CN1558237A - Fast detection device and method for HIV antibody in urine - Google Patents
Fast detection device and method for HIV antibody in urine Download PDFInfo
- Publication number
- CN1558237A CN1558237A CNA2004100042443A CN200410004244A CN1558237A CN 1558237 A CN1558237 A CN 1558237A CN A2004100042443 A CNA2004100042443 A CN A2004100042443A CN 200410004244 A CN200410004244 A CN 200410004244A CN 1558237 A CN1558237 A CN 1558237A
- Authority
- CN
- China
- Prior art keywords
- lateral flow
- flow device
- hiv
- antibody
- urine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides device and method for fast detection of endogenous antibody, especially HIV envelope protein resisting antibody, in urine. Other pathogens, including known pathogen of sexually transmitted disease, capable of causing the raising of endogenous antibody may be also detected with the device and method of the present invention.
Description
Technical field
The present invention relates to immunochemistry and Methods Biochem Anal, a kind of apparatus and method that are used for the fast detecting urine endogenous antibody specific antibody of HIV virus envelope protein (particularly at) are provided.Other can cause the pathogen that the urine endogenous antibody raises, and comprises the pathogen of at present known sexually transmitted disease, also can use by the present invention and detect.
Background technology
There have been at present many kinds of lateral flow detection methods to be applied to detect various materials as in the body fluid such as blood or urine.These detection methods comprise: antigen-antibody reaction, and with enzyme, fluorescent material or the crosslinked complex of visable indicia thing, the reaction chamber of particular design.In this class detection method of great majority, a special acceptor at specific antigen (as antibody) is arranged, and a cover detects the antigen-antibody complex appearance and/or reaches a certain amount of means.This detection design mostly is a kind of detection by quantitative, but only need obtain the result of male/female under many circumstances.The example of this qualitative detection comprises: blood grouping, early pregnancy detects and some other urine detection.In qualitative detection, visual label preferably appears, such as aggegation or color change occurring.
The detection of male/female must be very responsive, because the concentration of purpose part is often very low in testing liquid.The positive of mistake will produce trouble, particularly aggegation experiment and other fast detecting means, as: be stained with the experiment of rod (dipstick) and color change.At these problems, developed the responsive detection means of sandwich method and use metal-sol or other coloured particle, yet these means do not solve all problems that runs in the method for quick.The method that still needs now low concentration target material in not only responsive but also the specific detection body fluid is as detecting in the urine endogenous antibody at pathogen.
Summary of the invention
The invention provides a kind of fast detecting urine endogenous antibody that is used for, particularly at the apparatus and method of the specific antibody of HIV virus envelope protein.The inventor finds, under the situation of rising pH value, can obtain susceptibility and the selectivity higher than existing method.Accordingly, provide a kind of lateral flow method to detect the urine detection of antibodies device that hits in one embodiment of the invention, this device comprise can specificity in conjunction with the antigen of target antibody with when contacting urine, can make the pH value of solution value reach 8 the sample area that comprises damping fluid at least.Damping fluid among the embodiment can contain carbonate, also can contain carbonate and hydrocarbonate.The saleratus in the damping fluid in the preferred device and the mol ratio of sal tartari are 2: 1.
The antigen of the foregoing description can be the albumen of human immunodeficiency virus (HIV).HIV can be HIV-1, also can be HIV-2.In this embodiment, not only can use a kind of antigen, can also be many antigens of HIV-1 and/or HIV-2.In this embodiment, proteantigen can also be recombinated.Preferred HIV albumen is envelope protein, and albumen also can be the non-natural peptide of the gp36 of the gp120 of HIV-1 or gp41 or HIV-2, and these peptides have the described amino acid sequence of sequence table SEQ ID No.:1-5.Preferred then comprise of containing in the described polypeptide of sequence table SEQ ID No.:1-5 and/a plurality of HIV albumen of a plurality of combination.In this embodiment, antigen can be by the biotin labeling thing, and by avidin-or Avidin-biotin link thing be connected on the matrix of device.
In the above-described embodiments, sample area can contain birds serum, preferred chicken serum.Can also include cross-linking zone, it contains the labelled reagent crosslinked with label.Preferred labelled reagent is a Protein G, is more preferably albumin A.Preferred label is a collaurum.Can also comprise the contrast district of containing capture agent in the above-described embodiments.Preferred capture agent comprises the antibody of anti-human IgG, and preferred anti-human IgG antibody is the goat anti-human igg antibody.
Another one embodiment of the present invention detects the device of antibody in the urine for using lateral flow method, and this device contains: (a) cross-linking zone: contain albumin A/collaurum cross-linking agent; (b) check plot: contain capture agent, wherein antibody in capture agent and the human urine, ability that albumin A/the collaurum cross-linking agent combines are greater than the binding ability of antibody, albumin A/collaurum cross-linking agent in albumin A and the human urine.Embodiment is the same as described above, and capture agent is the antibody of anti-human IgG, is preferably the goat anti-human igg antibody.Present embodiment can also increase the sample area by pH is at least 8 damping fluid and the antigen that combines with the target antibody specificity is formed.The preferred damping fluid of present embodiment comprises carbonate and/or hydrocarbonate, preferably their sylvite or ammonium salt.Can comprise in the damping fluid of present embodiment that mol ratio is 2: 1 potassium bicarbonate and a sal tartari.
The antigen of the foregoing description can be human immunodeficiency virus's albumen.HIV can be HIV-1, also can be HIV-2, and this depends on selects for which concrete application.Also can use many antigens of HIV-1 and/or HIV-2.In certain embodiments, antigen is envelope protein, and antigen is selected from SEQ ID NO:1-5 among other embodiment.As described in the previous embodiment, these antigens can be acquisitions that separate from nature or recombinant expressed.The foregoing description also can comprise the sample area of birds serum, preferred chicken serum.
Above-mentioned two embodiment of this invention, and all feasible patterns of these embodiment, all the form with kit provides, and attaches one or multinomial supplementary provision in packing, as described herein.
Another embodiment of the present invention is the method that detects antibody from patient's urine, and this method comprises the sample area that sample is added to the lateral flow device, and wherein the pH of buffer value of sample area is at least 8.This method can also be used identical damping fluid and the antigen with preceding two embodiment.
It also is the method that detects antibody from patient's urine that the present invention also has an embodiment, and this method comprises the sample area that sample is added to the lateral flow device, and this device is made up of land and check plot, and albumin A/collaurum cross-linking agent is contained in the land; The check plot comprises capture agent, and wherein antibody in capture agent and the human urine, ability that albumin A/the collaurum cross-linking agent combines are greater than the binding ability of antibody, albumin A/collaurum cross-linking agent in albumin A and the human urine.Aspect antibody, antigen and damping fluid, the embodiment basically identical of this embodiment and front.
