CN1768267A - Multiple-channel test device, method for producing the same and use thereof - Google Patents
Multiple-channel test device, method for producing the same and use thereof Download PDFInfo
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- CN1768267A CN1768267A CN 200480008672 CN200480008672A CN1768267A CN 1768267 A CN1768267 A CN 1768267A CN 200480008672 CN200480008672 CN 200480008672 CN 200480008672 A CN200480008672 A CN 200480008672A CN 1768267 A CN1768267 A CN 1768267A
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Abstract
The object of the invention is a multiple-channel test devise based on immunodiffusion and immunochromatography, which enables the simultaneous or parallel determination of several analytes. In the test devise, it is possible to group together different combinations of markers recognizing allergens, myocardial infarction markers, venereal disease analytes, blood screening analytes, respiratory infection producing agents, IgG, IgA and IgM antibodies, other infectious disease producing agents as well as various cancer markers. The multiple-channel test devise comprises a porous carrier material on which a channel network has been formed by etching the carrier material by laser to form a shaped figure that contains several channels. In the channels, various specific binding reagents have been immobilized, which enable the diagnoses of a target illness and/or syndrome. The sample application point is optionally provided with a filter and optionally contains a label mobilizable by the analyzable sample and a specific binding reagent. Also the method for the production of the test device and its use are disclosed in the invention.
Description
Technical field
The present invention relates to the multichannel test device based on immunodiffusion, this proving installation can be simultaneously or is parallelly carried out a plurality of different analyses.The invention also discloses the preparation and the purposes of described device.
Background technology
Known multiple method and proving installation based on immunodiffusion, disclosed in for example following patent and the patented claim: US 4,757,002, and US 3,990,852, and US 4,562, and 147 and EP 0 250 137.Also disclose the immune chromatography method based on immunodiffusion, disclosed in for example following patent and the patented claim: EP 0 291 194, EP 0 284 232 (FI 93150) and WO 86/03839.
For above-mentioned proving installation based on immunodiffusion and immunochromatography, modal is that each proving installation only is used to carry out an analysis.Under many circumstances, make a definite diagnosis and select suitable methods of treatment may need to carry out a plurality of different analyses suffering from specified disease or syndromic patient.If identification causes the cause of disease of symptom and disease or gets rid of specified disease and need carry out a plurality of different tests, diagnose so and will become very expensive, to such an extent as to only carry out one or the seldom analysis of number usually, these analyses are based on treatment doctor's judgement and select at random, or test commonly used, in this case, other optional test possibly can't be carried out.
Use more than a test channel is known, and for example US B1 6,171,870 and US A12003/0040021 in disclosed.US B1 6,171,870 relates to the method for producing this type of passage, this method relate to scold water (water-repellent) material for example wax be applied on the porous carrier.Processed zone has formed the blocking part between the passage.In the method that US A12003/0040021 describes, sample moves along the passage of handling, and untreated zone is stayed between the passage.
Compare with proving installation with method of the prior art, advantage of the present invention comprises the decline of minimizing, sample volume and the load of reagent and material consumption, and the improvement of environment friendly, shelf life and user friendly.The raising of environment friendly is because the caused environmental loads of used proving installation descends.Because the saving of reagent consumption and human cost, method of the present invention can be with extremely low cost production test device.
With obtained solution in the present invention based on the relevant problem of the conventionally test device of immunodiffusion and immune chromatograph, feature of the present invention is disclosed in the following claim.
Summary of the invention
Proving installation based on immunodiffusion of the present invention comprises the specific bond reagent (immunoreagent) of the form of porous carrier materials and district's band (zone) wherein or spot (blot).Be applied in the described porous carrier separately or in advance with the detectable mark of second kind of specific bond reagent (immunoreagent) coupling, and can be moved by described sample.The optional sample that provides filtrator applies the site and also is positioned on this proving installation.The porous carrier materials of this proving installation is preferably made by cellulose nitrate, and comprises channel network, and it is by etching in the described porous carrier materials with laser treatment.This channel network comprises two or more passages (1), separate by the zone of handling (2), be fixed in one or more specific bond reagent (3) wherein, place the contiguous sample of described passage to apply site (5) or be positioned at sample to apply the gauge point of choosing wantonly (4) in the site (5) itself, the mode of its placement makes described sample can be evenly distributed in each passage.
Proving installation of the present invention can be diagnosed different diseases or syndrome based on while or the parallel identification of carrying out to syndromic reagent of multiple generation or analyte.This proving installation can be used for discerning simultaneously the multiple irritated reagent that produces, miocardial infarction label, the reagent of generation venereal disease, screening of blood analyte, the reagent of generation infection in respiratory system, the reagent and/or the cancer markers of generation infectious disease.
The example of useful specific bond reagent is antibody, antibody fragment, recombinant antibodies, recombinant antibody fragment, antigen, agglutinin, acceptor and/or part.Useful labelled reagent is for example latex, gold, metal or coloring agent particulate or fluorescent material.
The invention discloses a kind of method of producing this proving installation, wherein said specific recognition immunoreagent is fixed in the porosint, used material to make it become inertia, and wherein sample applies that the site is optional to provide filtrator and detectable mark, is combined with specific bond reagent on this detectable mark.By on described porosint, forming channel network with anticipated shape and size with the laser-induced thermal etching porosint.The zone (2) that this network comprises two or more passages (1) and handled; Be fixed in each passage (1) as district's band or spot (3) with one or more identification specific bond reagent.
The useful material that makes described porosint become inertia is a potpourri, it comprises natural or synthetic polymer, for example albumin and casein or PEG (polyglycol) and PVA (polyvinyl alcohol (PVA)), the nonionic scaling agent is hexane sulfonic acid and TRITON-X-100, BRIJ and antiseptic for example, for example sugared, as sucrose and trehalose, or their derivant.
After applying described reagent, described proving installation is dried and remains on relative humidity and is no more than 8% and sealed packing, thereby has improved the storage stability of described proving installation greatly.
