detect product.Therefore, these Enhancement Method have departed from the conventional single stage method mensuration that adopts coloured particle label and colloidal sol to carry out, because matrix must contact with imaging liquid (silver salt or substrate solution) before certification mark thing.
In a most preferred embodiment, the invention provides a kind of inspection test-strips, recombinant protein or synthetic peptide that it contains the immune determining area that represents HIV-1gp41 and HIV-2gp36 envelope protein, and mountain goat anti-human igg F (ab ')
2the contrast of fragment antibody prize procedure, they are respectively fixed on the nitrocellulose filter of detection zone and check plot.
Fig. 1 illustrates the program that mouth cavity fluid fast immune chromatographic according to a preferred embodiment of the invention detects, and wherein Fig. 1-1 illustrates the step of detection to 1-15.For testing, with the gums up and down of polyester swab wiping object, then described swab be placed in to test tube approximately 1000 μ l mouth cavity fluid sample buffers (0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin950 of pH7.2+/-0.2 potassium phosphate buffering) and mix at test tube.Extrude the fluid in swab and discard, this sample potpourri of 200 μ l is transferred to clean tube to measure.By measuring examination bar, put into vertically the test tube that contains 200 μ l sample potpourris.When the sample of dilution is when measuring examination bar and upwards move, it makes to try blush protein A-colloidal gold reagent rehydration on bar, and in sample, IgG is combined with albumin A/colloid gold particle and is formed IgG/ conjugate compound.Described IgG/ conjugate compound continues along the upwards migration of examination bar, first arrives on the antigen line of measuring the detection zone (described detection zone contain can with the HIV antigen that anti-HIV antibody is combined) of examination bar and being fixed in detection zone and occurs the erythroid colo(u)r streak that has.This Indicator Reaction or positive findings.The intensity of line not with sample in the antibody amount that exists proportional.In detection zone, lack and have colo(u)r streak indicateing arm originally not contain anti-HIV antibody.IgG/ conjugate compound continues to make progress migration until it arrives check plot along measuring examination bar.Check plot is contained to be fixed on and is measured the goat anti-human F of examination bar in reaching the standard grade (ab ')
2igG fragment antibody.Remaining IgG/ conjugate compound and immobilization F (ab ')
2fragment combination, and there is the erythroid colo(u)r streak that has.The appearance of control line is the evidence that detects normal operation and contain IgG.No matter sample is reactive or non-reacted to the antibody of HIV-1 or HIV-2, all will occur in check plot carrying out blush control line between all effective detection periods.Sample continues migration and enters final absorption pad through check plot, and described absorption pad helps to pull IgG/ conjugate compound by examination bar and removes any background color.In mensuration examination bar is introduced to dilution sample 20 minutes but after being no more than 45 minutes, result is made an explanation.After detection starts early than 20 minutes or be later than and within 45 minutes, read testing result and may give the result make mistake.Remaining dilute sample can be used for other and detects, such as authenticity test.
Fig. 2 illustrates three kinds of possible results that mouth cavity fluid fast immune chromatographic according to a preferred embodiment of the invention a detects, and wherein Fig. 2 A, 2B and 2C represent respectively feminine gender, the positive and invalid result.
Fig. 2 A shows non-reacted result, wherein only in check plot, occurs single line, and prompting lacks active anti-HIV-1 or anti-HIV-2 antibody in mouth cavity fluid sample.This testing result is interpreted as HIV negative antibody.
Fig. 2 B shows reactive result, and wherein detection line and control line all occur, in inspection test-strips, two lines appear at respectively detection zone and check plot.In these lines one comparable another is darker.Reactive result means and anti-HIV-1/2 antibody in mouth cavity fluid sample, detected.This testing result is interpreted as preliminary HIV antibody positive.
Fig. 2 C shows null result, wherein in check plot, there is no control line.Even there is detection line in detection zone, result is also invalid.Invalid test should adopt new inspection test-strips to carry out repeated test.
Above-mentioned embodiment can design with alternative, and it comprises substituting label outside aurosol.For example, other label includes but not limited to that element or metal-sol are such as selenium, silver, iron or carbon, and other pearl particle is such as coloured latex, liposome and dyed particles.Can specificity catch other first capture agent of antibody in mouth cavity fluid sample and include but not limited to the antibody of anti-IgM or IgA; Protein G, or concanavalin A.The second capture agent also can be mixed with and include but not limited to substitute part, such as albumin A or Protein G.