Embodiment
Apparatus and method provided by the invention are suitable for fast detecting urine endogenous antibody, particularly direct antibody at the HIV virus envelope protein.Other causes the pathogen that the urine endogenous antibody raises, pathogeny as sexually transmitted disease and infectious diseases, also can use the present invention to detect, for example Chlamydia, herpesviral, gonorrhoea, syphilis, pylorus bacillus, hepatitis A, hepatitis B, third liver, Epstein-Barr virus, cytomegalovirus, herpes simplex virus, malaria, influenza, West Nile Virus, rubella, dengue fever, Lyme disease, look into Gus's disease, tuberculosis, tokoplasmosis, Ebola virus etc.Use suitable damping fluid to make urine sample pH remain on 8 or higher, because when low pH value, can't normally detect.Urine sample pH remains on 8 or when higher, sensitivity of the present invention and specificity significantly improve, and the optical signal of institute's mark significantly strengthens during than low pH.The invention provides a present detection method of ratio technology more accurately.
Apparatus and method provided by the invention are economical because in immunoassays, use reagent cheaply as far as possible, such as non-specific antibody in conjunction with albumen such as albumin A, Protein G or hemagglutinin.Find a wonderful situation at work: can detect and in immune complex of the present invention, exist this antibody-like of a part in conjunction with albumen.
Assay device of the present invention comprises a series of different district's bands, and the reagent difference that these district's bands are comprised is reacted at different district's bands respectively during operation.These district's bands can be the different pieces of a continuous matrix, or respectively attached on the pad that separates, make liquid flow by the absorption of filling up.
For the term among the present invention easier to understand, below provide the definition of some terms:
The I definition
" analysis area " is meant flow of liquid through the zone, comprises the immobilized antigen of endogenous target antibody in the tested urine of specific bond.Antigentic specificity combining target antibody makes target antibody and any molecule that is attached thereto be trapped in analysis area.
" antigen " fingering stimulates it to produce the material of antibody when going into mammal or birds.Therefore, antibody recognition and specificity are in conjunction with the antigen that excites its generation.Preferred antigens of the present invention comprises human immunodeficiency virus (HIV) albumen, the particularly gp120 of virus envelope protein HIV-1 and gp41, the gp36 of HIV-2.Antibody among the present invention is to use target antibody to stimulate in the species different with patient to produce, so the endogenous target antibody in patient's urine also is the antigen that antibody is discerned among the present invention.Such as, the target antibody in the human urine is the antigen by anti-people's antibody specific recognition of inhuman species generation.
" antibody " refer to by immunoglobulin gene or fragment encoding, the specificity combination and identification antigen polypeptide.Known immunoglobulin gene comprises κ, λ, alpha, gamma, δ, ε and μ constant region gene and countless variable region genes at present.Light chain is divided into κ or λ again, and heavy chain is divided into γ, μ, and α, δ or ε, the kind of their decision immunoglobulin (Ig)s is respectively IgG, IgM, IgA, IgD and IgE.Antibody of the present invention can be polyclone or monoclonal.
Antibody exists with two kinds of forms: complete immunoglobulin (Ig) or produce the segment of a fixed structure through multiple peptide enzymic digestion.For example, behind the pepsin digested antibody, hinge region cuts off antibody and produces F (ab) ' below disulfide bond
2(dimer of Fab), Fab by light chain by disulfide bond and VH-CH
1Connect.Under the condition of gentleness, can interrupt the disulfide bond degraded F (ab) ' of hinge region
2Dimer makes it to become Fab ' monomer.Fab ' monomer mainly is made up of Fab and part hinge region and (is seen: Fundamental Immunology, Paul ed., 3
RdEd., 1993)).Disconnected by antibody fragment or antigen binding fragment that the difference of digestion complete antibody is different, those skilled in the art can use chemical method or DNA recombinant technique to synthesize to come again synthetic these segments.Therefore, the definition of antibody, should comprise the antibody fragment that produces behind the whole antibody modification, perhaps use the synthetic again segment (as strand Fv) of DNA recombinant technique or (see McCafferty et al. by the antibody that phage display library produces, Nature, 348:552-554 (1990) and Zapata et al., Protein Eng.8 (10): 1057-1062[1995]).Antibody among the present invention can be polyclone or monoclonal.The technology for preparing these antibody is that general knowledge known in this field is (referring to Kohler and Milsten, Nature, 256:495-497 (1975); Kozbor et al., ImmunologyToday, 4:72 (1983); Cole et al., pp.77-96 Monoclonal Antibodies and CancerTherapy, Alan R.Liss, Inc (1985); United States Patent (USP), 4,946,778).
" target antibody " refers to the endogenous antibody in the tested urine, can detect by device provided by the invention.Target antibody may be aforesaid antibody fragment, but mostly are the whole immunoglobulin (Ig)s that contain function-variable district and constant region.The function-variable district of target antibody is specific in conjunction with corresponding antigen, the function constant region then with the antibody of anti-target antibody (anti-) specific bond as two, or with albumin A or G or other and target antibody specific bond family generation specific bond.
" suction " refers to that adsorbent is supported the ability of different solute migrations when liquid passes through adsorbent.Therefore the adsorbent of suction can carry out chromatography according to the physical/chemical of solute.
" capture agent " refer to can with any molecule of target antibody specific bond.Capture agent of the present invention preferably is fixed on the medium with deterministic model, and is vertical with flowing through channel usually.Preferred capture agent is antibody, albumin A and the Protein G of anti-target antibody.
" collaurum " refers to tiny gold grain colloidal sol, exists with uncertain aqueous suspensions form.
" albumin A " refers to the high stability surface receptor by the staphylococcus aureus generation, it can combine (Boyle, M.D.P.and K.J.Reis.Bacterial Fc Receptors.Biotechnology 5:697-703 (1987)) with the Fc section of the immunoglobulin (Ig) (particularly IgG) of a large amount of species.An albumin A molecular energy is simultaneously in conjunction with at least two IgG molecule (Sj quist, J., Meloun, B.and Hjelm, H.Protein A is isolated from Staphylococcus aureus after digestion withlysostaphin.Eur J Biochem 29:572-578 (1972)).
" Protein G " refers to streptococcic thalline surface associated protein, affinity height with IgG, the IgG that three height homologies are arranged is in conjunction with territory (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).
" label " refers to can detected atom or family of molecule, and it and analyte are by direct or indirect mode specific bond.Label of the present invention can detect by physics or chemical method.Answer naked eyes as seen when preferred label should be for the indication positive findings.