This multichannel test device at the same time or parallel carrying out in a plurality of detections be useful, target disease and/or syndrome that this makes people to diagnose may to be difficult to diagnose under the situation of only using a label.What described sample was applied to this proving installation applies site (5), but it contains the mark that can be moved by analytic sample and second kind of specific bond reagent of coupling with it, described sample and reagent evenly move to this passage by the channel network of laser treatment formation from this site, they and described specific bond reagent are located reaction or are not reacted at its point of fixity (3) herein, and the positive or negative result can directly read from here.
The invention still further relates to and be used for simultaneously or the parallel proving installation that is used for recognition objective disease and/or syndromic a plurality of detections.What sample was applied to this proving installation applies site (5), described sample moves to the passage from this site, it is with the mark and the second kind of specific bond reagent mix that combines with it that can be moved by described sample and react herein, after this, described sample and reagent evenly move in this passage by the channel network of laser treatment formation, they and described specific bond reagent are located reaction or are not reacted at its point of fixity (3) herein, and the positive or negative result can directly read from here.
The invention still further relates to described proving installation at the same time or the parallel purposes that is used for recognition objective disease and/or syndromic a plurality of detections.Described sample mixes with independent mark (this mark has second kind of specific bond reagent of coupling with it), and what this potpourri was applied to this proving installation applies site (5), described sample and reagent evenly move to this passage by the channel network of laser treatment formation from this site, they and described specific bond reagent are located reaction or are not reacted at its point of fixity (3) herein, and the positive or negative result can directly read from here.
Description of drawings
Fig. 1 shows the network with eight passages, and this is to be used to reach eight different preferred proving installations that detect.Gauge point 4 applies site 5 as sample simultaneously and works, and passage 1 processed zone 2 separates, and contains specific binding agents and is with 3 as the district.
Fig. 1 a shows the better embodiment that is used to carry out maximum eight different multichannel test devices that detect according to Fig. 1.Gauge point 4 applies site 5 as sample simultaneously and works, and passage 1 processed zone 2 separates, and contains specific binding agents and is with 3 as the district.This device is marked with the information about this test and manufacturer.
Fig. 2 shows a kind of proving installation, and this proving installation can be used for carrying out six different detections and control reaction thereof, perhaps randomly, carries out maximum 12 different detections by six different marks.One of passage 1 branches into two passage 1a, 1b, and their processed zones 2 separate.Sample applies the centre that the site is positioned at channel network, and each passage 1 comprises gauge point 4 and one or more bonds district band 3.
Fig. 3 shows a kind of proving installation, and this proving installation can be used for carrying out four different detections and control reaction thereof, perhaps randomly, carries out maximum eight different detections by four different marks.
Fig. 4 shows a kind of proving installation, and this proving installation can be used for carrying out four different detections and control reaction thereof, perhaps randomly, carries out maximum eight different detections by four different marks.
Fig. 5 shows a kind of proving installation, and this proving installation can be used for carrying out maximum six different detections, perhaps randomly three detections, and control reaction.
Fig. 6 shows a kind of proving installation, and this proving installation can be used for carrying out 12 different detections by four different marks.
Fig. 7 shows the network with eight passages, and this is to be used to carry out maximum 24 different preferred proving installations that detect.Each passage 1 comprises three bond district bands 3.
Fig. 8 shows and can be used for carrying out simultaneously 16 different proving installations that detect.
Fig. 9 shows a kind of proving installation, and this proving installation can be used for carrying out 20 different detections by five different marks.
Figure 10 shows the proving installation that can be used for carrying out two detections and control reaction.This device is marked with the information about this test and manufacturer.
Figure 11 shows the proving installation that can be used for carrying out two detections and control reaction.This device is marked with the information about this test and manufacturer.
Figure 12 shows a kind of proving installation of producing by laser-induced thermal etching, and this proving installation can be used for carrying out three parallel detections, perhaps three each and every one other detection and control reaction thereof randomly.This device is marked with the information about this test and manufacturer.
Embodiment
Target of the present invention is based on the multichannel test device of immunodiffusion, this device comprises porous carrier materials, with district's band in this material or one or more specific bond reagent (immunoreagent) of spot form, and sample applies the site, this sample applies the optional filtrator that disposes in site, for example be used to remove haemocyte, but and the optional mark that can be moved by analytic sample that comprises, it is by second kind of specific bond reagent (immunoreagent) bag quilt.This mark is arranged on the proving installation or is added into sample.
Proving installation of the present invention is characterised in that it comprises channel network, this channel network be by on porous carrier materials by etching, for example by laser, and preparation.Passage to this proving installation provides one or more identical or different specific binding agents, and this specific binding agents is to select from different label groups, can use together, and be that the diagnosis particular integration is levied needed.Therefore, by being used to discern specific objective disease and/or syndromic a plurality of detection simultaneously, make that diagnosis is carried out.Sample applies the form that the site can be point or spot or line or district's band, is positioned at the centre or the other end of for example rectangular (strip), makes sample to be distributed to equably in each passage.If identical specific bond reagent is used for passage with different concentration, can obtain semiquantitative result so.
Proving installation of the present invention comprises a plurality of passages, can be used for simultaneously or the replicate determination multiple analytes.In proving installation, can randomly divide into groups: the reagent miocardial infarction label of discerning the pathogenic agent (for example allergen) relevant with various syndromes to following reagent, the suitable agent that is used to discern venereal disease and is used for screening of blood, identification produces the label of the reagent of infection in respiratory system, IgG, IgA and IgM antibody, identification produces the label and the various cancer markers of other infectious diseases, when being used for them or replicate determination.
Another object of the present invention is the method for producing described proving installation.This production method is characterised in that, by laser technology, etches the multichannel network with anticipated shape and size (seeing Fig. 1-12) on porosint, and described laser technology produces certain shape on substrate.Different identification specific bond reagent is for good and all connected (just fixing) in the porous mass of each passage.The analysis result of test reaction can be read from these points.