Aspect the 4th, the invention provides the kit for detection of mouth cavity fluid analyte, it comprises above-mentioned disposable lateral flow immunochromatography inspection test-strips.Inspection test-strips is closed in dry container.Each kit optionally comprises sample buffer, mouth cavity fluid gatherer, the package insert of appended inspection test-strips operation instruction is provided, is equipped with for inspection test-strips being carried out to the positive of quality inspection and the tubule of negative control, can be used for timer and/or bio-hazard product container handling that when definite mensuration of the present invention completes.
In a preferred embodiment in this regard, sample buffer is the solution of pH7.2+/-0.2 potassium phosphate buffering, and it also contains 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin950.
Although understand for knowing and being convenient to, the mode of explanation and example has described foregoing invention in detail by way of example, but apparent to those skilled in the art, according to instruction of the present invention, can it be made a variety of changes and be revised and without prejudice to the spirit and scope of claims.
Except diagnose HIV by HIV antibody in detection mouth cavity fluid, the present invention can easily be configured for the diagnosis of many illnesss, and these illnesss need to be carried out immune detection to the analyte in mouth cavity fluid.Especially easily use concept of the present invention to detect for sexually transmitted disease, such as syphilis antibody and hepatitis virus antibody.
embodiment
Following examples will illustrate the present invention but not be limited.
embodiment 1: the production of immunochromatography inspection test-strips
Inspection test-strips for quick HIV-1/2 mouth cavity fluid antibody test is provided in the present embodiment, wherein use glass fiber material as the matrix of sample pad, use polyester material as the matrix of conjugate pad, use nitrocellulose filter as the matrix detecting and contrast is padded.
The S & S S-33 glass fiber material of 1 inch is soaked with sealing damping fluid, described sealing damping fluid is by forming below: 40% normal chicken serum (hot deactivation), 0.25M saleratus, 0.05M dipotassium hydrogen phosphate, 0.1%Tween80,100mM potassium stannate and 0.2% carbamide peroxide, its pH is 8.2 to 8.5, under room temperature, (15-30 ℃) is dried 8 hours in low humidity room, then in drying receptacle, spend the night in 50 ℃, and kept dry.
By using gasoloid tip pAg conjugate to be drawn on pad to striped, from polyester film, prepare conjugate pad.Before drawing striped, by conjugate stabilization in 20% sucrose, 5% trehalose, 100mM potassium stannate and 0.1% urea peroxide.Then described pad is immersed in the damping fluid that contains polyvinylpyrrolidone, chicken serum, bovine serum albumin(BSA) and carbonate buffer agent and at 50 ℃ and uses forced draft dry 50 minutes.
HIV antigen can be coupled to detection zone pad, and it comprises use streptavidin/biotin connexon.In order to draw striped, with 300ng/ examination bar and 0.15ng/ examination bar, synthetic HIV-1 peptide and synthetic HIV-2 peptide (for example SEQ ID No:1 to 5) are put on to detecting pad respectively.The solution being comprised of 1.2mg/mlHIV-1,0.06mg/ml HIV-2,4.36mg/ml affinity element and 0.05% isopropyl alcohol is used to draw striped on detecting pad.1mg/ml mountain goat anti-human igg Fc F (ab) 2 is put on to contrast pad.
For the inspection test-strips of use Recombinant HIV-2, Recombinant HIV-1/, the gp36 albumen of the gp41 albumen of the 0.4-0.7 μ g/ examination bar in 0.001%Tween80,5% sucrose and 2% methyl alcohol and 0.04-0.08 μ g/ examination bar is put on to detection zone, and by the mountain goat anti-human igg F of 0.175 μ g/ examination bar in 5% sucrose, 2% methyl alcohol and 0.01M sal tartari pH8.4 (ab ')
2put on check plot.
Detection zone and the check plot of as above processing are sealed with sealer, and described sealer is comprised of 0.15% bovine serum albumin(BSA), the 0.075%Tween20 in the kaliumphosphate buffer of pH7.8+/-0.1.Then gained pad is alignd to make it mutual fluid and be communicated with, make conjugate pad be positioned at the downstream of sample pad; Detection and check plot pad are positioned at the downstream of conjugate pad, so that check plot is positioned at the downstream of detection zone.