" matrix " refers to support the insoluble substance of moving phase.Host material can be nature or synthetic, can absorb water maybe to absorb water fibrous or graininess.The continuous strip that matrix of the present invention can be made up of same material, or is made up of the potpourri of the consistent different materials that distributes along band, or the material of inconsistent distribution forms, there are different physics, chemical property in other district on the material distinguished and the band as forming.Equally, the pad of series of discrete also is made up of identical or different host material, is added with detectable on each pad.Then pad is placed on the liquid flow position and forms continuous flow path.Consider that material of the present invention keeps insoluble when detecting, might take place with reagent or not react.
" downstream " passes through the direct flowing through channel of matrix from application of sample after referring to that liquid adds.
" upstream " refers to that liquid flows to the flowing through channel at application of sample place by matrix.
Approach when " flowing through channel " refers to that urine is passed through matrix.Flowing through channel is single channel preferably, but also can comprise several approach, and every approach wherein can be simultaneously, order for other approach or independently supported liquid flow.
The II brief introduction
Just as summarized above, device of the present invention is the endogenous target antibody that is used for detecting in patient's urine sample.The target antibody that apparatus of the present invention are at first discerned is and HIV albumen the antibody of the gp120 of preferred HIV-1 or the gp36 combination of gp41 and HIV-2.
Device of the present invention comprises a series of district band, and each district band is difference owing to use different chemical reagent, reacts and/or reciprocation takes place respectively not in the same district.Each device comprises three districts at least: sample area, land and analysis area.Device of the present invention can also comprise the check plot, and whether be used to refer to operation correct and effective experimental result is provided.The band position, area postrema that can also be chosen in the device flowing through channel keeps the waste liquid district, and excess sample can remain in the there.The existence in waste liquid district makes and adds the excess volume sample where necessary and become possibility.Absorbing material is contained in typical waste liquid district, can be identical with the host material of forming arbitrary district band or all district's bands.
Hereinafter the not same district band to apparatus of the present invention provides detailed description and these district's bands is how longshore current obtains the effective diagnosis result through the approach arrangement.The method of operating that helps to understand this device by these descriptions.
The III apparatus structure
Generally speaking, the present invention is a kind of device that urine sample is crossed along the flowing through channel sequential flow of each district's band composition.Therefore after urine sample is added to sample area, will runs into each district successively and be with interior reagent, so predetermined reaction takes place in sample meeting and reagent in each district's band.Flow of liquid is to control by the host material with multiple function through device, as described below.Host material also can optionally place shell, and this discusses hereinafter.After host material and shell have been discussed, will bring line description into to each district on the device by flowing through channel order after adding urine sample.
A. matrix
The suitable matrix that the present invention uses is to support the insoluble material of liquid flow.Host material can be the fiber or the particle of nature or synthetic, the characteristics that possess porous, suction or do not absorb water.It also can form continuous strip by identical materials or along the potpourri of the different materials of a band continuous distribution or at the discontinuously arranged potpourri that different banded zones form different physics or chemical property.Equally, matrix also is made up of two or more host material pads, and these pads water conservancy diversion liquid in device makes it mobile in a certain direction.When needing a lot of zones (different reagent is all contained in each zone) in the device or preparing to use unified approach, very useful by the liquid flowing through channel that a series of pads are formed.Consider that the material of forming matrix keeps insoluble state when detecting, just may take place with one or more reagent or not react.
The host material that is fit to is generally hydrophilic; Maybe can compensate possess hydrophilic property, comprise inorganic powder, as silicate and aluminium oxide; Glass fiber filter paper; Natural polymer, preferred cellulose-based analog as filter paper, chromatographic paper etc.; The natural polymers of synthetic or modified is as cellulose nitrate, cellulose acetate, Polyvinylchloride, polyacrylamide, crosslinked dextran, agarose etc.; These materials can be used alone or as a mixture.Host material can also comprise functional group or have and invent in antigen or the function of reagent covalent cross-linking.
Mesostroma material direct urine is mobile along certain approach detecting, and the reagent that uses in therefore detecting mainly directly is adsorbed on the host material with the form of particle or solution, and is as described below.
1. damping fluid
Characteristics of the present invention are Laemmli buffer system Laemmlis, and it makes urine pH remain on 8 at least in operating process, preferably between 8-11, and 8.2-11 more preferably, 8.2-10 preferably again, 8.5-9.5 preferably most preferably is 9-9.5 again.It is well-known to make urine remain on the production technology of damping fluid of appropriate pH scope, as " biological buffer " described in the Sigma catalogue in 2002.Preferred damping fluid comprises carbonate, hydrocarbonate, borate, especially its sylvite and ammonium salt.Other useful damping fluid of the present invention comprises acetate, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, MOPS, CHES, CAPSO, AMP, CAPS, CABS and some other.
The damping fluid that uses among the present invention preferably is applied to the matrix of device before operating.Can add damping fluid by any way, as long as can when urine contacts with matrix, make it to form solution with ideal pH.For example, can damping fluid be joined on the matrix perhaps preferred form, freeze-drying on matrix then with the form of dry powder with solution.
2. closed reagent
Although can use absorbent material to do matrix in the present invention, had better use the natural material that does not absorb water.
The no longer suction that absorbent material may become behind the use closed reagent.These reagent can be detergents, sugar or albumen, and they can reduce the interaction force that produces water-absorption characteristics.Typical protein blocking reagent comprises the bovine serum albumin(BSA) of native state or methylation state or succinyl state, animal's whole blood such as horse serum or hyclone or other haemproteins.Preferred closed reagent is the birds serum, such as goose or turkey serum, most preferably chicken serum.Other protein blocking reagent comprises casein and skimmed milk power.Multi-form selections such as the characteristics according to sealing matrix never dissociate, acid, alkalescence and both sexes contain the sealer of detergent.Polysorbas20 is very useful when closing membrane.Typical steamed bun stuffed with sugar as sealer is drawn together sucrose and fructose.
During sealing water absorptivity matrix, use the confining liquid of effective concentration to handle host material and do not want the reaction that takes place with what remove stromal surface.In a word, this method is to use confining liquid to seal, and as the protein solution of 1-20mg/ml, finishes in by several hours in a few minutes under the room temperature.By air drying, freeze-drying or other drying means encrusting substance matter is adsorbed onto on the matrix lastingly.
Itself has water absorptivity use, but changes the matrix that does not absorb water afterwards into, and is very effective when immobilized antigen and capture agent.For example, before the sealing capture agent is added on the matrix, can carries out anchored in place.Can after being fixed to matrix, seal again capture agent.
B. shell
Matrix of the present invention can be placed in the shell of functional and protectiveness.In a preferred embodiment, the canonical form of shell is for having a port at least, so that hold urine sample and guided liquid-flow contacts with sample area.Shell also can be designed as the form with window, thereby allows to select different port can select the liquid flowing through channel, flow through analysis area and/or check plot.Embodiment with this shell is called as " cassette arrangement ".