After this, employing can make the free responding site become the mass treatment porosint of inertia, that is to say, they do not react in the undesired mode of people, for example by slowing down or stoping analyte or mark to move in the reagent area band or spot of permanent fixation.Combine second kind of specific bond reagent on detectable, the optional visible mark, this mark is applied to sample and applies on the precalculated position of site or test channel.The mark of this binding reagents institute combination also be introduced in the sample, and in this case, when sample was added into sample and applies the site, this mark was transferred in the device.
In addition, the present invention has also described proving installation at the same time or parallelly carry out a plurality of detections to discern the purposes in some disease and/or the syndrome.The passage of branch also can be used to contrast the suitable function of this device, adopts one of passage as test channel, and other passage is as functional or contrast (control) passage.Also can in proving installation, add the functional passage that independently contrasts that indicates device and reagent.
The difference of disclosed proving installation of the prior art is one or more different or identical parallel testings in multichannel test device of the present invention and the application in diagnostic method thereof and above-mentioned patent and the patented claim, preferred 2-40, more preferably 4-30, most preferably 8-24 parallel testing can adopt the minimum object that makes from porosint to carry out.
Adopting laser to produce channel network is the method for optimizing of making proving installation of the present invention.The machine that uses in this production method is not expensive, and operating cost mainly is made of power consumption.Scold for example hot wax phase ratio of water substance with use,, can avoid in manufacture process, using chemicals by using laser.By using laser, can obtain accurate etching result (printing), and owing to print the levels of precision of (impression), if necessary, proving installation can be labeled in manufacture process, thereby reduce the danger that handle assembly is mixed up.
Can be by a large amount of test of following method manufacturing: single passage, parallel channels or the channel network of etching on roller bearing, and needed reagent of adding and processing on same production line, in the mode of expectation test being separated from each other at last becomes single test and is used for one or more detections, perhaps becomes the test combination of a plurality of parallel testings.
In addition, proving installation can be used to analyze patient's sample of small size, for example urine, blood, blood plasma, serum, saliva, tissue fluid, ight soil, environmental sample etc.Little sample volume, though little of each proving installation 1.0-50 μ l, preferred 2.0-40 μ l, more preferably 4.0-20 μ l, or most preferably 5.0-10.0 μ l is just enough, and this has many good qualities, and is particularly carrying out context of detection at the sample from children's or finger tip blood sample collection.
The production testing device
The production of pick-up unit comprises six key steps.Form channel network, apply and fixing specific reagent, make porous channel become inertia, apply the mark with the second species specificity pack quilt, production sample applies site and steady testing device and guarantees its storage properties.Between the key step of mentioning, can dry test strip.If mark is joined in the sample, the step that so mark is applied to proving installation can be omitted.
In proving installation of the present invention, by form passage in proving installation, a plurality of similar and different parallel testings (Fig. 1-12) can carry out on very intensive porosint project (item).
Produce passage
The channelizing of reagent and sample can scold water or part to scold the material of water by using, for example wax, polyolefin, polyacrylamide pigment or its potpourri, handle the seepy material of porous, for example cellulose nitrate, polysulfonates (polysulphonate), nylon or paper, and obtain.Scolding applying of water material on the porosint is to carry out on their fluxing temperature, the preferred temperature between 50 ℃ and 80 ℃, advantageously print (printing), brush or spray technique by using, thereby on porosint, form and scold the water figure, this figure has the shape of expection and surrounds channel network, described channel network comprises several passages that separated by processed zone each other, and sample is because capillary action and diffusion and move along described passage.After applying, immediately substrate is cooled to room temperature.It is possible in the mode that keeps reagent reacting it being applied in the channel network.
Nature, described passage also can be produced by the carrier material of suitable size being suppressed the channel network that has anticipated shape and a size with formation.
But, according to the present invention, channelizing preferably by with laser treatment porous and permeable material for example cellulose nitrate usually carry out.By adopting suitable laser energy, do not wish that fluid flow to the cellulose nitrate of cellulose nitrate substrate region wherein can be etched, and the zone of handling is only covered by the plastic foil (Mylar film) below the cellulose.Difform figure (Fig. 1-12) is to be formed by the passage that contains cellulose nitrate or certain other porosint and channel edge or the zone of handling, the zone of described processing extends to substrate edge fully, and adopted the suitable computer program of programming in advance to carry out laser-induced thermal etching, wherein adopted the 10-90% of equipment peak power output, preferred 10-80%, more preferably 12-60%, 15-40% most preferably, etching speed preferably arrives 1500mm/s at 100mm/s, more preferably 400mm/s is to 1000mm/s, resolution is 50/1000 to 1000/1000, preferably 100/1000 to 800/1000, more preferably 150/1000 to 500/1000, most preferably 200/1000 to 300/1000.
The zone that separates the processing of passage can extend to the edge of proving installation fully, and perhaps randomly, untreated zone can be stayed on the pattern edge.Proving installation can be used about the information of manufacturer or test by etching and carry out mark, for example passes through laser treatment.
Reagent is applied in the carrier
To test required all ingredients, to apply also that (if necessary) be fixed in the channel network be possible.Can use reagent with very little volume.Required reagent comprises at least two kinds of specific bond reagent, wherein a kind of being fixed, another kind of with can be combined and at least a detectable mark by the mark that sample solution moves.
Specific bond reagent
Suitable material comprises various binding reagents, specific immunity chemical reagent, for example antibody, antibody fragment, recombinant antibodies, recombinant antibody fragment, antigen, and other part for example acceptor, agglutinin, biotin, avidin etc.Specially suitable is monoclonal and polyclonal antibody, antigen and fragment thereof.As for allergy test, suitable allergen comprises the extract that the pollen by tree, grass or weeds makes, the extract that is made by spore, acarid, house dust, zoodermic epidermis, insect, latex, parasite, medicine or the food of mould etc.
Can binding reagents be fixed or adopt chemistry or physical means are connected to expection site on the porous carrier structure by known method, the binding reagents of control reaction also can randomly be fixed or adopt chemistry or physical means to be connected on the passage of same passage or branch.