embodiment 2: mouth cavity fluid fast immune chromatographic detects
Provide the mouth cavity fluid fast immune chromatographic that uses the present invention to try bar to detect, if Fig. 1-1 is to as shown in Fig. 1-15.First, by inverted bottle lightly, take biased sample damping fluid (with 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30ug/ml affinity element, 0.2%Tween80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin950 of pH7.2+/-0.2 potassium phosphate buffering) for approximately 3 times.From bottle, take off bottle cap (Fig. 1-1) and damping fluid is filled to dropper to groove (Fig. 1-2).The entire contents of dropper is dispensed to (Fig. 1-3) in test tube.Then a clean swab being provided by bag is provided.Catch swab handle.Avoid touching the fabric end of swab.Subsequently, apply suitable pressure and gently with swab fabric end gums line in wiping to and fro.One jiao at mouth starts softly and lentamente wiping until arrive another angle (Fig. 1-4) of mouth, then along the wiping of upper gums line, returns to starting point (about 5-6 second) (Fig. 1-5).Then rotate swab with gums (Fig. 1-6) under the opposite side wiping of use swab.The opposite side that uses swab is gums line under wiping back and forth softly and lentamente.One jiao at mouth starts (Fig. 1-7), at another angle of mouth, finishes, and then along the wiping of lower gums line, returns to the place of setting out (about 5-6 second) (Fig. 1-8).Immediately swab is put in to (Fig. 1-9) in the pipe that fills sample buffer.
Hold swab handle.Swab is back and forth inserted in sample buffer pipe 6-8 time, be close to the both sides (Fig. 1-10) of pipe friction swab.From pipe, take out swab (Fig. 1-11).Sample has been ready for detection now.Transferase 12 00 this sample of μ l is to empty test tube.To use following mensuration examination bar to detect this sample.If sample is carried out to the detection of more than one inspection test-strips, a plurality of 200 μ l samples can be transferred to single empty test tube.Open the tank of measuring examination bar is housed.From tank, taking out one measures examination bar and again builds at once tank.Keep away the film surface that manual-free touches examination bar central authorities.By measuring examination bar, be placed in the pipe that contains 200 μ l dilution samples, make to measure under the arrow points on examination bar (Fig. 1-12).Set the timer to 20 minutes, or record tries the time (Fig. 1-13) that bar adds to sample by measuring.Within 20 minutes, read testing result (Fig. 1-14) later.Then examination bar, pipe and swab are abandoned in bio-hazard product waste canister (Fig. 1-15).
embodiment 3: determine sensitivity, specificity and the accuracy of kit
Measure in the present embodiment sensitivity, specificity and accuracy that mouth cavity fluid detects.In the anonymous HIV clinic of the Thailand Red Cross (Thai Red Cross Anonymous HIV Clinic) of Bangkok, THA, carry out the external certificate test that mouth cavity fluid HIV side direction detects, on-test,, in April, 2004, is completed in June, 2004.986 are gone to the anonymous HIV clinic of the Thailand Red Cross and the current object of not accepting retrovirus treatment to accept voluntary HIV antibody test and consulting.Adopt the sequence detection of object carry out this research, do not know in advance result.In addition, the object of 37 known positives and antiretroviral therapy (ARV) is also accepted voluntary HIV antibody test and consulting.Give object chance to agree to voluntarily providing extra sample to be detected by these tests.
The reference method using in anonymous HIV clinic is the Orgenics Rapid HIV-1/-2Blood Test for preliminary screening, Doublecheck
tMiI.Use Bio-Rad GenScreen
tMhIV-1/2Version2ELISA and/or Fujirebio
(HIV-1only) Particle Agglutination Test confirms the reactive sample of this detection.
Sensitivity table is shown the number positive that detects with reference divided by the percentage of the number positive gained that mouth cavity fluid detects fast.Specificity is expressed as the negative number that detects with reference divided by the percentage of the negative number gained that mouth cavity fluid detects fast.Accuracy be expressed as object sum divided by quick mouth cavity fluid detect detect with reference between the percentage of the consistent number gained of result.Result sees following table.
Table 1. is the synthetic HIV-2 peptide in HIV-1/2 mouth cavity fluid detection-Recombinant HIV-1/ fast
Table 2. fast HIV-1/2 mouth cavity fluid detects-synthesizes the synthetic HIV-2 peptide of HIV-1/
The invention still further relates to the following embodiment (they are corresponding to the claim of original application):
1. for detection of a lateral flow immunochromatography inspection test-strips for analyte in mouth cavity fluid, it is comprised of sample pad, conjugate pad, detection zone and check plot pad substantially, and described pad is made by least one host material, wherein
Described conjugate pad is positioned at the downstream of described sample pad, and draws striped thereon with conjugate;
Described detection zone and check plot pad are positioned at the downstream of conjugate pad, and contain detection zone and check plot, wherein
Described detection zone is fixed with the specificity combinating reagent of specific binding target analyte; And
Described check plot is fixed with the second capture agent.