Accordingly, matrix can not have holder, or long life material is arranged as holder, and this long life material is preferably antiseep and can keeps the physical integrity of matrix.Embodiment with this type of architectural feature is called as " being stained with rod (dipstick) ".
The 3rd embodiment of this device comprises a protectiveness shell similar to cassette arrangement, but sample area extends and surmounts shell, forms a wick structure, and this structure can immerse in the urine sample.Other version of this aspect all is to be template with basic structure, thereby can be discerned by those skilled in the art easily.
Shell can use any suitable material well known in the art to make up.Case member in the liquid contacts with flowing through channel, but can not stop liquid to flow in the footpath on the way, so its material water wettability can not be too strong.On the contrary, if the shell material water wettability that contacts with the liquid flowing through channel is too strong, then sample may be absorbed a part or see through the shell wall seepage by shell.
C. device is distinguished
Device of the present invention has three districts at least, comprise in each district can with the reactant of endogenous antibody effect in the urine sample that adds on the auto levelizer.Sample area holds urine sample, and the urine sample application of sample can pass through fluid means in sample area, or collects earlier urine sample again by dropping, sample injector or topple over the mode application of sample to sample area.Urine sample is not preferably diluted, and uses immediately after collection.If necessary, the urine sample of using apparatus of the present invention to detect can be deposited the limited time (as reaching a week) in room temperature after collecting, if freezing, can deposit the longer time.Sample area preferably has enough damping fluids, makes urine sample pH value between the device detection period raise and/or remain on about 8.As mentioned above, damping fluid should be by the urine sample solubilising, and enough surge capabilities are arranged, and the pH value is remained on about 8 between the device detection period.
Application of sample at first moves in the urine sample of sample area and enters cross-linking zone, and endogenous antibody in this section urine sample and the labelled reagent effect on the label of being connected in that describes below form the labelled antibody cross-linking agent.
The migration of labelled antibody cross-linking agent enters analysis area, and at analysis area, antigen combines with the target antibody specificity and is fixed on the matrix.If the antibody in the labelled antibody cross-linking agent is target antibody, fixing antigen combines with the target antibody specificity, and the antibody linked thing of fixation mark is on matrix.In this way, label is assembled at analysis area, thereby can be detected, and the existence of target antibody in the urine sample is described.If the labelled antibody cross-linking agent does not comprise target antibody, then their longshore current roads continue migration, do not assemble at analysis area.
Device of the present invention can optionally comprise the check plot, is the capture agent that is fixed on the matrix in the check plot.Capture agent forms control line in the check plot, and the antibodies in nonspecific and the labelled antibody cross-linking agent.In this way, the labelled antibody cross-linking agent is assembled in the check plot, illustrated that device is working properly.When comprising the check plot, the check plot is positioned at the downstream of cross-linking zone and analysis area.
In the downstream of above-mentioned section, device also alternative comprises abandoned stope.Abandoned stope can be the simple extension of matrix, but preferably is made of the water absorptivity material, thereby can be so that the sample size of application of sample on device reaches maximum.
In order better to describe the present invention, this part each section above-mentioned will be discussed in more detail below.
Sample area
Sample area is used for the operator and adds urine sample.Sample area generally is made of the material of target antibody low hold-up.Accordingly, the closed reagent that is used for sample area sealing needs to stop target antibody to interact with host material during operation.The preferred birds serum of reagent that is used for the sample area sealing, more preferably chicken serum.In a preferred embodiment, sample area is made by fiberglass packing, and earlier (containing polyvinylpyrrolidone, bovine serum albumin(BSA), birds serum, borate and/or carbonate buffer solution (～0.5M), Triton X-100 or Tween-20 detergent) soaks into solution with it.Then pad is pressed dry and remove unnecessary damping fluid, 30 ℃ of dried overnight.The advance of this method has been to increase the water absorptivity and the wick swabbing action of sample area.In an embodiment, sample area also can play filtrator, removes those unwanted particles.
Cross-linking zone
Cross-linking zone is positioned at the downstream of sample area, and includes the mark part of the labelled reagent formation that directly or indirectly is connected with label.Here the linkage flag thing of mentioning and the method for labelled reagent are the known technologies of this area.
Mark part is positioned at the cross-linking zone on the matrix, and it is contacted with fluid sample when fluid sample (as urine sample) upwards flows.Finish this process, the matrix of cross-linking zone needs be made up of the dacron yarn fabric, and with its be immersed in contain polyvinylpyrrolidone, bovine serum albumin(BSA), birds serum, borate and/or carbonate buffer solution (～0.5M), seal in the damping fluid of Triton X-100 or Tween-20 detergent.Dry in 50 ℃ of ventilated environments at last.Mark part is cut into strip, is fixed on the pad by cohesive terminus,cohesive termini or gasoloid end.Before being treated to strip, the best advanced line stabilization processing of mark part can place single or compound sugar juice (as the trehalose of the sucrose and 5% (w/v) of 20% (w/v), or the dextrin of 10% (w/v)) as: mark part.
When urine sample was flowed through cross-linking zone, mark part dissolved and flows the device of flowing through together with liquid.Suitable labelled reagent and label will be discussed further below.
A) labelled reagent
The preferred energy of labelled reagent of the present invention specificity is in conjunction with the endogenous antibody in the urine sample.The suitable labelled reagent that can combine with all endogenous antibody in the urine sample comprises bacterioprotein (as Protein G and albumin A) and can discern the antibody of specific antibodies type that for example: the goat anti-human igg can be used for combination from any IgG antibody in patient's urine sample.
No matter use which kind of labelled reagent, even there is not target antibody in the urine sample, always the labelled antibody cross-linking agent generates at cross-linking zone.If there is not target antibody, the labelled antibody cross-linking agent of Xing Chenging will comprise the common endogenous antibody in the urine sample so.Contain enough endogenous antibody in the urine sample and form the labelled antibody cross-linking agent that can be detected.
B) label
The label that is suitable among the present invention can be visible also can be sightless, if but they assemble at surveyed area, then must be detected.The label that is suitable among the present invention includes but not limited to: particle family and enzyme.Visible label can be a visible painted polymer under dyestuff or the capacity situation, preferred label is a particulate matter, as painted latex bead, liposome, metal, organism, inorganics, dye solution, fluorescent grain, painted or coloured cell or tissue, erythrocyte etc.Metal-sol particle, painted or fluorescently-labeled particulate can with the naked eye see or use suitable instrument to read (spectrophotometer, fluorescence readout instrument etc.).Accordingly, also can use radioactive isotope.