Make porous mass become inertia
After having produced channel network and having applied specific bond reagent, by using so-called closed reagent that the porous carrier material is become inertia, thereby by the suitable free responding site of carrier mass and the non-specific bond site of specific bond reagent of removing, the described potpourri of potpourri in contain natural and/or synthetic polymkeric substance, for example albumin or casein and/or PEG (polyglycol) and PVA (polyvinyl alcohol (PVA)), the nonionic scaling agent, for example hexane sulfonic acid and TRITON-X-100, BRIJ, and antiseptic, for example sugared, as sucrose and trehalose, or their derivant.After this, proving installation is carried out drying.
If desired, can after preparing, proving installation add closed reagent again, and relevant with applying of sample.
The preparation gauge point
Suitable labelled reagent comprises the various plastics or the metal particle that can be moved by sample flow; the for example particulate of latex, gold, liposome, coloring agent, fluorophore, fluorescent dye or other this type of metallics or coloring agent, its can bound analyte and with specific binding agents for example antibody, acceptor, agglutinin or other part combine.Fluorescent grain, fluorescent dye or super paramagnetic particle also can be used as mark.
The sample that mark can be fixed on the proving installation in the porous carrier applies the site or another applies on the different site, site with sample.But but labelled reagent also can join in the analytic sample before analytic sample is applied on the test strip.
The preparation sample applies the site
Sample applies the site can randomly be furnished with filtrator, places filtrator and realizes by applying at sample on the site, and this filtrator for example stops that haemocyte is carried in the passage.Sample applies the site can be furnished with mark, has combined second kind of specific bond reagent on this mark.
The steady testing device is also guaranteed its storage properties
Can for example be lower than 8% relative humidity for it provides by handling proving installation, after this, pack this proving installation and be stored in dry place, make the proving installation anti-storage that becomes thereby be lower than 8% with the relative humidity of keeping proving installation by drying.Like this, this device keeps it functionally to reach 24-36 month, without any tangible decline, comprises susceptibility or specificity on functional.
In case of necessity, this proving installation can be placed in the chest of being made by cardboard or plastics, and when needing, it can be equipped with the operation of description proving installation and the instructions of use.
The structure of proving installation
The structure of proving installation and possible variation thereof are described among Fig. 1-12.
Proving installation comprises a plurality of passages 1, and preferred 2-10, more preferably 3-8, passage 1 can be further divided into the passage of a plurality of branches on take-off point, preferred 2-5 (1a, 1b), more preferably 2-3.The zone 2 of handling separates each other passage 1, and guides sample moving in proving installation.
The passage of passage 1 or branch (1a, 1b) contains one or more binding reagents as bond district band or spot 3.In addition, proving installation comprises one or more gauge points 4, and it can randomly apply site 5 with sample and combine.Sample applies site 5 can randomly be furnished with filtrator.
Fig. 1 shows a kind of proving installation, wherein have the expection size for example the nitrocellulose filter of 25 * 25mm be divided into eight identical passages 1.This passage is by at porosint printing curve on the nitrocellulose filter for example, and on the repellency district band 2 of the processing of nitrocellulose filter, prepare with the imprinting apparatus heat of transfer, fusion, about 60 ℃ polyolefin.Each passage all contains specific binding agents 3.Be positioned at gauge point 4 in the middle of the proving installation and contain second kind of labelled reagent at test analyte.Sample applies site 5 and works as the gauge point 4 of proving installation simultaneously.Be characterised in that according to the proving installation of Fig. 1 and can contain different specific binding agents (for example allergen) in each passage, and the particulate mark all identical to every kind of bond (for example anti human IgE) is positioned at the centre of passage.
Fig. 1 a shows a kind of proving installation, and wherein nitrocellulose filter has been divided into eight identical passages 1.This passage is by for example preparing by the laser-induced thermal etching figure on the nitrocellulose filter at porosint, make the zone 2 of handling stay between the passage, in laser treatment process, on the zone of described processing, added the mark relevant with test and manufacturer.Each passage all contains specific binding agents 3.Be positioned at gauge point 4 in the middle of the proving installation and contain second kind of labelled reagent at test analyte.Sample applies site 5 and works as the gauge point 4 of proving installation simultaneously.Be characterised in that according to the proving installation of Fig. 1 and can contain different specific binding agents (for example allergen) in each passage, and the particulate mark all identical to every kind of bond (for example anti human IgE) is positioned at the centre of passage.
Fig. 2 shows a kind of proving installation, and wherein one of passage 1 comprises the passage 1a and the 1b of branch, and it contains specific binding agents on bond district band 3.Other passage 1 comprises two bond district bands 3, and wherein another can be the check plot band.All passages 1 contain independent gauge point 4.The center of this device comprises independent sample and applies site 5.
Fig. 3 shows a kind of proving installation, wherein four identical or different passages 1 comprise the passage 1a and the 1b of branch, it contains specific binding agents on bond district band 3, and contain four independent particulate gauge points 4 in the downstream of channel branch point, this particulate gauge point 4 is with specific binding agents bag quilt.This device comprises independent sample and applies site 5.
Fig. 4 shows a kind of proving installation, it has four identical or different passages 1, described passage 1 branches into passage 1a and 1b, it is corresponding to contain specific binding agents on bond district band 3, and contain four independent particulate gauge points 4 in the downstream of channel branch point, this particulate gauge point 4 is with specific binding agents bag quilt.This device comprises independent sample and applies site 5.
Fig. 5 shows a kind of proving installation, and wherein sample applies the site and is positioned at this proving installation or a rectangular end.This device comprises three passages 1, has two bond district bands 3 in each passage 1.It is identical that gauge point 4 and sample apply site 5.
Fig. 6 shows a kind of proving installation, and wherein four different passages 1 further branch into three passage 1a, 1b and 1c, in 12 of described passage 1 independent bond district bands 3, contains maximum 12 kinds of different bonds.And this device comprises four independent gauge points 4.This proving installation comprises independent sample and applies site 5.
Fig. 7 shows a kind of proving installation, comprises the zone 2 of eight identical passages 1 and the processing between them.Contain three kinds of specific binding agents in each passage 1, optional at different analytes, be arranged in three independent bond district bands 3.Be positioned at gauge point 4 in the middle of the proving installation and contain another kind of labelled reagent at test analyte.