2. according to the inspection test-strips of embodiment 1, wherein said analyte to be detected is selected from the antibody of anti-following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
3. according to the inspection test-strips of embodiment 1, wherein said host material is selected from inorganic powder, such as silicon dioxide and aluminium oxide; Glass fiber filter paper; Natural polymerization material is especially based on cellulosic material, chromatographic paper; The natural polymer of synthetic or modification is such as cellulose nitrate, cellulose acetate, Polyvinylchloride, polyacrylamide, cross-link dextran, agarose; With and combination.
4. according to the inspection test-strips of embodiment 3, the wherein said host material for sample pad is glass fiber filter paper.
5. according to the inspection test-strips of embodiment 3, the wherein said host material for conjugate pad is polyester material.
6. according to the inspection test-strips of embodiment 3, the wherein said host material for detection of pad and contrast pad is nitrocellulose filter.
7. according to the inspection test-strips of embodiment 1, wherein said conjugate comprises the label with the first capture agent coupling, and described the first capture agent is caught the endogenous antibody of mouth cavity fluid.
8. according to the inspection test-strips of embodiment 7, wherein said label is selected from colloid gold particle; Element or metal-sol particle, comprise selenium, silver, iron or carbon; Other pearl particle, comprises coloured latex, liposome and dye granule.
9. according to the inspection test-strips of embodiment 7 or 8, wherein said label is colloid gold particle.
10. according to the inspection test-strips of embodiment 7, wherein said the first capture agent is selected from the antibody of anti-IgG, IgM or IgA, albumin A, Protein G and concanavalin A.
11. according to the inspection test-strips of embodiment 8, and wherein said the first capture agent is albumin A.
12. according to the inspection test-strips of embodiment 1, and wherein said specificity combinating reagent is selected from following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
13. according to the inspection test-strips of embodiment 12, and the antigen of wherein said communicable disease is restructuring or the synthetic peptide that represents HIV protein immunization determining area.
14. according to the inspection test-strips of embodiment 13, wherein said HIV albumen is HIV envelope protein.
15. according to the inspection test-strips of embodiment 14, and wherein said HIV envelope protein is selected from the gpl20 of HIV-1 and the gp36 of gp41 and HIV-2.
16. according to the inspection test-strips of embodiment 1, and wherein said the second capture agent is selected from the antibody of anti-IgG, IgM or IgA, albumin A, Protein G and concanavalin A.
17. according to the inspection test-strips of embodiment 16, and wherein said anti-IgG antibody is mountain goat anti-human igg antibody.
18. produce the method for the lateral flow immunochromatography inspection test-strips that any one defined in embodiment 1-17, and it comprises:
A) described conjugate is drawn on described conjugate pad to striped;
B) described specificity combinating reagent is fixed on the detection zone of described detection zone and check plot pad;
C) described the second capture agent is fixed on the check plot of described detection zone and check plot pad;
D) with sealer, seal each pad; With
E) alignment gained pad makes it mutual fluid connection.
19. according to the method for embodiment 18, and wherein said conjugate is stabilized in simple or glycoconjugate solution before drawing striped.
20. according to the method for embodiment 19, and wherein said sugar juice contains sucrose, trehalose, potassium stannate and carbamide peroxide.
21. according to the method for embodiment 18, wherein uses biotin/streptavidin connexon that described specificity combinating reagent is fixed on detection zone.
22. according to the method for embodiment 18, wherein uses biotin/streptavidin connexon that described specificity combinating reagent is fixed on check plot.
23. according to the method for embodiment 18, the detergent that wherein said sealer contains nonionic, cationic, anionic and amphoteric ion type; Sugar, comprises sucrose, fructose; Or protein, comprise that bovine serum albumin(BSA), animal's whole blood are clear, casein and skimmed milk power.
24. according to the method for embodiment 19, and wherein said animal's whole blood is clearly hyclone.
25. according to the method for embodiment 19, and wherein said animal's whole blood is clearly birds serum.
26. according to the method for embodiment 21, and wherein said birds serum is selected from goose serum, turkey serum and chicken serum.