Preferred label of the present invention is the colloid gold particle of diameter greater than 20nm, is more preferably 20-100nm, most preferably is 20-40nm.The collaurum that uses among the present invention can use present well-known technology manufacturing, as: G.Frens, Natrure, 241,20-22 (1973).Except collaurum, particle also can be to be made by the alloy or the metal composite of platinum, gold, silver, selenium, copper or other expression characteristics color.The poly nuclear technology of the alloy among the present invention, metal composite and metal or metal composite parcel is at United States Patent (USP) 4,313, associated description arranged in 734.Also have other that analyte is adsorbed in method on the gold grain in the prior art.These methods comprise by covalent bond and hydrophobic bond and adsorbing, but are not limited thereto.
Analysis area
Analysis area is arranged in the downstream of flow path cross-linking zone.Analysis area contains the specificity that is fixed on the matrix antigen at target antibody, by combining with target antibody the labelled antibody cross-linking agent is fixed on the matrix.Immobilized antigen can be any antigen of energy specific bond target antibody, but preferred HIV albumen, HIV envelope protein more preferably, again preferably from HIV-1gp120 or gp41, perhaps from one or more peptide sections of HIV-2 gp36, at least one antigen of from SEQ ID NO:1-5, selecting most preferably.
The antigen that the present invention is fit to use can be any source of existing method, comprises product natural, chemosynthesis or reorganization.For example, preferred SEQ ID:1-5 peptide section can use the solid-phase peptide synthetic technology to come chemosynthesis, perhaps can connect the nucleotide of coding appropriate peptide section and it is imported expression vector, then recombinant vector is imported suitable hosts, recombinant production.After the separation and purification of peptide section, just can use known technology biotin labeling.
Suitable antigen can use present known technology to be fixed on the matrix, but can not destroy the site that antigen combines with the target antibody specificity.Preferred fixing means is for using biotin/strepavidin connexon, and more preferably antigen and biotin coupling combines with the strepavidin that is coupled in advance on the matrix then.Before water absorptivity matrix is sealed, carry out the coupling of strepavidin and matrix earlier, this technology is known now.Coupling effect can be at least acquisition in 2: 1 by regulator solution streptococcus intermedius avidin biotin binding site and biotin ratio preferably, other ratio was as 0.5: 1,1: 1,3: 1,4: 1 and 5: 1, and other some ratios, all within the scope of the invention.For water absorptivity matrix, final compound can simply be added on the host material, and suitable confining liquid sealing is used in dry back.Can be used among the present invention has description among the amino acid sequence SEQ ID:1-5 below of the antigenic peptide section of imitating.SEQ ID:1,3,4 is connected cysteine residues with interchain in 5 and shows, the side chain of these residues oxidation reaction takes place easily forms halfcystine.
The fixing antigen labelled antibody cross-linking agent of specificity that can concentrate on the matrix in conjunction with them.By the labelled antibody cross-linking agent that concentrates, strengthened signal that label generates, improved susceptibility, simultaneously also the reduction of maximum possible produce the probability of error result.
Typical label signal can be observed in 15-60 minute after urine sample is added on sample area, preferably at 15-45 minute, most preferably at 15-30 minute.As mentioned above, the signal that coloured label produces can detect on device without further handling directly.Fluorescent marker then needs fluorophotometer to detect.The signal that the metal-sol label produces can use silver salt solution to strengthen by means commonly known in the art.Same, the thing if the use enzyme serves as a mark then needs to produce the product that can be detected with corresponding substrate-function.These reinforcement means all are to come from the use colored particle of routine and the development of colloidal sol label single step detection method, add in the strong method at these, and matrix needs same imaging liquid (as silver salt solution or substrate solution) effect, thereby produces the signal that can detect.
The check plot
Can optionally comprise the check plot in the device of the present invention.When it existed, the check plot was positioned at the downstream of flowing through channel cross-linking zone, the downstream of preferred analysis area.
The capture agent that can combine with endogenous antibody specificity in the urine sample is contained in the check plot, and capture agent preferably is fixed in the check plot and forms control line, their can in conjunction with and the urine sample that concentrates in all labelled antibody cross-linking agents.Capture agent can be the albumen that affinity is arranged with a certain type antibody, as: Protein G or albumin A, but in the present invention, to have only when this antibody-like binding molecule is not used as labelled reagent, they just can be used as capture agent.Preferred capture agent is in operating period of the present invention, should have than albumin A bigger with affinity endogenous urine antibody, they comprise the anti-IgG antibody from different urine samples source species as mentioned above.
The capture agent that is suitable among the present invention can be fixed in by known technology on the matrix, comprising the above-mentioned technology that is used for fixing antigen.Preferred fixing means more preferably as top said capture agent and biotin coupling, combines with the strepavidin that is coupled in advance on the matrix then for using biotin/strepavidin connexon.For water absorptivity matrix, host material needs sealing after immobilized capture reagent, as: use the solution that contains 0.01M potassium phosphate, 0.25%BSA and 0.025%Tween-20 to seal, film is 50 ℃ of dried overnight then.
Under the correct condition of using, capture agent will be continuously in conjunction with all labelled antibody cross-linking agents, and unconjugated labelled antibody cross-linking agent or capture agent are saturated until no longer including.Because in general from all containing endogenous IgG in the health mammal urine sample, and the labelled reagent that combines with label surpasses fixing antigen on molecular amounts, so always underlined antibody linked thing and capture reagent bind, and in the check plot shows signal.So if can not detect signal in the check plot, it is wrong or improper to illustrate that then the device operation exists.
IV. kit
The invention provides the kit that comprises one or more said apparatus.Each kit can optionally comprise the explanation how the explanation packing device uses, whether effectively positive the bottle that is used for pick-up unit and negative control solution be housed, be used to the timer that determines when detection of the present invention finishes, collection tank (as: small test tube), one or more transfer pipets and/or disposable biological dangerous goods container.
Publication of quoting in the instructions and patented claim be all here as list of references, and every individual publication and patented claim are quoted document for referencial use respectively especially.
Aforementioned invention is by explaining and have been described in detail for example so that its clear more and easy to understand, those skilled in the art do not deviate from claim of the present invention spirit and scope some change and revise still in teachings of the present invention.
As the above abundant description of carrying out, the present invention has very big variation range in application.Accordingly, following Example is provided purpose of the present invention, but has been not to limit the invention to this for better illustrating.Those are applied to technology of the present invention many nonessential parameters, obtain similar result, can make amendment or changes these parameters.
Embodiment 1 estimates the immunoassay device to freezing and selectivity and sensitivity fresh sample
Present embodiment shows that immunoassay device constructed in accordance has very high sensitivity and specificity to the AIDS sample of separate sources.