Fig. 8 shows the proving installation that comprises eight passages 1, and each passage 1 branches into two passage 1a and 1b, and contains bond district band 3.Mark and sample that this device comprises combination apply site 4,5.
Fig. 9 shows the proving installation that comprises five passages 1, and each passage comprises four bond district bands 3.This device comprises four independent gauge points 4 and sample applies site 5.
Figure 10 shows the proving installation that is used to detect viral antigen, and wherein sample applies site 5 and is positioned at this proving installation or a rectangular end.This device comprises three passages 1, contains bond district band and the gauge point 4 that separates in each passage 1.This device is marked with the information about manufacturer and test.
Figure 11 shows and is used for the proving installation that miocardial infarction detects, and wherein sample applies site 5 and is positioned at this proving installation or a rectangular end.This device comprises three passages 1, contains bond district band 3 and the gauge point 4 that separates in each passage 1.This device is marked with the information about manufacturer and test.
Figure 12 shows three kinds of proving installations, and wherein sample applies the end that site 5 is positioned at this proving installation.This device comprises gauge point 4 and bond district band 3, and wherein another bond district band is a control point.
The form of above-mentioned proving installation and size can only be regarded as the example of the proving installation that method of the present invention can make.
Proving installation is used to carry out while or parallel detection
The present invention is based on porosint for example the fluid in the cellulose nitrate flow because diffusion and capillary action, described fluid flows and in fact takes place with identical speed in all directions, this point is known.Described radial or lateral flow makes sample can move in a plurality of passages, as long as the channelizing of porosint is carried out in reliable mode, wherein contains analyzable analyte and test agent in this sample, and described passage carries out different tests simultaneously.Be applied in the proving installation sample with join in the sample labeled reactant or with one or more labeled reactants that are applied in this device, this compound reacts with the bond of fixing subsequently, and described bond further is placed in the different passage of proving installation.Immunoreagent also can be fixed, and does not disturb it functional.Sample is applied in the proving installation by small amount of sample or its dilution being applied to sample applies on the site 5 and carry out, sample applies site 5 from sample and moves to gauge point 4, and further entering test section band 3 along passage, the specific binding agents of mark, the analyte in the sample and other occur in herein with reaction between the specific binding agents that solid carrier combines.If mark is coloured particulate, this reaction can be seen by bore hole; Perhaps, this reaction can be used as luminous point, spot, line or other shape and is read by bore hole under UV-light; Perhaps this reaction can be passed through suitable device, and the device of photometer, photofluorometer or measurement changes of magnetic field is read and explained.
In another embodiment, gauge point 4 is arranged in sample and applies site 5, and in this case, the analyte in the sample and the specific binding agents of mark formed compound at sample before channel flow.
In another embodiment, mark applying add sample in proving installation before in sample carried out.
This proving installation is useful especially in the allergy test, wherein needs a large amount of relatively serum to determine the irritated specific antibody of I gE class at present.When application is of the present invention, by several different allergens being assigned in each passage of proving installation, can be by very a spot of sample, even have only serum, blood plasma or the whole blood of 10-40 μ l, detect the patient simultaneously to 24 kinds of as many as or the allergenic sensitization of more kinds of difference with same proving installation.
In a preferred embodiment of the present invention, several different marks are connected in the proving installation.Therefore, a little sample moves this mark, and this mark is optional to differ from one another, and places different passages.The immunoglobulin (Ig) specific antibody that such proving installation is well suited for the anti-different agent of causing a disease detects.
The embodiment of application testing
Following embodiment should be counted as example, should be appreciated that other possible application of the present invention is apparent to those skilled in the art.
Figure described in Fig. 1 a is by Domino DGM-1 " High Resolution LaserMarker " device, will not wish cellulose nitrate in the zone that fluid flows therein to etch away and prepares on the cellulose nitrate carrier of porous.On proving installation, formed the network that comprises eight passages 1, separated by the zone 2 of handling between the described passage 1.For fear of the danger mixed up of device, form mark about product and manufacturer by not etching cellulose nitrate.
20% laser (85-132V/170-260V, I import the output with 20W mutually) and 250/1000 the resolution that are pre-programmed into expection figure in the computer program and are signature velocity by 700mm/ second of adopting 10-2500mm/ second, peak power output 20W are carried out etched.
The proving installation module (matrix) with channel network of Sheng Chaning is used for carrying out a plurality of detections simultaneously according to the following examples like this.
Specificity can be analyzed in allergen joins the proving installation of producing according to embodiment 1 as solution (concentration 1-5mg/ml) the passage 1 of network, thereby when they are connected to sample and apply the cellulose nitrate in site, has formed soluble district and be with 3.Apply different allergen (extract that makes by the pollen of tree, grass or weeds, the extract that makes by spore, acarid, house dust, zoodermic epidermis, insect, latex, parasite, medicine or the food of mould) in each passage.After this, the reactive cellulose nitrate of closed channel 1 promptly makes it to become inertia, wherein adopt contain bovine serum albumin(BSA) (BSA) (0.1-5.0%), hexane sulfonic acid and trehalose (1.0-3.0%).Solution (10-100 μ l) is applied to the centre of proving installation, and after this, because diffusion and capillary action, it moves in each passage, and combines (promptly sealing) with the free responding point of cellulose nitrate.
After this, in the drying at room temperature proving installation, be lower than 8% up to its relative humidity.
After the drying, the manual aqueous solution that applies 1.0-10.0 μ l in the middle of proving installation, wherein contain useful resisting-IgE and wrap the coloured latex particle (0.2-1.0%) of quilt, also contain the BSA of 0.1-1.0% in this potpourri, 0.01-0.05% polysorbas20 and 0.5-1.5% trehalose.Particle colloidal sols is dried to the centre of proving installation.
When use test, the sample that is applied in the middle of the proving installation from the 10-50 μ l serum of suffering from irritated people or its dilution applies on the site 5.Sample dissolution in the middle of mark.Anti--IgE antibody and irritated specific IgE molecular reaction of mark, and owing to capillary action is diffused in the test channel.
But, will in the reaction channel that contains allergen antibody to be measured, form coloured district band so if contain in the analytic sample at one or more the allergenic specific IgE antibodies in the proving installation district band 3.
Adopt suitable marking press, with the graphic printing of formation shown in Figure 1 to the cellulose nitrate carrier of porous, wherein formed channel network, by coloured about 60 ℃ polyolefin is shifted on porous carrier, thereby it has sealed the hole of the carrier in the expected areas, and formed network on proving installation, this network contains eight passages 1 that processed zone 2 separates.
Specific allergen to be measured (concentration 1-5mg/ml) is joined in the passage of this network, thereby they have formed and are connected to the soluble district that sample applies the cellulose nitrate in site and are with 3.Apply different allergen (extract that makes by the pollen of tree, grass or weeds, the extract that makes by spore, acarid, house dust, zoodermic epidermis, insect, latex, parasite, medicine or the food of mould) in each passage.After this, the reactive cellulose nitrate of closed channel 1 promptly makes it to become inertia, wherein adopt contain bovine serum albumin(BSA) (BSA) (0.1-5.0%), hexane sulfonic acid and trehalose (1.0-3.0%).Solution (10-100 μ l) is applied to the centre of proving installation, and after this, because diffusion and capillary action, it moves in each passage, and combines (promptly sealing) with the free responding point of cellulose nitrate.
After this, in the drying at room temperature proving installation, be lower than 8% up to its relative humidity.After the drying, in the middle of proving installation, apply the aqueous solution of 1.0-10.0 μ l, wherein contain useful resisting-IgE and wrap the coloured latex particle (0.2-1.0%) of quilt, also contain the BSA of 0.1-1.0% in this potpourri, 0.01-0.05% polysorbas20 and 0.5-1.5% trehalose.Particle colloidal sols is dried to the centre of proving installation.
When use test, the sample that is applied in the middle of the proving installation from the 10-50 μ l serum of suffering from irritated people or its dilution applies on the site.Sample dissolution the mark in the middle of being placed on.Anti--IgE antibody and irritated specific IgE molecular reaction of sample, and owing to capillary action is diffused in the test channel.
But, will in the reaction channel that contains allergen antibody to be measured, form coloured district band so if contain in the analytic sample at one or more the allergenic specific IgE antibodies in the proving installation district band 3.
By the embodiment 1 described multichannel network that prepared, this proving installation is further prepared in the proving installation shown in Fig. 2, thereby makes the first passage 1 that branches into two parts be formed for the test channel of HIV1 and HIV2 antibody test.The typical polypeptide of virus to be measured or recombinant antigen are placed these passages (0.1-0.5 μ l) by the form with line (Line), and concentration is 0.5-5.0mg/ml.Coloured gauge point 4 places passage 1, is to be produced by for example gold grain by the third polypeptide or recombinant antigen bag with identification HIV1 and HIV2 virus.
The second channel 1 of same proving installation is used to detect the HIV viral antigen, the line or the spot (0.5-5.0mg/ml) of the monoclonal antibody by the anti HIV-1 virus p24-antigen that will for example prepare place this passage, and so-called contrast agents district band placed same not branched bottom 1, wherein, described contrast agents district band comprises the monoclonal antibody at same labelled antibody to be measured.
In the same way, will place four-way 1 by the reagent area band 3 of the special recombinant antigen preparation that is suitable for detecting microspironema pallidum (Treponema pallidum) bacterial antibodies.
In five-way road 1, placed the test macro that detects hepatitis B virus surface antigen, adopted the specific antibody at surface antigen of two kinds of productions, a kind of in test section band 3, another kind of in gauge point 4.
Detecting the required reagent of hepatitis C virus is placed in the 6th passage 1.The typical recombinant antigen of HCV places p-wire 3, and gauge point 4 is by being prepared by gold grain with anti-human IgG bag.
With contain albumin (BSA) (0.1-5.0%), the so-called lock solution of TRITON-X-100, BRI and sucrose handles all passages 1 of proving installation, the sample that joins proving installation by this solution with capacity applies site 5 to carry out, this solution applies site 5 from sample and is diffused into each passage, and fills the reflecting point of cellulose nitrate.Dry proving installation is to quicken drying in vacuum tank.
After this, by using suitable automatic allocation device, the distinctive required above-mentioned mark of every kind of test is joined the predetermined labels point 4 of proving installation.By the described drying of carrying out of the foregoing description.
When analyzing patient samples, serum, blood plasma or the whole blood of 10-50 μ l is applied to the centre of proving installation, the result can read after 1-10 minute reaction time.When the result was positive, the test section was taken the line or the spot of existing color out of, and second kind of coloured district band occurred in the check plot of those passages or branched bottom band (if any).
In order to detect the HIV viral antigen, carried out channelizing by embodiment 1 is described in the proving installation shown in Fig. 3, this proving installation is by adopting following means preparation: the spot of polyclonal antibody (0.5-5.0mg/ml) preparation of the anti-HI V virus p24 antigen that will produce places first passage 1, and so-called contrast agents district band placed same not branched bottom, wherein, described contrast agents district band comprises the polyclonal antibody at labelled antibody to be measured.
In the same way, will place second channel 1 by the reagent area band 3 of the special recombinant antigen preparation that is suitable for detecting the microspironema pallidum bacterial antibodies.
In third channel 1, placed the test macro that detects hepatitis B virus surface antigen, adopted the specific antibody at surface antigen of two kinds of productions, a kind of in test section band 3, another kind of in gauge point 4.
Detecting the required reagent of hepatitis C virus is placed in the four-way 1.The typical recombinant antigen of HCV places p-wire 3, and gauge point 4 is by being prepared by gold grain with anti-human IgG bag.Handle all passages 1 of proving installation with the so-called lock solution that contains casein (0.1-5.0%), hexane sulfonic acid and trehalose (1.0-3.0%), the sample that joins proving installation by this solution with capacity applies site 5 to carry out, this solution applies site 5 from sample and is diffused into each passage, and fills the reaction zone band of cellulose nitrate.Dry proving installation is to quicken drying in vacuum tank.
After this, the distinctive above-mentioned required mark with every kind of test joins predetermined labels point 4.By the described drying of carrying out of the foregoing description.
The fine example of having described the present invention's application is the proving installation that is used for being detected by patient's whole blood, blood plasma or blood serum sample miocardial infarction.
The proving installation of having produced according to Fig. 4 is used for four kinds of different analytes.Proving installation as production as described in the embodiment 1 comprises four identical passages, and each passage further branches into two independent passages.Proving installation just can be used for being measured simultaneously by a duplicate samples existence of patient samples Troponin I, myoglobins, creatine kinase mb isoenzymes (CKMB) and c reactive protein (CRP) like this.
As for the passage 1a and the 1b of proving installation, be that the antibody of the Troponin I of 1.0-5.0mg/ml is applied among the passage 1a with 0.1-0.5 μ l concentration, be that the anti-mouse antibodies of 1.0-5.0mg/ml is applied among the passage 1b with 0.1-0.5 μ l concentration.Passage 1a forms so-called test channel, and passage 1b forms so-called internal functionality contrast passage.
Another reaction pair is by coloured granuloplastic, and this particle is by the antibody sandwich of producing at Troponin I, and by the embodiment 1 described predetermined labels point 4 that is applied in the passage 1.
Except Troponin I, also be prepared in the proving installation in the above described manner at the special test of myoglobins, CKMB and c reactive protein (CRP).
Handle all passages 1 of proving installation with the so-called lock solution that contains polyglycol (PEG), TRITON-X-100 and trehalose (1.0-3.0%), the sample that joins proving installation by this solution with capacity applies site 5 to carry out, this solution applies site 5 from sample and is diffused into each passage, and fills the reaction zone band of cellulose nitrate.Dry proving installation is to quicken drying in vacuum tank.
After this, the characteristic mark of every kind of test is applied to the predetermined labels point 4 of proving installation.By the described drying of carrying out of the foregoing description.
Sample is applied in the middle of the proving installation, and sample is radial from here to be diffused in each identical passage, if contain the analyte corresponding to the miocardial infarction label in the patient samples, will start test and control reaction so.Analysis is undertaken by serum, blood plasma or whole blood sample, and when adopting whole blood, suitable filtering system is removed red blood cell and leucocyte from whole blood sample.
Embodiment 7 is used for the proving installation of miocardial infarction test
As the proving installation of production as described in the embodiment 1, wherein contain mark relevant for the information of Troponin I and the test of myoglobins specificity according to Figure 11.
Specificity test as generation Troponin I as described in the embodiment 6 and myoglobins.As described in embodiment 6, also carry out the sealing of passage and the adding of mark.One of passage is passage in contrast, is correctly stored and reagent has function to guarantee to test.
Embodiment 8 is used for the proving installation of infection in respiratory system test
Below case description the application of the present invention in infection in respiratory system, wherein expect to measure classification specific antibody at bacterium that is sought or viral antigen by patient's serum, blood plasma or whole blood sample.
As the proving installation of production as described in the embodiment 1 according to Fig. 6.Place the point 3 of test channel 1 by the antigen of every kind of analyzable bacterium or virus preparation, concentration is 0.1-0.5mg/ml, and the cumulative volume of every kind of reagent is 0.1-0.5 μ l.The non-reaction zone band of passage 1 is closed, and presses embodiment 1 described dry proving installation.In the gauge point 4 of the take-off point of each test channel, place conjugate respectively by anti--IgG, anti--IgM or anti--IgA antibody is made.By the drying of carrying out mentioned above.
The sample that patient's sample (serum, blood plasma or whole blood) is applied in the middle of the proving installation applies site 5, because capillary action, sample spreads from here and at first transfers to conjugate (Conjugate) point, and in gauge point to be measured 4 with the particulate labeled reactant, further move subsequently to test section band 3, if contain the subclass specific antibody to be measured at analyzable bacterium or virus in the sample, 3 places will react at the test section band.Detect positive findings in mode similar to the above embodiments.
Embodiment 9 is used for the proving installation of cancer diagnosis
Embodiment 9 has described the application of the present invention in the cancer diagnosis proving installation.As the proving installation of production as described in the embodiment 1 according to Fig. 9.This proving installation comprises five practically identical passages.The monoclonal antibody CA125 at cancer markers (cancer antigen 125), PSA (prostate specific antigen), pKAc (pKAc), CEA (carcinomebryonic antigen), the AFP (alpha-fetoprotein) that 0.1-0.5 μ l is produced joins each test point of each passage (from left to right), and concentration is 0.1-5.0mg/ml.After applying antibody, carry out sufficient drying, utilize polyvinyl alcohol (PVA) (PVA), hexane sulfonic acid and sucrose to seal then.Then proving installation is carried out drying, after this promptly can be used for the adding of mark.
Add the specific marker at the preparation of corresponding cancer markers in the mark zone of each test channel section start band 4 respectively, in this specific marker, it is described press the foregoing description, with production at the second antibody bag of label to be measured by gold grain.After adding, dry proving installation, and it is packaged in the protectiveness plastic casing.
The patient samples (serum, blood plasma, whole blood etc.) of 10 μ l is applied to sample applies site 5, sample moves in the passage 1 corresponding to each cancer markers from here, from gauge point 4 dissolving marks, and further to test section band 3.If contain analyzable cancer markers in the sample, in passage to be measured visible test result will appear.
Embodiment 10 is used for the proving installation that viral antigen detects
Embodiment 10 has described the application of the present invention at the proving installation that is used for the viral antigen detection.
In order to detect Rota virus, by inserting spot in the test section band 3 in first passage 1, produce proving installation shown in Figure 10, this proving installation has carried out channelizing by embodiment 1 is described, described spot is made by the specific antibody of producing at viral antigen, and this antibody is the antibody at the used labelled antibody of gauge point 4.
The third channel 1 of same proving installation is used to detect adenovirus antigen, and by putting into line or spot carries out in the test section of this passage band 3, this line or spot comprise at the specific antibody that is placed on the labelled antibody to be measured in the mark zone band 4.
The second channel of this proving installation is with comparing passage, to test the functional of this proving installation.In gauge point, add non-specific conjugate, and in the band of test section, add anti-mouse antibodies.
It is described to press embodiment 1, the reflecting point of closed channel 1 and dry proving installation.The characteristic mark that in the gauge point 4 of the take-off point of each test channel, adds every kind of detection.By the drying of carrying out mentioned above.
The sample that patient's sample (fecal specimens of dilution) is applied to proving installation applies site 5, because capillary action, sample spreads from here and at first moves in the coupling object point, and in gauge point to be measured 4 with the particulate labeled reactant, continue subsequently to move to test section band 3, if contain analyzable viral antigen in the sample, 3 places will react at the test section band.Detect positive findings in mode similar to the above embodiments.
Claims (12)
1. proving installation based on immunodiffusion, it comprises porous carrier materials, and wherein the form with district's band or spot has applied specific bond reagent (immunoreagent); The detectable mark that can be moved by sample, itself and the coupling and being applied to separately or in advance in the described porous carrier mutually of second kind of specific bond reagent; And the optional sample that provides filtrator applies the site, it is characterized in that, it comprises the channel network in the described porous carrier materials, this channel network is by forming with laser treatment etching porous carrier materials, this channel network comprises two or more passages (1), separate by the zone of handling (2), be fixed in one or more specific bond reagent (3) wherein, place the contiguous sample of described passage to apply site (5) or be positioned at sample to apply optional gauge point (4) in the site (5) itself, the mode that sample applies site (5) placement makes described sample can be evenly distributed in each passage.
2. the proving installation of claim 1 is characterized in that, it can be based on while or parallel diagnosing one or more identifications that produce syndromic reagent or analyte of carrying out.
3. the proving installation of claim 1 is characterized in that, it is used for discerning simultaneously the reagent that produces allergy more than a kind of, the miocardial infarction label, the reagent of generation venereal disease, screening of blood analyte, produce the reagent of infection in respiratory system, other produces the reagent and/or the cancer markers of infectious diseases.
4. the proving installation of claim 1 is characterized in that, described porous carrier is a cellulose nitrate.
5. the proving installation of claim 1 is characterized in that, described specific bond reagent is antibody, antibody fragment, recombinant antibodies, recombinant antibody fragment, antigen, agglutinin, acceptor and/or part.
6. the proving installation of claim 1 is characterized in that, the described reagent that is labeled is latex, gold, metal or coloring agent particulate or fluorescent material.
7. method of producing the proving installation of claim 1, wherein said specific recognition immunoreagent is fixed in the porosint, with making this porosint become this porosint of mass treatment of inertia, and wherein sample applies optional filtrator and the detectable mark of providing in site, be combined with specific bond reagent on this detectable mark, it is characterized in that
(a) by on described porosint, forming channel network with the laser-induced thermal etching porosint, the zone (2) that this network comprises two or more passages (1) and handled with anticipated shape and size;
(b) one or more identification specific bond reagent are fixed in each passage (1) as district's band or spot (3).
8. the method for claim 7, it is characterized in that, the material that makes described porosint become inertia is a potpourri, it comprises natural or synthetic polymer, for example albumin and casein or PEG (polyglycol) and PVA (polyvinyl alcohol (PVA)), the nonionic scaling agent is hexane sulfonic acid and TRITON-X-100, BRIJ and antiseptic for example, for example sugar, as sucrose and trehalose, or their derivant.
9. the method for claim 7 is characterized in that, after applying described reagent, described proving installation is dried to relative humidity and is no more than 8% and sealed packing.
10. the proving installation of claim 1 at the same time or parallelly be used for diagnosing the disease sought and/or the purposes of syndromic a plurality of detections, it is characterized in that, what described sample was applied to this proving installation applies site (5), but its contain the mark that can be moved by analytic sample and with second kind of specific bond reagent of mark coupling, described sample and reagent evenly move to this passage by the channel network of laser treatment formation from this site, they and described specific bond reagent are located reaction or are not reacted at its point of fixity (3) herein, and the positive or negative result can directly read from here.
11. the proving installation of claim 1 at the same time or parallelly be used for discerning the disease sought and/or the purposes of syndromic a plurality of detections, it is characterized in that, what described sample was applied to this proving installation applies site (5), described sample moves to the passage from this site, it is with the mark and the second kind of specific bond reagent mix that combines with mark that can be moved by described sample and react herein, after this, described sample and reagent evenly move in this passage by the channel network of laser treatment formation, they and described specific bond reagent are located reaction or are not reacted at its point of fixity (3) herein, and the positive or negative result can directly read from here.
12. the proving installation of claim 1 at the same time or parallelly be used for discerning the disease sought and/or the purposes of syndromic a plurality of detections, it is characterized in that, described sample and independent mark and with second kind of specific bond reagent mix of mark coupling, and what this potpourri was applied to this proving installation applies site (5), described sample and reagent evenly move to this passage by the channel network of laser treatment formation from this site, they and described specific bond reagent are located reaction or are not reacted at its point of fixity (3) herein, and the positive or negative result can directly read from here.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI20030463 | 2003-03-28 | ||
| FI20030463A FI20030463A0 (en) | 2003-03-28 | 2003-03-28 | Multichannel test instrument, method of its manufacture and its use |
| FI20030921 | 2003-06-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1768267A true CN1768267A (en) | 2006-05-03 |
| CN100451652C CN100451652C (en) | 2009-01-14 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB2004800086721A Expired - Fee Related CN100451652C (en) | 2003-03-28 | 2004-03-29 | Multiple-channel test device, method for producing the same and use thereof |
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| CN (1) | CN100451652C (en) |
| FI (1) | FI20030463A0 (en) |
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| US5569608A (en) * | 1995-01-30 | 1996-10-29 | Bayer Corporation | Quantitative detection of analytes on immunochromatographic strips |
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| Publication number | Publication date |
|---|---|
| CN100451652C (en) | 2009-01-14 |
| FI20030463A0 (en) | 2003-03-28 |
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