27. detect the lateral flow immune chromatography method of analyte in mouth cavity fluid, and it comprises:
(a) use with the lateral flow immune chromatograph testing that any one defined in embodiment 1-17 and try the gatherer collection mouth cavity fluid that bar separates;
(b) gatherer dropped in the sample buffer of certain volume to mouth cavity fluid is discharged in damping fluid and obtain potpourri;
(c) the lateral flow immune chromatograph testing examination bar that in previous embodiments, any one defined is put into potpourri; With
(d) detecting in 15-60 minute that starts, by observing the existence of check plot signal, determining the validity detecting, and by observing the existence of detection zone signal, determine the existence of analyte.
28. according to the method for embodiment 27, and wherein the gatherer in step (a) is untreated polyester swab.
29. according to the method for embodiment 28, and wherein said swab is Texwipe Large Alpha Swab TX714A.
30. according to the method for embodiment 27, and wherein said sample buffer is pH7.2+/-0.2 buffer solution of potassium phosphate.
31. according to the method for embodiment 30, and wherein said solution also contains 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin950.
32. according to the method for embodiment 27, and the volume of wherein said sample buffer is 1000 μ l.
33. according to the method for embodiment 27, and wherein said method is also included in the step of taking out the sample of potpourri between step (b) and step (c).
34. according to the method for embodiment 33, and the volume of wherein said sample is 200 μ l.
35. according to the method for embodiment 27, and wherein said signal is to have colo(u)r streak.
36. according to the method for embodiment 35, and wherein said to have colo(u)r streak be blush line.
37. according to the method for embodiment 27, wherein in 20-45 minute, observes described signal.
38. kits for detection of analyte in mouth cavity fluid, it comprises the defined lateral flow immunochromatography inspection of at least one any one in embodiment 1-17 test-strips.
39. according to the kit of embodiment 38, and wherein said inspection test-strips is closed in dry container.
40. according to the kit of embodiment 38, and wherein said kit also comprises sample buffer and at least one is for collecting the gatherer of mouth cavity fluid.
41. according to the kit of embodiment 40, and wherein said sample buffer is the buffer solution of potassium phosphate of pH7.2+/-0.2.
42. according to the kit of embodiment 41, and wherein said solution also contains 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin950.
43. according to the kit of embodiment 40, and wherein said gatherer is untreated polyester swab.
44. according to the kit of embodiment 43, and wherein said swab is Texwipe Large Alpha Swab TX714A.
45. according to the kit of any one in embodiment 38 to 44, wherein said kit also comprise be equipped with for to inspection test-strips carry out the positive of quality inspection and the tubule of negative control.
Claims (20)
1. for detection of a lateral flow immunochromatography inspection test-strips for analyte in mouth cavity fluid, it is comprised of sample pad, conjugate pad, detection zone and check plot pad substantially, and described pad is made by least one host material, wherein
Described conjugate pad is positioned at the downstream of described sample pad, and draws striped thereon with conjugate;
Described detection zone and check plot pad are positioned at the downstream of conjugate pad, and contain detection zone and check plot, wherein
Described detection zone is fixed with the specificity combinating reagent of specific binding target analyte; And
Described check plot is fixed with the second capture agent.
2. according to the inspection test-strips of claim 1, wherein said analyte to be detected is selected from the antibody of anti-following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
3. according to the inspection test-strips of claim 1, wherein said host material is selected from inorganic powder, such as silicon dioxide and aluminium oxide; Glass fiber filter paper; Natural polymerization material is especially based on cellulosic material, chromatographic paper; The natural polymer of synthetic or modification is such as cellulose nitrate, cellulose acetate, Polyvinylchloride, polyacrylamide, cross-link dextran, agarose; With and combination.
4. according to the inspection test-strips of claim 3, the wherein said host material for sample pad is glass fiber filter paper.
5. according to the inspection test-strips of claim 3, the wherein said host material for conjugate pad is polyester material.
6. according to the inspection test-strips of claim 3, the wherein said host material for detection of pad and contrast pad is nitrocellulose filter.
7. according to the inspection test-strips of claim 1, wherein said conjugate comprises the label with the first capture agent coupling, and described the first capture agent is caught the endogenous antibody of mouth cavity fluid.
8. according to the inspection test-strips of claim 7, wherein said label is selected from colloid gold particle; Element or metal-sol particle, comprise selenium, silver, iron or carbon; Other pearl particle, comprises coloured latex, liposome and dye granule.
9. according to the inspection test-strips of claim 7 or 8, wherein said label is colloid gold particle.
10. according to the inspection test-strips of claim 7, wherein said the first capture agent is selected from the antibody of anti-IgG, IgM or IgA, albumin A, Protein G and concanavalin A.