Immunoassay device of the present invention comprises following component:
Use contains the solution sealing of polyvinylpyrrolidone, bovine serum albumin(BSA), birds serum, carbonate buffer solution (0.5M) and Triton-X 100 and the glass fiber sample district pad that loads.The extruding sample pad is removed excess liq, 30 ℃ of dried overnight.
The pad of analysis area comprises that employing strepavidin/biotin crosslinking chemical is linked to the HIV antigen on the polyester nonwoven film.In brief, prepare the avidin solution of 100mg/ml, concentration is HIV-1 polypeptide, the HIV-2 polypeptide solution of 10mg/ml.Avidin solution and HIV solution are according to the ratio mixing of 2.1 avidin binding sites corresponding to 1 biotin molecule.Room temperature (25 ℃) reaction 5 minutes is diluted to final volume with deionized water/5% aqueous isopropanol with it.Use linear charger will dilute good solution and be added on the film, 50 ℃ of dryings are after 4 hours, and 50 ℃ are sealed in confining liquid (the 0.01M potassium phosphate solution that contains 0.25%BSA and 0.025% polysorbas20) and spend the night.
The pad of cross-linking zone is to use the aerosol suction nozzle that label is added on the polyester nonwoven film and makes.Before adding that the label bound fraction of pad is stable at the dextrin solution of the sucrose solution and 5% (w/v) of 20% (w/v).To fill up then and immerse the damping fluid that contains polyvinylpyrrolidone, bovine serum albumin(BSA), birds serum and carbonate, the following 50 ℃ of dryings of ventilation condition 50 minutes.
The control line of check plot is that this fragment is special at people's antibody Fc section by F (ab) produced in fragments of goat-anti body in the dilute solution.Use the aerosol suction nozzle that dilute solution is sprayed on the polyester nonwoven film, 50 ℃ of dryings 4 hours.50 ℃ are sealed in confining liquid (the 0.01M potassium phosphate solution that contains 0.25%BSA and 0.025% polysorbas20) and spend the night.
Through film series arrangement in the liquid flowing through channel of above-mentioned processing, sample area is positioned at the cross-linking zone upstream, and cross-linking zone is positioned at the analysis area upstream, and analysis area is positioned at the upstream, check plot.
Add 4 (50-100 μ l) urines at sample area, room temperature was placed after 20 minutes, read the result from device.If occur positive signal (as coloured band occurring) in the check plot, illustrate that detecting operation is correct.If positive signal does not appear in the position, check plot, illustrate that mistake appears in this device, should discard, and use new device to carry out immune detection.
The suppose device operation is correct, and positive signal appears in the position of the corresponding antigen of analysis area, illustrates to have direct antibody at antigen in the urine.In present analysis, this result shows that the urine donor has infected HIV.If positive signal do not occur at analysis area, illustrating does not have direct antibody at antigen in the urine, and promptly the urine donor does not have infected by HIV.
Table 1 is depicted as the result of the urine use said method analysis of 10 separate sources.
| The source | Preservation condition | Estimate the result | Actual result |
| Low danger negative sample | Fresh sample | Negative | 16/16 feminine gender |
| Positive | Receive, preserve, detect and all carry out in room temperature | Positive | 11/12 positive |
| Low danger negative sample | Receive, preserve, detect and all carry out in room temperature | Negative | 17/17 feminine gender |
| The HIV-1 positive | Receive, preserve, detect and all carry out in room temperature | Positive | 9/9 positive |
| The HIV-1 positive | Receive, preserve, detect and all carry out in room temperature | Positive | 12/12 positive |
| Negative sample | Receive, preserve, detect and all carry out in room temperature | Negative | 11/11 feminine gender |
| Ghanan HIV-1 positive | 2-8 ℃ of preservation, room temperature detects | Positive | 29/29 positive |
| Negative sample | Receive, preserve, detect and all carry out in room temperature | Negative | 6/7 feminine gender |
| Positive | Receive, preserve, detect and all carry out in room temperature | Positive | 15/15 positive |
| Ghanan patient AIDS | 2-8 ℃ of preservation, room temperature detects | Positive | 24/24 positive |
Embodiment 2 tachysynthesis chromatogram (RIC) single stage method testing results
Present embodiment explanation boxlike detection method is fit to detect the HIV antibody in the urine.Although the antibody horizontal in the urine is lower than blood or serum, this method is to having carried out successfully detecting through the urine of EIA and Western Blot conclusive evidence.Tachysynthesis chromatography (RIC) is used for detecting the HIV antibody in blood/serum, plays important effect to reducing the HIV spreading rate.Research described below uses HIV-1 RIC analytic approach to detect the potential ability of HIV-1 antibody in the urine.
Method: the assessment to fast detecting is undertaken by the file urine sample that detects 11 parts of freezing preservations, by using Calypte HIV-1 urine EIA method to detect, use Cambridge BiotechHIV-1 urine Westem Blot method to confirm, have 6 parts to be the anti-HIV-1 antibody positive in 11 parts of urine samples, 4 parts is the anti-HIV-1 negative antibody.Urine sample is at application of sample at room temperature simple earlier mixing before the RIC pick-up unit.150ml urine sample application of sample is used for analyzing in device, and determines the intensity at the signal of sample wire that installs and control line appearance subsequently.The ratio of calculation sample and contrast is as the index of relative intensity.If ratio is greater than 1.0, then interpretation is the HIV positive.The control line that occurs on the device for fast detecting has also confirmed its performance, shows that simultaneously sample is effective.In the time of 10,20 and 30 minutes, use above-mentioned boxlike analytical equipment to read the result respectively.
The result: the RIC testing result shows that 6 parts of known HIV-1 positive have all provided HIV-1 antibody response result, and 4 parts of whole anergy results of negative sample relatively obtain 100% susceptibility and specificity with EIA method and WesternBlot result.Generally speaking, the accuracy rate of this detection is 100%.
Table 2 urine HIV-1 fast detecting result-freezing sample
| The urine sample numbering | Estimate the result | Sample and contrast ratio | Actual result |
| ??FDA31 | The HIV positive | ????4.196 | The HIV positive |
| ??FDA32 | The HIV positive | ????1.529 | The HIV positive |
| ??FDA33 | The HIV positive | ????1.894 | The HIV positive |
| ??FDA34 | The HIV feminine gender | ????0.345 | The HIV feminine gender |
| ??FDA35 | The HIV feminine gender | ????0.408 | The HIV feminine gender |
| ??FDA36 | The HIV feminine gender | ????6.494 | The HIV feminine gender |
| ??FDA37 | The HIV positive | ????3.122 | The HIV positive |
| ??FDA38 | The HIV feminine gender | ????0.325 | The HIV feminine gender |
| ??FDA39 | The HIV feminine gender | ????0.325 | The HIV feminine gender |
| ??FDA40 | The HIV positive | ????2.439 | The HIV positive |
Conclusion: in the blind test detection of single stage method RIC detection mode to freezing sample, the testing result of all samples is consistent with the expectation result, illustrates that this detection method is applicable to anti-HIV-1 antibody in the detection urine.