11. inspection test-strips according to Claim 8, wherein said the first capture agent is albumin A.
12. according to the inspection test-strips of claim 1, and wherein said specificity combinating reagent is selected from following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
13. according to the inspection test-strips of claim 12, and the antigen of wherein said communicable disease is restructuring or the synthetic peptide that represents HIV protein immunization determining area.
14. according to the inspection test-strips of claim 13, wherein said HIV albumen is HIV envelope protein.
15. according to the inspection test-strips of claim 14, and wherein said HIV envelope protein is selected from the gpl20 of HIV-1 and the gp36 of gp41 and HIV-2.
16. according to the inspection test-strips of claim 1, and wherein said the second capture agent is selected from the antibody of anti-IgG, IgM or IgA, albumin A, Protein G and concanavalin A.
17. according to the inspection test-strips of claim 16, and wherein said anti-IgG antibody is mountain goat anti-human igg antibody.
18. produce the method for the lateral flow immunochromatography inspection test-strips that any one defined in claim 1-17, and it comprises:
A) described conjugate is drawn on described conjugate pad to striped;
B) described specificity combinating reagent is fixed on the detection zone of described detection zone and check plot pad;
C) described the second capture agent is fixed on the check plot of described detection zone and check plot pad;
D) with sealer, seal each pad; With
E) alignment gained pad makes it mutual fluid connection.
19. detect the lateral flow immune chromatography method of analyte in mouth cavity fluid, and it comprises:
(a) use with the lateral flow immune chromatograph testing that any one defined in claim 1-17 and try the gatherer collection mouth cavity fluid that bar separates;
(b) gatherer dropped in the sample buffer of certain volume to mouth cavity fluid is discharged in damping fluid and obtain potpourri;
(c) the lateral flow immune chromatograph testing examination bar that in aforementioned claim, any one defined is put into potpourri; With
(d) detecting in 15-60 minute that starts, by observing the existence of check plot signal, determining the validity detecting, and by observing the existence of detection zone signal, determine the existence of analyte.
20. kits for detection of analyte in mouth cavity fluid, it comprises the defined lateral flow immunochromatography inspection of at least one any one in claim 1-17 test-strips.
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| CN201410158273.9A CN103983773A (en) | 2005-05-20 | 2005-05-20 | Fast immunochromatographic detection of oral fluid |
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| CN201410158273.9A CN103983773A (en) | 2005-05-20 | 2005-05-20 | Fast immunochromatographic detection of oral fluid |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738622A (en) * | 2015-10-23 | 2016-07-06 | 北京玛斯玛克生物科技有限公司 | Human urine HIV1/2 antibody detection test paper through colloidal gold chromatographic method and preparation method thereof |
| CN106970219A (en) * | 2017-04-28 | 2017-07-21 | 北京金豪制药股份有限公司 | One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent |
| CN109633179A (en) * | 2019-02-02 | 2019-04-16 | 金华市安口生物科技有限公司 | A kind of simple and direct detection method of human oral cavity liquid immunoglobulin total content |
| CN111164095A (en) * | 2017-08-08 | 2020-05-15 | 奥瑞许科技公司 | Assay methods for improved analyte detection |
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2005
- 2005-05-20 CN CN201410158273.9A patent/CN103983773A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738622A (en) * | 2015-10-23 | 2016-07-06 | 北京玛斯玛克生物科技有限公司 | Human urine HIV1/2 antibody detection test paper through colloidal gold chromatographic method and preparation method thereof |
| CN106970219A (en) * | 2017-04-28 | 2017-07-21 | 北京金豪制药股份有限公司 | One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent |
| CN111164095A (en) * | 2017-08-08 | 2020-05-15 | 奥瑞许科技公司 | Assay methods for improved analyte detection |
| CN111164095B (en) * | 2017-08-08 | 2024-06-04 | 奥瑞许科技公司 | Assay methods for improved analyte detection |
| CN109633179A (en) * | 2019-02-02 | 2019-04-16 | 金华市安口生物科技有限公司 | A kind of simple and direct detection method of human oral cavity liquid immunoglobulin total content |
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Address after: 101407 Beijing city Huairou District Yanqi Industrial Development Zone No. 25 Applicant after: Beijing Marr Bio-Pharmaceutical Co., Ltd. Address before: 100600, No. 8-24, 9 Avenue, Jianguo, Beijing, Chaoyang District Applicant before: Beijing Marr Bio Pharmaceutical Co., Ltd. |
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Application publication date: 20140813 |