The advantage of this detection mode is to be easy to use, and needs few Special Training, equipment and sampling apparatus, i.e. the freezing preservation of right and wrong also has stability preferably, and this makes it be easy to use in the popular backcountry of AIDS.There is not the feasible dangerous significantly reduction that is exposed under the HIV of infectious HIV in the urine sample.In addition, urine sample also be easy to provide in a large number carry out that HIV infect to detect and other disease and state detection.
Sequence table
＜110〉Beijing Kelipute Biopharmaceutical Technology Co., Ltd.
＜120〉device and the detection method of the HIV antibody in the fast detecting urine
<130>MP040001
<160>5
<170>PatentIn?Ver.2.1
<210>1
<211>39
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: biotin labeling mono-lysine connexon HIV-1 (gp41 peptide section)
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=biotin labeling Lys
<220>
<221>DISULFID
<222>(20)...(26)
<400>1
Xaa?Ile?Leu?Ala?Val?Glu?Arg?Tyr?Leu?Lys?Asp?Gln?Gln?Leu?Leu?Gly
1???????????????5??????????????????10??????????????????15
Ile?Trp?Gly?Cys?Ser?Gly?Lys?Leu?Ile?Cys?Thr?Thr?Ala?Val?Pro?Trp
20??????????????????25??????????????????30
Asn?Ala?Ser?Gly?Lys?Leu?Ile
35
<210>2
<211>22
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: biotin labeling HIV-1 gp120 peptide section
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=biotin labeling Lys
<400>2
Xaa?Thr?Glu?Pro?Leu?Gly?Val?Ala?Pro?Thr?Lys?Ala?Lys?Arg?Arg?Val
1???????????????5??????????????????10??????????????????15
Val?Gln?Arg?Glu?Lys?Arg
20
<210>3
<211>35
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: biotin labeling mono-lysine connexon HIV-2 (gp36 peptide section)
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=biotin labeling Lys
<220>
<221>MOD_RES
<222>(35)
<223>Xaa＝serinamide
<220>
<221>DISULFID
<222>(20)...(26)
<400>3
Xaa?Val?Thr?Ala?Ile?Glu?Lys?Tyr?Leu?Gln?Asp?Gln?Ala?Arg?Leu?Asn
1???????????????5??????????????????10??????????????????15
Ser?Trp?Gly?Cys?Ala?Phe?Arg?Gln?Val?Cys?His?Thr?Val?Pro?Trp?Val
20??????????????????25??????????????????30
Asn?Asp?Xaa
35
<210>4
<211>40
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: the two lysine connexon HIV-1 (gp41 peptide section) of biotin labeling
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=biotin labeling Lys
<220>
<221>DISULFID
<222>(21)...(27)
<400>4
Xaa?Lys?Ile?Leu?Ala?Val?Glu?Arg?Tyr?Leu?Lys?Asp?Gln?Gln?Leu?Leu
1???????????????5??????????????????10??????????????????15
Gly?Ile?Trp?Gly?Cys?Ser?Gly?Lys?Leu?Ile?Cys?Thr?Thr?Ala?Val?Pro
20??????????????????25??????????????????30
Trp?Asn?Ala?Ser?Gly?Lys?Leu?Ile
35??????????????????40
<210>5
<211>37
<212>PRT
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: the two lysine connexon HIV-2 (gp36 peptide section) of biotin labeling
<220>
<221>MOD_RES
<222>(1)
＜223〉Xaa=biotin labeling Lys
<220>
<221>MOD_RES
<222>(37)
<223>Xaa＝serinamide
<220>
<221>DISULFID
<222>(21)...(27)
<400>5
Xaa?Lys?Val?Thr?Ala?Ile?Glu?Lys?Tyr?Leu?Gln?Asp?Gln?Ala?Arg?Leu
1???????????????5??????????????????10??????????????????15
Asn?Ser?Trp?Gly?Cys?Ala?Phe?Arg?Gln?Val?Cys?His?Thr?Thr?Val?Pro
20??????????????????25??????????????????30
Trp?Val?Asn?Asp?Xaa
35
Claims (51)
1. lateral flow device that is used for detecting the target antibody of urine, this device contain and can specificity make the pH value of solution value be at least the sample area of 8 damping fluid in conjunction with the antigen of target antibody when contacting with urine with presetting.
2. lateral flow device as claimed in claim 1, wherein said damping fluid contains carbonate.
3. lateral flow device as claimed in claim 2, wherein said damping fluid also contains hydrocarbonate.
4. lateral flow device as claimed in claim 3, the saleratus that contains in the wherein said damping fluid and the mol ratio of sal tartari are 2: 1.
5. lateral flow device as claimed in claim 1, wherein said antigen are HIV albumen.
6. lateral flow device as claimed in claim 5, wherein said HIV are HIV-1.
7. lateral flow device as claimed in claim 5, wherein said HIV are HIV-2.
8. lateral flow device as claimed in claim 5, wherein said albumen are recombinant protein.
9. lateral flow device as claimed in claim 5, wherein said HIV albumen is envelope protein.
10. lateral flow device as claimed in claim 9, wherein said albumen are what select from the albumen of SEQ IDNO:1-5.
11. lateral flow device as claimed in claim 1, wherein said sample area also contains birds serum.
12. lateral flow device as claimed in claim 11, wherein said birds serum is chicken serum.
13. lateral flow device as claimed in claim 1, it also contains the cross-linking zone that constitutes with the crosslinked labelled reagent of label.
14. lateral flow device as claimed in claim 13, wherein said labelled reagent are albumin A.
15. lateral flow device as claimed in claim 13, wherein said label are collaurum.
16. lateral flow device as claimed in claim 1, it also has the check plot of containing capture agent.
17. lateral flow device as claimed in claim 16, wherein said capture agent are anti-human IgG antibody.
18. lateral flow device as claimed in claim 17, wherein said anti-human IgG antibody is goat anti-human igg's 2 antibody.
19. a lateral flow device that detects antibody in the urine, this device contains:
A. the cross-linking zone that contains albumin A/collaurum;
B. the control line that contains capture agent, wherein said capture agent has affinity with the urine endogenous antibody that albumin A/collaurum cross-linking agent combines, and this affinity is greater than the affinity of albumin A in albumin A/collaurum cross-linking agent with the urine endogenous antibody.
20. lateral flow device as claimed in claim 19, wherein said capture agent are anti-human IgG antibody.
21. lateral flow device as claimed in claim 20, wherein said anti-human IgG antibody is goat anti-human igg's 2 antibody.
22. lateral flow device as claimed in claim 19, it also comprise one contain the pH value be at least 8 damping fluid sample area and can specificity in conjunction with the antigen of target antibody.
23. lateral flow device as claimed in claim 22, wherein said damping fluid contains carbonate.
24. lateral flow device as claimed in claim 23, wherein said damping fluid also contains hydrocarbonate.
25. lateral flow device as claimed in claim 24, the saleratus that wherein said damping fluid contains and the mol ratio of sal tartari are 2: 1.
26. lateral flow device as claimed in claim 22, wherein said antigen are HIV albumen.
27. lateral flow device as claimed in claim 26, wherein said HIV are HIV-1.
28. lateral flow device as claimed in claim 26, wherein said HIV are HIV-2.
29. lateral flow device as claimed in claim 26, wherein said albumen are recombinant protein.
30. lateral flow device as claimed in claim 26, wherein said HIV albumen is envelope protein.
31. lateral flow device as claimed in claim 30, wherein said albumen are to select from the albumen of SEQ IDNO:1-5.
32. lateral flow device as claimed in claim 22, wherein said sample area also contains birds serum.
33. lateral flow device as claimed in claim 32, wherein said birds serum is chicken serum.
34. kit that contains claim 1 or the described lateral flow device of claim 19.
35. a method that detects antibody in patient's urine sample, this method contain the step that the sample area of useful lateral flow device contacts with urine sample, wherein said sample area contains the pH value and is at least 8 damping fluid.
36. method as claimed in claim 35, wherein said damping fluid contains carbonate.
37. method as claimed in claim 36, wherein said damping fluid also contains hydrocarbonate.
38. method as claimed in claim 37, the saleratus that contains in the wherein said damping fluid and the mol ratio of sal tartari are 2: 1.
39. method that detects antibody in patient's urine sample, this method contains the step that the sample area of useful lateral flow device contacts with urine sample, the cross-linking zone and the check plot of containing capture agent that contain albumin A/collaurum cross-linking agent in this device, wherein said capture agent has affinity with the urine endogenous antibody that albumin A/collaurum cross-linking agent combines, and this affinity is greater than the affinity of albumin A in albumin A/collaurum cross-linking agent with the urine endogenous antibody.
40. method as claimed in claim 39, wherein said capture agent are anti-human IgG antibody.
41. method as claimed in claim 40, wherein said anti-human IgG antibody is the goat anti-human igg antibody.
42. containing pH, method as claimed in claim 39, wherein said sample area be at least 8 damping fluid.
43. method as claimed in claim 42, wherein said damping fluid contains carbonate.
44. method as claimed in claim 43, wherein said damping fluid also contains hydrocarbonate.
45. method as claimed in claim 44, the saleratus that contains in the wherein said damping fluid and the mol ratio of sal tartari are 2: 1.
46. as claim 39 or the described method of claim 35, wherein said patient is suspicious HIV the infected, and described lateral flow device contains HIV antigen.
47. method as claimed in claim 46, wherein said HIV is HIV-1.
48. method as claimed in claim 46, wherein said HIV is HIV-2.
49. method as claimed in claim 46, wherein said albumen are recombinant protein.
50. method as claimed in claim 46, wherein said HIV albumen is envelope protein.
51. method as claimed in claim 50, wherein said albumen are what select from the albumen of SEQ ID NO:1-5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100042443A CN1558237A (en) | 2004-02-12 | 2004-02-12 | Fast detection device and method for HIV antibody in urine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100042443A CN1558237A (en) | 2004-02-12 | 2004-02-12 | Fast detection device and method for HIV antibody in urine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1558237A true CN1558237A (en) | 2004-12-29 |
Family
ID=34350806
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2004100042443A Pending CN1558237A (en) | 2004-02-12 | 2004-02-12 | Fast detection device and method for HIV antibody in urine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1558237A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109613257A (en) * | 2018-12-01 | 2019-04-12 | 山东博科生物产业有限公司 | A kind of transferrins (TRF) detection kit |
-
2004
- 2004-02-12 CN CNA2004100042443A patent/CN1558237A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109613257A (en) * | 2018-12-01 | 2019-04-12 | 山东博科生物产业有限公司 | A kind of transferrins (TRF) detection kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1902489A (en) | Diagnostic test for analytes in a sample | |
| EP3084441B1 (en) | Improved diagnostic test for csfv antibodies | |
| CN1564945A (en) | Rapid diagnostic device, assay and multifunctional buffer | |
| CN1842707A (en) | Prion protein binding materials and methods of use | |
| CN1254844A (en) | Improved by-pass immunoassay | |
| AU2013329025B2 (en) | Peptides, devices, and methods for the detection of Ehrlichia antibodies | |
| CN1527943A (en) | Allergen-microarray assay | |
| CA2614332A1 (en) | Method for the preparation of prrs virus and proteins of and diagnostic test kits for detecting them | |
| JP2010502187A5 (en) | ||
| CN1105181A (en) | Immunoassay | |
| CN1505759A (en) | Detection of candida | |
| CN107299111A (en) | Detect the preparation of cell climbing sheet component, CBA detection kits and its application of MOG IgG CBA kits | |
| CN106632691B (en) | HIV recombinant antigen, expression gene, expression vector and HIV detection kit | |
| CN103983781A (en) | Porcine pseudorabies virus gE IgM antibody colloidal gold immunochromatograohic assay test strip as well as preparation method and application thereof | |
| CN103364561A (en) | Porcine circovirus 2 (PCV2) antibody colloidal gold immunity chromatography detection test paper and making method thereof | |
| CN112946294A (en) | Novel coronavirus 2019-nCoV antibody detection test strip and preparation method and application thereof | |
| CN101793897A (en) | Method for improving screening efficiency of hybridoma | |
| KR101317417B1 (en) | Antigen for detecting of classical swine fever virus, antibody detecting method and test kit using thereof | |
| US9632086B2 (en) | Method and kit for determining-antibody sensitivity and clone cell strain | |
| CN105759030A (en) | Colloidal gold test strip for testing toxoplasma gondii antibodies | |
| CN109187969A (en) | Detect immune chromatography test paper, kit and the purposes of antibody against swine fever virus | |
| AU754309B2 (en) | Diagnosis of feline immunodeficiency virus infection using env/gag polypeptide markers | |
| CN109280644B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof | |
| CN1558237A (en) | Fast detection device and method for HIV antibody in urine | |
| HUE030824T2 (en) | Method for diagnosing and differentiating hiv-2 infections |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |