CN1786712A - Method and system for quantitatively fast level testing flow - Google Patents
Method and system for quantitatively fast level testing flow Download PDFInfo
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- CN1786712A CN1786712A CN 200410077363 CN200410077363A CN1786712A CN 1786712 A CN1786712 A CN 1786712A CN 200410077363 CN200410077363 CN 200410077363 CN 200410077363 A CN200410077363 A CN 200410077363A CN 1786712 A CN1786712 A CN 1786712A
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Abstract
The invention relates to quantitative fast horizontal side flow method and system. It includes the following steps: practicing each detecting item for liquid state biological specimen on biochemical reaction platform; using filtration to separate cell from liquid state biological specimen to reach the need liquid volume; the biochemical reaction platform is made up test strip and testing cassete shell; the biological specimen is added at any one of the application of sample port of the shell; forming effective capillary chromatography differential pressure to make the filtered liquid and the dissolved measured matter do orientation flow on the platform; judging detecting result according to optical density. Thus the invention can do quantitative fast detection for whole blood or other cell biology, and realize relative volume non dependence of detecting result to ensure its accuracy.
Description
Technical field
A kind of qualitative and quantitative quick horizontal effluent method and system have been the present invention relates to, be applicable to the material to be checked of analyzing (biological sample that especially contains whole blood or red blood cell or leucocyte or other cell) in the biological sample, to obtain qualitative or quantitative analysis result.
Background technology
Quantitatively, fast, Ling Min diagnostic method is current " diagnosis in time before the bed " (Point of care test, developing direction POCT) of popularizing day by day.And real " fast " requires sample to be checked is left intact and directly detects.Yet, contain cell in the biological samples more, contain the cell of a large amount of red blood cells, leucocyte and other type such as blood, go to toward requiring the separation step consuming time poly-, to remove the cell in the sample to be checked implementing to detect.And, because the difference of hematid specific volume between the individuality, make the whole blood of equal volume can obtain the plasma volume of different volumes because of different hematid specific volumes, promptly may make the result because of application of sample amount difference difference to some extent.On the other hand, come out, popularize gradually, be widely used in the early diagnosis of early pregnancy, drug abuse, infectious disease based on the tachysynthesis detection method (quick detection test paper bar) of capillary chromatography principle (horizontal effluent) from the eighties.These detect the biological sample that test paper is used for having removed cell more, only do qualitative detection.
Require any horizontal side streaming system and method really can be applied to fast quantification and detect, must meet the following conditions:
1, can automatically separate cell in the sample to be checked;
2, (5～60 minutes) are implemented in the liquid biochemical reaction that just can finish at short notice;
3, testing result satisfies the not celliferous sample of specific usefulness (as blood plasma and serum) same sensitivity and specificity requirement;
4, detection method relatively is not subjected to the influence of hematid specific volume in the sample, i.e. relative volume dependent/non-dependent.
5, can scale production.
Existing many trials set up the quick horizontal lateral flow methods that can be used for whole blood test (for example U.S. Pat 6,136,610; U.S. Pat 6,528,323, WO03/3008933, U.S. Pat 5,766,552 etc.), but all fail because of not satisfying above condition simultaneously.They or can not intercept cell effectively, red blood cell is spilt or haemolysis, cause that background (background) is too high can not to satisfy the sensitivity/specificity requirement; Perhaps the filtration efficiency because of liquid sample is low excessively, makes its volume very few and be not enough to finish required biochemical reaction; Perhaps cross slowly and can not finish fast detecting at short notice because of filter velocity; Perhaps because of the strict volume dependence of its detection method itself, and can not overcome the difficulty of hematid specific volume difference in the individual whole blood sample; Perhaps its principle of work of discussion/elaboration not is under suspicion its result's the repeatability and the scale productivity of structure thereof.
Summary of the invention
At above-mentioned defective, fundamental purpose of the present invention is to provide a kind of whole blood that can be used for, or other contain the cell biological sample quantitatively/quick qualitatively horizontal lateral flow methods.
Another object of the present invention is to provide a kind of whole blood that can be used for, or other contains the quantitative quick horizontal side streaming system of cell biological sample.
To achieve these goals, the invention provides a kind of quantitative quick horizontal lateral flow methods, platform with biochemical reaction, enforcement is made various projects to be checked to being not limited to human liquid biological sample to be checked, intercept cell contained in the liquid sample with filtration, obtain finishing the required liquid volume of its biochemical reaction; And by this platform structure, form effective capillary chromatography pressure reduction, make filtered liquid and be dissolved in wherein material to be checked on this platform through capillary chromatography effect directed flow; And according to material to be checked in the biochemical reaction and contrast material produced can be detected optical density judge testing result.
The platform of the biochemical reaction of the inventive method is made up of test-strips and testing cassete shell, and the structure of test-strips has two ends and to guarantee effective cell filtration, capillary chromatography and biochemical reaction; Guarantee application of sample result's the observation and the fixing of test-strips physical arrangement of liquid biological sample with the testing cassete shell; Its biological sample can be added in an one or both ends of application of sample mouth in two ends on the testing cassete shell.
The cell filtration structure of the test-strips of the inventive method is made up of the filtering membrane in the different apertures of single or multiple lift, and the filtering membrane aperture of cells contacting is less than cell dia to be intercepted in its direct and sample.
The cell filtration structure of the test-strips of the inventive method has been used double faced adhesive tape, impels filtered solution to be advantage towards preset direction and flows.
The cell filtration structure of the test-strips of the inventive method and capillary chromatography pressure reduction require to be consistent, to guarantee the flow rate of liquid in effective liquid filtration and the chromatography process.
The cell filtration structure of the inventive method, require consistent with capillary chromatography pressure reduction, by membrane material with different apertures, capillary attraction, set up cell filtration---chromatograph test strip, make celliferous liquid state biological sample to be checked VOLUME LOSS in filtration minimum, and after by physical filtering, liquid is being collected initial point place capillary chromatography pressure reduction less than biochemical reaction zone, thereby makes the liquid after the filtration be subjected to capillary attraction to drive directed moving on the test chromatography strip.
The cell filtration structure of the inventive method, the available pretreated filter membrane of chemical reagent that makes cell aggregation to be filtered makes thing to be filtered increase because of the gathering volume, and improves the barriering effect of filtering membrane pair cell.
The inventive method makes the chemical reagent of cell aggregation to be filtered adopt anti erythrocyte antibody or vegetable protein agglutinin, and anti erythrocyte antibody can be monoclonal antibody or polyclonal antibody at the antigenic determinant that the erythrocyte membrane surface exposes.
The inventive method is to comprise with surfactant Tween-20 handling film to increase the absorption of antibody to filtering membrane with the pretreated filter membrane of anti erythrocyte antibody.
The capillary chromatography pressure reduction of the test-strips of the inventive method comes from the material and the package assembly thereof of assembling test bar; According to the biochemistry detecting item requirement, but celliferous liquid biological sample application of sample is in the one or both ends of appointing of testing cassete; The different sample loading mode that add have different cell filtration-chromatographies-biochemical reaction structure.
The to be checked liquid biological sample of the inventive method is celliferous whole blood, but is not limited to whole blood sample; Celliferous liquid biological sample to be checked can contain anti-coagulants or not contain anti-coagulants, and it is required that the liquid volume that obtains behind the filtration through the reaction platform satisfies biochemical reaction.
The biochemical reaction process of the inventive method has utilized the background in the two-way horizontal effluent reduction biochemical reaction; And the relative optical density value RI analyzing and testing result of the ratio of the optical density value that is produced with thing to be checked in the biochemical reaction and tester, and improve the sensitivity that detects, it is poor to reduce between box, makes testing result be non-volume dependence relatively.
The biochemical reaction of the inventive method utilizes the material of the optical density that colloid gold particle or silver-colored particle or fluorescent dye or other generation can detect by the light reflection principle thing that serves as a mark, and makes the ratio of the optical density that the result of biochemical reaction can produce by thing to be checked and tester determined.
The biochemical reaction of the inventive method is based on immunology principle, but be not limited to the detection of the big intermolecular marriage relation of immunology principle, be used for detecting the mankind, but be not limited to the big molecule in people's fluid-like state sample, as antigen, antibody, polysaccharide, nucleic acid, polypeptide, pathogenic microorganism etc.
The present invention also provides a kind of quantitatively/qualitatively quick horizontal side streaming system, platform with biochemical reaction, enforcement is made various projects to be checked to being not limited to human liquid biological sample to be checked, the platform of biochemical reaction is made up of test-strips and testing cassete shell, and the structure of test-strips has two ends and to guarantee effective cell filtration, capillary chromatography and biochemical reaction; Biological sample can be added in an one or both ends of application of sample mouth in two ends on the shell.
Cell filtration-the chromatography of system of the present invention-biochemical reaction structure, partly or entirely overlapping according to the order of sequence between each group; Its lap is 0.1～20 millimeter.
Cell filtration-the chromatography of system of the present invention-biochemical reaction structure, cell filtration film are hydrophobic glass fibre (but not limitting the what glass fibre) or hydrophilic composite fibre.
The liquid biological sample of system of the present invention liquid collection membrane after filtering is hydrophobic glass fiber (but not limitting the what glass fibre) or hydrophilic composite fibre, and has the aperture that is not less than the cell filtration film.
The liquid biological sample of the system of the present invention liquid collection membrane aperture after filtering and ratio＞1 in filtering membrane aperture.
Label that the biochemical reaction of system of the present invention is required such as collaurum by also dry on hydrophobic glass fibre (but not limitting the what glass fibre), are positioned at an end of chromatography strip by pre-bag; Collaurum is lower than the reaction zone of biochemical reaction at the chromatography pressure reduction of present position on the chromatography strip when discharging in that collaurum is dissolved, so collaurum is can coating antigen point mobile to the biochemical reaction zone orientation.
The biochemical reaction zone of system of the present invention is made up of hydrophilic NC Nitroncellulose film (but not limitting what NC Nitroncellulose film), and have in first time of two-way horizontal flow measurement during liquid flow, the highest capillary attraction in this structure makes liquid move to its reaction zone is directed from sample application zone or liquid collection region.
The other end of the chromatography strip of system of the present invention, the position that corresponds to chemical coupling thing pad is equipped with hydrophilic suction pad, its effect is soaked at the NC Nitroncellulose film, after the water-intake capacity decline collaurum dissolving, provide with its height water-intake capacity and to impel liquid to move required capillary chromatography pressure reduction to suction pad end is directed from the collaurum end.
When celliferous liquid biological sample application of sample during at suction pad one end, its cell filtration film directly contacts with the NC Nitroncellulose film but does not contact with the pad that absorbs water; When celliferous liquid biological sample application of sample during at collaurum one end, its cell filtration film contacts with whole with liquid collection membrane part, but does not directly contact with chemical coupling thing pad; When containing the liquid biological sample of cell application of sample be in two ends application of sample mouth at the same time or separately, the combination of its cell filtration structure during for application of sample respectively.
The liquid biological sample of system of the present invention liquid collection membrane after filtering makes with a kind of test strips structure and both can be used for immune indirect method detection, can be used for immune sandwich method again and detects.
The quantitative quick horizontal effluent method and system of the present invention compared with prior art, intercept the cell that contained in the liquid sample by the filtration on the biochemical reaction platform to obtain required volume, and form effective capillary chromatography pressure reduction, make test substance through capillary chromatography effect directed flow; And according to material to be checked and contrast material produced can be detected optical density judge testing result, thereby the present invention can be used for whole blood, or other contain the cell biological sample and carry out quantitatively/qualitative, detect fast, and realize the relative volume dependent/non-dependent of testing result, guaranteed the accuracy of testing result.
Brief Description Of Drawings
Below in conjunction with accompanying drawing,, will make technical scheme of the present invention and other beneficial effect apparent by detailed description to preferred embodiment of the present invention.
In the accompanying drawing,
Fig. 1 is the vertical view of testing cassete loam cake of the present invention.
Fig. 2 is the stereographic map of testing cassete lower cover of the present invention.
Fig. 3 is test-strips of the present invention and testing cassete principle of work vertical view.
Fig. 4 is a two-way horizontal lateral flow test strip fundamental diagram of the present invention.
The side view of test-strips structure when Fig. 5 is the port one application of sample of testing cassete of the present invention.
Test-strips structure side view when Fig. 6 is port 2 application of samples of testing cassete of the present invention.
Test-strips structure side view when Fig. 7 is the port one of testing cassete of the present invention and port 2 two ends application of samples.
The capillary pressuring difference of test-strips when Fig. 8 is port 2 application of samples of testing cassete of the present invention.
Fig. 9 is a filtration design drawing of the present invention (containing double faced adhesive tape).
Figure 10 is filtration design drawing of the present invention (not containing double faced adhesive tape).
Embodiment
Hereinafter, will describe the present invention in detail.
The present invention is achieved through the following technical solutions the separation of the red blood cell in the sample to be checked (or other cell): the testing cassete that adopts the horizontal effluent of special construction is as biochemical platform, implements to make various projects to be checked to being not limited to human liquid biological sample to be checked (for example being not limited to macromolecular antigen, antibody, polysaccharide, nucleic acid, polypeptide, pathogenic microorganism etc. in people's fluid-like state sample).This testing cassete is made up of test-strips and shell, extremely shown in Figure 2 as Fig. 1, the testing cassete shell is made up of loam cake and lower cover, the observation window 3 that loam cake provides sample/damping fluid application of sample port one and port 2 and is used to observe testing result, wherein port one is used as the application of sample port of sample in two-way immunochromatography detects, and port 2 is used as the application of sample port of sample or damping fluid in two-way immunochromatography detects; Lower cover provides the carrying platform of test-strips, to guarantee the fixing and capillary chromatography process of test-strips physical arrangement.The upper and lower cover of testing cassete and the design of inner structure can be by realizing plastic mould design, and are used for large-scale production.
Test-strips of the present invention provides cell filtration, capillary chromatography and detects the platform of required biochemical reaction.Shown in Fig. 3～4, the port one place of this test-strips is provided with suction pad 16; Port 2 places of test-strips are provided with chemical coupling thing pad 12, and this chemical coupling thing pad 12 contains the antibody or the antigen of collaurum coupling, also can adopt the material of the optical density that silver-colored particle or fluorescent dye or other generation can detect by the light reflection principle thing that serves as a mark; The place is provided with two contrasts and is with 4, two contrasts to be with to be provided with in the middle of 4 calibration tapes 5 in the middle of the test-strips, contrast with 4 and the position of calibration tape 5 corresponding to the view window 3 of testing cassete shell, observe to make things convenient for the user.
Different requirements according to project to be checked, celliferous sample (as whole blood) can be added in application of sample port one or port 2, or be added in port one and port 2 two ends according to the order of sequence, particular filter structure by test-strips, shown in Fig. 5～7, cell (as red blood cell) is blocked in outside the biochemical reaction zone greater than pore size filter because of volume, utilization has the film (asking for an interview reference 1 and 3) of special pore size filter and can make the chemical substance (asking for an interview reference 2) of cell aggregation (as anti erythrocyte antibody), cell to be filtered is intercepted effectively at the application of sample initial point, and the material less than this aperture in the sample (as blood plasma or serum and be dissolved in wherein material to be checked) can effectively be filtered.
(5～60 minutes), the biochemical reaction finished at liquid state are achieved through the following technical solutions to fast in the present invention: biochemical reaction carries out on test-strips.According to the requirement of test item, sample can be added in an end or the port one and port 2 two ends of port one or port 2.The material of test-strips and ad hoc structure provide effective capillary chromatography required pressure reduction, make liquid sample can be on test-strips directed flow.In native system:
After the sample that contains cell (as whole blood) adds port one, because of making cell, filtration stays the application of sample initial point, and only liquid filters.At this moment, as shown in Figure 5, the bottom of test-strips is support pad 14 (5 * 60 millimeters), and it is a kind of plastic spacer, in order to the structure between each level of physical support test-strips; Support pad 14 is provided with the NC Nitroncellulose film 13 (5 * 48 millimeters) as the reaction platform, it is a water wettability, be coated with contrast agents (i.e. contrast be with 4) on it, and with liquid biological sample in the reagent (being calibration tape 5) of substance reaction to be checked, constitute the biochemical reaction zone of test-strips; NC Nitroncellulose film 13 is provided with chemical coupling thing pad 12 (5 * 13 millimeters) at port 2 places, it is the hydrophobic glass fiber, and the collaurum bag is by on it, and with NC Nitroncellulose film 13 overlapping 3mm; Chemical coupling thing pad 12 is provided with buffering fluid cushion 11 (5 * 12 millimeters), and it is the water wettability composite fibre, adds the sample or the damping fluid of inbound port 2 in order to absorption; NC Nitroncellulose film 13 is provided with suction pad 16 at the port one place, it is the water wettability composite fibre, is used for making up the two-way horizontal effluent required capillary chromatography pressure reduction of effluent for the second time, and absorbs excess liquid; Be pasted with filtering membrane 15 (5 * 7.5 millimeters) near suction pad 16 places with double faced adhesive tape 17 on the NC Nitroncellulose film 13, also can form filtration by the filtering membrane in the different apertures of single or multiple lift, filtering membrane 15 is made by water wettability composite fibre or water wettability composite fibre, replace sample pad, through 0.25 mg/ml rabbit anti-human erythrocyte antibody or 0.5 mg/ml mouse-anti human red blood cell antibody pre-service, be used for intercepting application of sample in the red blood cell of the whole blood sample of port one and filter plasma; Double faced adhesive tape 17 (5 * 5 millimeters) does not have permeability, can impel the plasma sample of filtration to flow to port 2 in whole blood test.Wherein, to press preamble section/all overlapping mode combinations, its lap is 0.1～20 millimeter between each structure, and can craft or the process of mechanization realize.
Because the capillary chromatography pressure reduction of port 2 is set to be higher than port one, and filtering membrane 15 and the NC Nitroncellulose film 13 that contacts with it as double faced adhesive tape 17 partial destruction between the NC Nitroncellulose film 13 of reaction platform, because of double faced adhesive tape 17 does not have permeability, when pasting NC Nitroncellulose film 13, the contact site capillary portion is destroyed with it to make NC Nitroncellulose film 13, thereby make the siphon funtion part forfeiture of contact portion, cause from NC Nitroncellulose film 13 and double faced adhesive tape 17 contacts site, the siphon power of port 2 ends of NC Nitroncellulose film 13 impels sample faster greater than the siphon power of suction pad 16 ends of NC Nitroncellulose film 13, move to port 2 ends more.Simultaneously, the right-hand member of double faced adhesive tape 17 (by suction pad 16 ends) is concordant with the right-hand member (by suction pad 16 ends) of filtering membrane 15, and the sample of having blocked filtering membrane 15 filtrations like this directly flows to suction pad 16 ends, also impels the filtration sample to move to port 2 ends.As shown in Figure 4, liquid sample flows to port 2 from port one; By way of the antigen or the antibody that can produce biochemical reaction that are coated in advance on the calibration tape 5, form immune complex (horizontal effluent A for the first time) with material to be checked.The port 2 of described test-strips is coated with the collaurum coupling, can with material to be checked, or the immune complex that has formed on calibration tape 5 produces the antigen or the antibody of biochemical reaction; And coupling, can with the antigen or the antibody of the tester qualitative response of observation window 3 districts pre-bag quilt.This moment, test-strips was soaked by the liquid of horizontal effluent A for the first time, the suction pad 16 that is placed in port one makes the capillary chromatography pressure reduction of port one be higher than port 2, when liquid sample or damping fluid add inbound port 2, soluble chemistry conjugates 12, flow (horizontal effluent B for the second time) to port one from port 2 by capillary pressuring difference, by way of view window 3; Can produce biochemical reaction and be deposited on the reaction initial point with the contrast material and/or the established immune complex of pre-bag quilt in the port 2 chemical coupling things 12 that dissolved.Contain collaurum in the sediment that for the second time horizontal effluent B mesophytization reaction produces, the redness of collaurum is being deposited into visible band of formation or zone; The suction pad 16 of the port one of test-strips absorbs excess liquid in for the second time horizontal effluent B reaction.Above process is called the two-way horizontal effluent.
As shown in Figure 6, when the sample that contains cell (as whole blood) when adding inbound port 2, stay the application of sample initial point because of filtration makes cell, only liquid and the material to be checked that is dissolved in liquid can filter.Different with Fig. 5 is that chemical coupling thing pad 12 is provided with the liquid collecting pad 18 (5 * 10 millimeters) with filtering function, also is provided with filtering membrane 15 on this liquid collecting pad 18.This liquid collecting pad 18 is the hydrophobic glass fiber, but do not wrap by the reagent of colloid gold label, through 0.25 mg/ml rabbit anti-human erythrocyte antibody or 0.5 mg/ml mouse-anti human red blood cell antibody pre-service, be used to collect the plasma of application of sample after the process filter membrane 15 of port 2 filters, and this liquid collecting pad 18 overlaps 3 millimeters with chemical coupling thing pad 12.Can before application of sample, add port one, to soak test-strips with damping fluid.As previously mentioned, port 2 contains bag quilt and dry chemical coupling thing 12 in advance, needs the liquid of q.s volume that it is dissolved.For reducing the loss of sample volume, select the hydrophobic film material to filter (data 3 sees reference).Because kapillary work chromatography requires certain pressure reduction, concentrate the kapillary work chromatography pressure reduction between the NC Nitroncellulose film 13 at chemical reaction place on place's chemical coupling thing pad 12 and the calibration tape 5 so need to guarantee filtration back liquid: chemical coupling thing pad＞NC Nitroncellulose film, liquid is flowed to port one from port 2, and the material (data 3 sees reference) of therefore selecting for use capillary chromatography ability (Capillary Rise) to be lower than NC Nitroncellulose film 13 constitutes port 2 filtrations.As previously mentioned, test-strips was cushioned liquid and soaked this moment, the suction pad 16 of port one makes the capillary chromatography pressure reduction of port one be higher than NC Nitroncellulose film 13, thereby kapillary work chromatography pressure reduction on the formation test-strips: the structure of liquid collecting pad＞chemical coupling thing pad＞NC Nitroncellulose film, as shown in Figure 8.Require to finish at short notice owing to detecting again, so need the flow rate of efficient liquid ground from port 2 → port one, so select for use the aperture after filtration, to filter (data 3 sees reference) from chemical coupling thing pad 12 to NC Nitroncellulose film 13 directions to guarantee liquid effectively as the liquid collecting pad greater than the material of cell filtration film, therefore, the membrane aperture of liquid collecting pad requires＞1 with the ratio in filtering membrane aperture.
As shown in Figure 7, when celliferous sample (as whole blood) simultaneously or when adding port one and port 2 in succession, what they were different with Fig. 5 is that chemical coupling thing pad 12 is provided with the liquid collecting pad 18 with filtering function, and also is provided with filtering membrane 15 on the liquid collecting pad 18.This structure satisfies the condition when containing cell sample separation adding port one and port 2 simultaneously: effective filtration cell; Minimum liquid volume loss; Effective capillary chromatography pressure reduction; Effective flow rate of liquid.
The present invention is to guaranteeing that the particular detection project obtains realizing by following scheme with same sensitivity and the specific requirement of not celliferous sample (as serum or blood plasma): sensitivity refers to the positive minimum threshold value that can be examined and determine of certain particular detection project, and the possibility of (false negative) appears failing to pinpoint a disease in diagnosis in representative; Specificity refers to that certain particular detection project judges by the percent high threshold value of default feminine gender, the probability of mistaken diagnosis (false positive) occurs.Therefore, reliable detection is to guarantee to avoid occurring mistaken diagnosis (false positive) under minimum rate of missed diagnosis (false negative) condition, determines that promptly positive minimum threshold value is crucial.Determining except detection technique itself of positive lowest threshold, maximum influence factor is the background (background) in the detection system; On the horizontal lateral flow test strip of native system capillary chromatography, then be that sample is painted in the background that detection zone forms.In native system, adopted the two-way horizontal lateral flow methods, when making sample be added in port one, the background that the NC Nitroncellulose of flowing through film 13 districts form is painted (for example: add the damping fluid washing of inbound port 2 in the time of the redness that red blood cell spills or haemolysis causes) can be by horizontal effluent for the second time and reduce background (seeing accompanying drawing 4).When containing cell sample (as whole blood) application of sample at port 2, or simultaneously or when being added in port one and port 2 two ends in succession (seeing accompanying drawing 6,7), owing to lacked the damping fluid washing step, need to strengthen cell filtration efficient, to guarantee that red blood cell (or other cell) is not by filtration and NC Nitroncellulose film 13 districts that flow through.That is: when containing cell sample (as whole blood) application of sample, need 1. double medium filtration film at port 2 or port one and port 2 two ends; 2. with the short pretreated filter membrane of the cell aggregation factor (as anti erythrocyte antibody or vegetable protein agglutinin), thing to be filtered (as red blood cell) volume is increased to improve filtration efficiency.The membrane structure protein combination of anti erythrocyte antibody (antigenic determinant that exposes at erythrocyte membrane surface can be monoclonal antibody or polyclonal antibody) and erythrocyte surface forms immune complex, makes erythrocyte aggregation and filtration efficiency is improved.Because the surface hydrophobicity character of filtering membrane 15, make anti erythrocyte antibody be difficult to be adsorbed on the filtering membrane 15, so need employed hydrophobicity filtering membrane 15 through surfactant (as Tween-20) pre-service, anti erythrocyte antibody makes it to have the part water wettability, so that can be adsorbed on filtering membrane 15 surfaces (data 2 sees reference).
As previously mentioned, the difference of hematid specific volume will cause the plasma sample that obtains different volumes with the whole blood of volume, make that difference appears in added sample volume in the detection.For guaranteeing the accuracy of testing result, must guarantee the relative volume dependent/non-dependent of testing result.The present invention realizes by following scheme the relative volume dependent/non-dependent of testing result: it is 1. to soak into test-strips in order to meet the following conditions to form liquid biochemical reaction condition that the lowest volume of detection requires; 2. dissolve port 2 pre-bags by also dry chemical coupling thing.Promptly require to soak under the liquid volume condition of film and soluble chemistry conjugates satisfied, in certain sample volume variation range, its testing result is answered no significant difference.In native system,, determine minimum sample (serum or the blood plasma) consumption of a certain particular detection project earlier with not celliferous sample (as blood plasma or serum); Guaranteeing under effective cell filtration and flow rate of liquid (as mentioned above) prerequisite, can to be checked contain cell sample (as whole blood) but liquid being filtered (as blood plasma or serum) volume by calculating, judge the minimum amount that contains cell sample, be not less than with the sample volume that guarantees filtration back participation biochemical reaction and detect required minimum flow.On the other hand, because native system adopts band to be checked and contrast to be with ratio (the relative optical density value of optical density, RI) judge testing result (US patent 6,136,610), promptly the error of testing process mesophytization condition (as sample volume) will be trend on year-on-year basis to the influence of band to be checked and contrast band absolute light density value (Dr), but not influence the result of RI, thereby reduced the dependence that detects sample volume, made the content of cell in the sample not cause tangible influence testing result.
Example:
Promptly changing, when causing absolute light density value error, still can obtain the judged result of RI accurately according to trend principle on year-on-year basis because of sample volume.
Embodiment 1:
The screening of whole blood sample port one filtering membrane and filtration ( subordinate list 1,2,3,4).
Material:
1, filtering membrane: Ahlstron Filtration (U.S.)
Grade 111: glass fibre
Grade 141 and Grade 142: glass fibre
Grade 1660, and Grade 1661, Grade 1662 and Grade 1663
2, rabbit anti-human erythrocyte antibody (Chinese Rui Taitesi)
3, use the pretreated filtering membrane of anti-human erythrocyte antibody (with reference to program)
4, HIV (-) whole blood and plasma sample
5, HIV (+) whole blood and plasma sample
6, HIV testing cassete
Press accompanying drawing 5 structures assembling HIV antibody test paper box.
Relatively:
1, the effect of separating red corpuscle behind the different filtering membranes of usefulness anti-human erythrocyte antibody treatment.
2, the migration velocity of the blood plasma that leaches on the NC film.
3, whole blood and plasma sample detect the difference of the S/CO of HIV (+).
Result: see attached list 1,2,3 and 4.
Conclusion:
1, only is not enough to effectively filter red blood cell, needs to add the pretreated filter membrane of anti-human erythrocyte antibody with filtering membrane.
2, port one is used different types of filtering membrane of crossing through the anti-human erythrocyte antibody treatment, all can well separating red corpuscle.
3, synthetic fiber (Cytosep) 1663 films can not effectively filter serum because of aperture too small (2 microns) soak speed low excessively (35 ml/min).
4, detect by the HIV paper box, add the whole blood and the plasma sample of sample port 1, and its testing result (signal to noise ratio (S/N ratio), S/CO) identical.
Subordinate list 1 filters erythrocytic effect without anti-human erythrocyte antibody treatment whole blood filtering membrane
| The whole blood filtering membrane | Sample | Application of sample amount (microlitre) | Have or not filtration | Migration velocity (ml/min) | Background | The film time on the red blood cell (minute) |
| Grade 111 | Whole blood | 100 | Have | ≤16 | Scarlet | 8 |
| 100 | Have | ≤16 | Scarlet | 8 | ||
| 100 | Have | ≤16 | Scarlet | 8 | ||
| 100 | Have | ≤16 | Scarlet | 8 | ||
| 100 | Have | ≤16 | Scarlet | 8 | ||
| Grade 141 | Whole blood | 100 | Have | ≤16 | Scarlet | 3 |
| 100 | Have | ≤16 | Scarlet | 3 | ||
| 100 | Have | ≤16 | Scarlet | 3 | ||
| 100 | Have | ≤16 | Scarlet | 3 | ||
| 100 | Have | ≤16 | Scarlet | 3 | ||
| Grade 142 | Whole blood | 100 | Have | ≤16 | Scarlet | 2 |
| 100 | Have | ≤16 | Scarlet | 2 | ||
| 100 | Have | ≤16 | Scarlet | 2 | ||
| 100 | Have | ≤16 | Scarlet | 2 | ||
| 100 | Have | ≤16 | Scarlet | 2 | ||
| Grade 1660 | Whole blood | 100 | Have | ≤16 | Scarlet | 2 |
| 100 | Have | ≤16 | Scarlet | 2 | ||
| 100 | Have | ≤16 | Scarlet | 2 | ||
| 100 | Have | ≤16 | Scarlet | 2 | ||
| 100 | Have | ≤16 | Scarlet | 2 | ||
| Grade 1661 | Whole blood | 100 | Have | ≤16 | Scarlet | 3 |
| 100 | Have | ≤16 | Scarlet | 3 | ||
| 100 | Have | ≤16 | Scarlet | 3 | ||
| 100 | Have | ≤16 | Scarlet | 3 | ||
| 100 | Have | ≤16 | Scarlet | 3 | ||
| Grade 1662 | Whole blood | 100 | Have | ≤16 | Scarlet | 4 |
| 100 | Have | ≤16 | Scarlet | 4 | ||
| 100 | Have | ≤16 | Scarlet | 4 | ||
| 100 | Have | ≤16 | Scarlet | 4 | ||
| 100 | Have | ≤16 | Scarlet | 4 | ||
| Grade 1663 | Whole blood | 100 | Have | ＜16 | Scarlet | 10 (stifled filtering membranes) |
| 100 | Have | ＜16 | Scarlet | 10 (stifled filtering membranes) | ||
| 100 | Have | ＜16 | Scarlet | 10 (stifled filtering membranes) | ||
| 100 | Have | ＜16 | Scarlet | 10 (stifled filtering membranes) | ||
| 100 | Have | ＜16 | Scarlet | 10 (stifled filtering membranes) |
The effect that subordinate list 2 filters red blood cell through the pretreated filtering membrane of anti-human erythrocyte antibody
| The whole blood filtering membrane | Rabbit anti-human erythrocyte antibody (mg/ml) | Sample | Application of sample amount (microlitre) | Have or not filtration | Migration velocity (mm/min) | Background | The film time on the red blood cell |
| Grade 111 | 0.25 | Whole blood | 100 | Do not have | ≤16 | Totally | After 1 hour |
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| Grade 141 | 0.25 | Whole blood | 100 | Do not have | ≤16 | Totally | After 1 hour |
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| Grade 142 | 0.25 | Whole blood | 100 | Do not have | ≤16 | Totally | After 1 hour |
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| Grade 1660 | 0.25 | Whole blood | 100 | Do not have | ≤16 | Totally | After 1 hour |
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| Grade 1661 | 0.25 | Whole blood | 100 | Do not have | ≤16 | Totally | After 1 hour |
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| Grade 1662 | 0.25 | Whole blood | 100 | Do not have | ≤16 | Totally | After 1 hour |
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| 100 | Do not have | ≤16 | Totally | After 1 hour | |||
| Grade 1663 | 0.25 | Whole blood | 100 | --- | --- | Stifled fully filtering membrane | --- |
| 100 | --- | --- | Stifled fully filtering membrane | --- |
| 100 | --- | --- | Stifled fully filtering membrane | --- | |||
| 100 | --- | --- | Stifled fully filtering membrane | --- | |||
| 100 | --- | --- | Stifled fully filtering membrane | --- | |||
| Remarks: 1. " have or not filtration " and refer to have or not red blood cell to filter | |||||||
| 2. " migration velocity " refers to filter the migration velocity of blood plasma on reaction film | |||||||
The erythrocytic effect of subordinate list 3 different filtering membrane filterings relatively
| The filter membrane kind | Anti-human erythrocyte antibody | Sample | Application of sample amount (microlitre) | Have or not filtration | Background | Detection time (minute) | HC | LC | TEST | S/CO | The film time on the red blood cell |
| 142 whole blood filtering membranes | Do not have | The positive whole blood of HIV | 50 | Have | Scarlet | 30 | --- | --- | --- | --- | 2 minutes |
| Do not have | 50 | Have | Scarlet | 30 | --- | --- | --- | --- | 2 minutes | ||
| Do not have | 50 | Have | Scarlet | 30 | --- | --- | --- | --- | 2 minutes | ||
| Do not have | 50 | Have | Scarlet | 30 | --- | --- | --- | --- | 2 minutes | ||
| Do not have | 50 | Have | Scarlet | 30 | --- | --- | --- | --- | 2 minutes | ||
| Common filtering membrane | Have | 50 | Have | Little red | 30 | --- | --- | --- | --- | 3 minutes | |
| Have | 50 | Have | Little red | 30 | --- | --- | --- | --- | 3 minutes | ||
| Have | 50 | Have | Little red | 30 | --- | --- | --- | --- | 3 minutes | ||
| Have | 50 | Have | Little red | 30 | --- | --- | --- | --- | 3 minutes | ||
| Have | 50 | Have | Little red | 30 | --- | --- | --- | --- | 3 minutes | ||
| 142 whole blood filtering membranes | Have | 50 | Do not have | Totally | 30 | 0.2713 | 0.2116 | 0.182 | 8.603 | After 1 hour | |
| Have | 50 | Do not have | Totally | 30 | 0.1422 | 0.1715 | 0.1761 | 10.2693 | After 1 hour | ||
| Have | 50 | Do not have | Totally | 30 | 0.1981 | 0.1921 | 0.2282 | 11.8776 | After 1 hour | ||
| Have | 50 | Do not have | Totally | 30 | 0.2037 | 0.1843 | 0.1899 | 10.3033 | After 1 hour | ||
| Have | 50 | Do not have | Totally | 30 | 0.2287 | 0.1899 | 0.1846 | 9.723 | After 1 hour | ||
| Remarks: 1, " have or not filtration " and refer to have or not red blood cell to filter | |||||||||||
The test result of subordinate list 4 port one application of sample whole bloods and blood plasma relatively
| Filtering membrane | The red blood born of the same parents of the anti-people of rabbit antibody (mg/ml) | Sample | Application of sample amount (microlitre) | Background | Detection time (minute) | HC | LC | TEST | S/CO | The result |
| Whole blood filtering membrane (Grade 142) | 0.25 | Normal whole blood | 50 | Totally | 30 | 0.1777 | 0.0862 | 0 | 0 | Negative |
| 0.25 | 50 | Totally | 30 | 0.2268 | 0.0967 | 0 | 0 | Negative | ||
| 0.25 | 50 | Totally | 30 | 0.073 | 0.1083 | 0 | 0 | Negative | ||
| 0.25 | 50 | Totally | 30 | 0.183 | 0.0728 | 0 | 0 | Negative | ||
| 0.25 | 50 | Totally | 30 | 0.3277 | 0.109 | 0 | 0 | Negative | ||
| Whole blood filtering membrane (Grade 142) | 0.25 | Normal plasma | 50 | Totally | 15 | 0.4033 | 0.1926 | 0 | 0 | Negative |
| 0.25 | 50 | Totally | 15 | 0.3587 | 0.1945 | 0 | 0 | Negative | ||
| 0.25 | 50 | Totally | 15 | 0.36 | 0.1919 | 0 | 0 | Negative | ||
| 0.25 | 50 | Totally | 15 | 0.3822 | 0.1955 | 0 | 0 | Negative | ||
| 0.25 | 50 | Totally | 15 | 0.3759 | 0.1964 | 0 | 0 | Negative | ||
| Whole blood filtering membrane (Grade 142) | 0.25 | The positive whole blood of HIV | 50 | Totally | 30 | 0.1213 | 0.1781 | 0.2014 | 11.311 | Positive |
| 0.25 | 50 | Totally | 30 | 0.1419 | 0.1441 | 0.2181 | 15.1325 | Positive | ||
| 0.25 | 50 | Totally | 30 | 0.1781 | 0.1408 | 0.2153 | 15.2911 | Positive | ||
| 0.25 | 50 | Totally | 30 | 0.074 | 0.075 | 0.1032 | 13.7626 | Positive | ||
| 0.25 | 50 | Totally | 30 | 0.1653 | 0.1605 | 0.2142 | 13.3458 | Positive | ||
| Whole blood filtering membrane (Grade 142) | 0.25 | The positive blood plasma of HIV | 50 | Totally | 15 | 0.2755 | 0.1423 | 0.2011 | 14.13 | Positive |
| 0.25 | 50 | Totally | 15 | 0.3628 | 0.18 | 0.2344 | 13.0244 | Positive | ||
| 0.25 | 50 | Totally | 15 | 0.3084 | 0.1451 | 0.2005 | 13.8166 | Positive | ||
| 0.25 | 50 | Totally | 15 | 0.3149 | 0.1468 | 0.1862 | 12.6852 | Positive | ||
| 0.25 | 50 | Totally | 15 | 0.3425 | 0.1647 | 0.2164 | 13.139 | Positive |
Embodiment 2:
The port one place add with do not add double faced adhesive tape to filter sample flow to influence
Purpose: 1, observe dissimilar NC Nitroncellulose films (NC film) port one place and add and do not add the influence that double faced adhesive tape flows to plasma sample
2, observe dissimilar NC film port ones place add with do not add double faced adhesive tape to whole blood filter sample flow to influence
Material: 1, the HF135NC film U.S. MILLIPORE of 5 mm wides * 100 millimeters long
2, the HF90NC film U.S. MILLIPORE of 5 mm wides * 100 millimeters long
3,5 millimeters * 5 millimeters double faced adhesive tape China
4, with the 141 whole blood sample filter pads (5 mm wides * 7.5 millimeters long) that have 0.2 mg/ml mouse-anti human red blood cell antibody treatment to cross
5, the long steel ruler of 30cm China
6, the clinical blood donor's sample of normal person's whole blood and plasma sample
Design: application of sample 50ul------clocks, and------measures
The filtration design drawing is seen Fig. 9, shown in Figure 10.
Step: 1, on sample pad, add 50 microlitre blood plasma, observations and measure the sample distance that flows after 10 minutes with steel ruler
2, on sample pad, add 50 microlitre whole bloods, observations and measure the sample distance that flows after 10 minutes with steel ruler
Experimental result: see Table 5
| Structure | The HF90 film | The HF135 film | |||||||
| Plasma sample | Whole blood sample | Plasma sample | Whole blood sample | ||||||
| The left end distance (centimetre) | The right-hand member distance (centimetre) | The left end distance (centimetre) | The right-hand member distance (centimetre) | The left end distance (centimetre) | The right-hand member distance (centimetre) | The left end distance (centimetre) | The right-hand member distance (centimetre) | ||
| Do not add double faced | 1 | 2.51 | 2.44 | 2.13 | 2.26 | 2.65 | 2.70 | 2.03 | 2.12 |
| 2 | 2.61 | 2.65 | 2.25 | 2.31 | 2.71 | 2.68 | 2.25 | 2.18 | |
| 3 | 3.18 | 3.03 | 2.08 | 2.14 | 2.69 | 2.80 | 2.09 | 2.06 | |
| 4 | 3.06 | 3.19 | 2.14 | 2.25 | 2.72 | 2.70 | 2.16 | 2.09 | |
| 5 | 2.81 | 2.62 | 2.10 | 2.26 | 2.66 | 2.76 | 1.94 | 2.03 | |
| All plant | 2.83 | 2.79 | 2.14 | 2.24 | 2.69 | 2.73 | 2.09 | 2.10 | |
| Add double faced | 1 | 3.00 | 2.80 | 2.60 | 1.58 | 2.60 | 2.60 | 2.52 | 1.84 |
| 2 | 2.61 | 2.55 | 2.64 | 1.49 | 2.70 | 2.50 | 2.51 | 1.53 | |
| 3 | 2.53 | 2.32 | 2.52 | 1.60 | 2.33 | 2.30 | 2.62 | 1.61 | |
| 4 | 2.98 | 2.87 | 2.58 | 1.64 | 2.62 | 2.58 | 2.42 | 1.76 | |
| 5 | 2.74 | 2.61 | 2.62 | 1.71 | 2.48 | 2.37 | 2.60 | 1.54 | |
| All plant | 2.77 | 2.63 | 2.59 | 1.60 | 2.55 | 2.47 | 2.53 | 1.66 | |
Experimental analysis: 1, add under the sample pad and can impel behind the double faced adhesive tape sample moving to the left end predominant current
2, adding the whole blood sample ratio, to add the blood plasma sample moving more obvious to the left end predominant current
Embodiment 3:
The selection of the filtering membrane during whole blood sample port 2 application of samples and filtration (subordinate list 6A, 6B, 6C).
Purpose:
Set up the best whole blood filtration that whole blood is applicable to application of sample what port 2 so that and blood can be used for the two-way horizontal effluent and detect.
Material:
1, separation of whole blood film: Ahlstrom Filtering, the U.S.
GRADE CYTOSEP catalog number (Cat.No.) 1661 lot numbers: 2050401
GRADE CYTOSEP catalog number (Cat.No.) 1660 lot numbers: 2050401
GRADE CYTOSEP catalog number (Cat.No.) 142 lot numbers: 2050401
GRADE CYTOSEP catalog number (Cat.No.) 141 lot numbers: 2050401
GRADE CYTOSEP catalog number (Cat.No.) 111 lot numbers: 2050401
2, chemical coupling pad (not wrapping): Co., Ltd among the Millipore by collaurum
3, NC Nitroncellulose film: Co., Ltd among the Millipore
4, mouse-anti human red blood cell antibody (Chinese Xiamen ripple is given birth to).
5, use the pretreated filtering membrane of anti-human erythrocyte antibody (reference 2).
6, hepatitis B surface antibody (HBsAg) test lid (needing application of sample to carry out sandwich method at port 2 detects).
7, normal person's whole blood.
Press accompanying drawing 6 assembling test boxes
Experimental procedure
1, on the testing cassete of HBsAg, replace former damping fluid absorption pad with one deck whole blood filter membrane (without the anti-human erythrocyte antibody treatment), add whole blood sample, the hemofiltration effect of test whole blood filter membrane.
2, if monofilm can not reach separating of red blood cell that key point is arranged and blood plasma, then further screening with two-layer whole blood filter membrane without the anti-human erythrocyte antibody treatment in the hope of improving branch rate efficient.
3, if above-mentioned two steps all can not reach the purpose of effective separating red corpuscle, then the best relatively filter membrane of hemofiltration effect in the above-mentioned material after anti-human erythrocyte antibody carries out pre-service to film, compares it and filters erythrocytic effect.
The result; 6A, 6B, 6C see attached list.
Subordinate list 6A uses the red blood cell filter effect of single filtering membrane
| The filtering membrane model | Application of sample volume (microlitre) | Blood plasma leaches the time (second) | Window time (second) on earth | Red blood cell spills the time (second) | Blood plasma migration rate (mm/min) | Whole blood sample remnants behind the 30min |
| 1661 | 200 | 240 | 1236 | 290 | 1.8 | Have |
| 1660 | 200 | - | - | - | - | |
| 141 | 200 | 90 | 320 | 280 | 16.8 | Have |
| 142 | 200 | 98 | 380 | 395 | 16.1 | |
| 111 | 200 | - | - | - | - | Have |
Annotate: "-" is meant that the blood plasma that does not have blood plasma to leach or leach is not enough to move to the NC Nitroncellulose film.
Subordinate list 6B uses the red blood cell filter effect of double medium filtration film
| The filter membrane model | Application of sample amount (microlitre) | The exit window time (second) | The red blood cell exit window time (second) | Rate of diffusion (mm/min) |
| 142+142 | 200 | 457 | 582 | 5.87 |
| 141+141 | 200 | 135 | 1015 | 1.95 |
| 141+142 | 200 | 113.3333 | 238 | 2.93 |
| 142+141 | 200 | 640 | 686 | 2.26 |
| 141+1661 | 200 | 111 | 236 | 5.74 |
| 142+1661 | 200 | 112 | 253 | 2.83 |
Analyze:
From subordinate list 6A and 6B as can be seen: the physical filtering that (1) only relies on membrane aperture is the elimination red blood cell effectively, and the double medium filtration film can not improve filter effect; (2) #111 and 1660 is because of membrane aperture too small (1～3 μ m), can not satisfy the liquid effective liquidate speed and is eliminated; (3) combination of 141+141 or 141+142 is eliminated by ditch because of low blood plasma migration; (4) satisfy at the same time under filtration red blood cell and the effective plasma flow rate condition, 141# and 142# whole blood filtering membrane show better filtration red blood cell and higher migration rate, therefore, select 141# and 142# whole blood filtering membrane, after erythrocyte antibody (EA) carries out pre-service (data 2 sees reference), erythrocytic filtration and blood plasma transport efficiency in the further more different duplicature combinations, the result is shown in table 6C.
The different filtering membrane combination of subordinate list 6C red blood cell filters and the blood plasma migration rate compares
| The filter membrane model | Application of sample amount (microlitre) | The exit window time (second) | The red blood cell exit window time (second) | Rate of diffusion (mm/min) | Background after 30 minutes on the NC |
| 142 *+CP | 200 | 140 | 1150 | ＞16 | Some RBC |
| 142 *+CP * | 200 | 126 | - | ＞16 | There is not RBC |
| 141 *+CP | 200 | 130 | 680 | ＞16 | Some RBC |
| 141 *+CP * | 200 | 124 | 1350 | ＞16 | Minority RBC |
| CPonly | 200 | 138 | 159 | ＞16 | A large amount of RBC |
Annotate: " * " represents the mouse-anti human red blood cell antibody pre-service with 0.5 mg/ml.
"-" represents that no red blood cell leaches in 30 minutes
CP: chemical coupling thing pad
Analyze:
When whole blood sample is used for sandwich method port 2 application of sample mouths, as can be seen from subordinate list 6A～6C:
1. only depending on the aperture of filtering membrane to be difficult to reach has crucial location to intercept erythrocytic purpose.
2. can improve the effect of filter membrane separating red corpuscle to the pre-service of filtering membrane through anti-human erythrocyte antibody.
3. Zui Jia double-deck filter membrane structure is the liquid collecting pad that the conduct of the hydrophobic chemical coupling thing of 142# film underlying pad has filtering function, and both are all through the anti erythrocyte antibody pre-service.
4. effective red blood cell filters and leaches rate and migrate to port one with effective blood plasma is the precondition that satisfies port 2 application of samples, so there is the aperture of liquid collecting pad 18 of filtering function can not be too little, and need to form effective kapillary work chromatography pressure reduction between filtration EA and the chemical coupling thing pad 12 NC Nitroncellulose films 13, as shown in Figure 8, on NC Nitroncellulose film 13, be provided with liquid absorption pad 19 (5 * 18 millimeters) near the port one end, this liquid absorption pad 19 is the water wettability composite fibre, is used to absorb the liquid of application of sample in port 2.
Embodiment 4:
Anti-coagulants is to the influence (subordinate list 7A, 7B) of red blood cell filter effect.
Purpose:
When understanding immunochromatography that anti-human erythrocyte antibody whole blood filtering membrane is applied to the two-way horizontal effluent and detecting, whether anti-coagulants is influential to the filtration efficiency of blood plasma and blood plasma migration rate on the NC Nitroncellulose film of filtering out.
Material:
1, whole blood filter membrane: #142 (Ahlstrom Filtration, USA), 0.25 mg/ml mouse-anti is from erythrocyte antibody (EA) pre-service (data 2 sees reference);
2,142 films of handling through different anti-coagulants;
3, the whole blood sample that adds different anti-coagulants.
Press accompanying drawing 6 assembling test boxes.
The result: 7A and 7B see attached list.
The different whole blood results of #142 membrane filtration that subordinate list 7A handles without anti-coagulants compare
| The whole blood filtering membrane is handled without anti-coagulants | Whole blood application of sample volume | ||||||||
| 50 microlitres | 75 microlitres | 150 microlitres | |||||||
| Red blood cell spills | Plasma flow rate (ml/min) | Red blood cell remains on 142 films | Red blood cell spills | Plasma flow rate (ml/min) | Red blood cell remains on 142 films | Red blood cell spills | Plasma flow rate (ml/min) | Red blood cell remains on 142 films | |
| Non-anticoagulated whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
| Add EDTANa 2Whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
| Adding citric acid trisodium whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
| Add the liquaemin whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
The different whole bloods of #142 membrane filtration that subordinate list 7B handles through anti-coagulants compare
| The whole blood filtering membrane is handled without anti-coagulants | Whole blood application of sample volume | ||||||||
| 50 microlitres | 75 microlitres | 150 microlitres | |||||||
| Red blood cell spills | Plasma flow rate (ml/min) | Red blood cell remains on 142 films | Red blood cell spills | Plasma flow rate (ml/min) | Red blood cell remains on 142 films | Red blood cell spills | Plasma flow rate (ml/min) | Red blood cell remains on 142 films | |
| Non-anticoagulated whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
| Add EDTANa 2Whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
| Adding citric acid trisodium whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
| Add the liquaemin whole blood | Not | ≤16 | Trace | Not | ＞16 | On a small quantity | Not | ＞16 | More |
By showing 7A and 7B as can be seen:
1, whether handles through anti-coagulants, the sample #142 film of what equal volume filter effect no significant difference to whole blood; Be anti-coagulants not the outstanding blood plasma of shadow through the filtration efficiency of 142 films;
2, whether add anti-coagulants in the whole blood, under equal volume and identical filtering membrane condition, the filtration effect no significant difference of blood plasma.
Prompting: non-anticoagulated whole blood can be with this detection system of what.
Embodiment 5:
Hematid specific volume is to the influence (subordinate list 8A, 8B) of detection by quantitative
Purpose:
Understanding is in two-way horizontal effluent chromatography, and whether hematid specific volume influences the result of detection by quantitative.
Material:
1, the positive anticoagulated whole blood sample of hepatitis B virus surface antigen (HBsAg), measuring its hematid specific volume through wynn's method is 44%, and it with the packing of 1 milliliter/pipe, is got 2ml whole blood centrifugal (3000 rev/mins, 15 minutes) and obtain blood plasma.If 44% hematid specific volume is 1, increase or reduce the content of blood plasma in the whole blood on this basis, then obtain the whole blood of different hematid specific volumes.
2, it is 28 little international unit/milliliters that thyropathy patient anticoagulated whole blood, radioimmunology record its TSH (thyrotropic hormone) value.The whole blood for preparing different hematid specific volumes with 1 described method.
3, through the #142 filtering membrane (Ahlstrom Filtration) of 0.25 mg/ml anti-human erythrocyte antibody treatment.
4, HBsAg testing cassete is pressed accompanying drawing 7 assemblings.
5, TSH testing cassete is pressed accompanying drawing 7 assemblings.
The result:
The HBsAg test result 8A that sees attached list; The TSH test result 8B that sees attached list.
From showing 8A and 8B as can be seen:
1, behind the change hematid specific volume, the detection by quantitative result of HBsAg is shown: contain difference＜15% between the whole blood of different hematid specific volumes and the blood plasma testing result, for native system can hold difference scope between box, prompting detects quantitative HBsAg, no tangible volume dependence in hematid specific volume+30% or-40% scope.
2, change that the detection by quantitative result to TSH shows behind the hematid specific volume: containing difference＜15% between the whole blood of different hematid specific volumes and the blood plasma testing result, is difference scope between native system tolerable box.Prompting detects no tangible volume dependence in hematid specific volume+30% or-40% scope to quantitative TSH.
The influence of subordinate list 8B hematid specific volume in the HBsAg detection by quantitative
| Hematid specific volume | Application of sample volume (microlitre) | Migration rate (mm/min) | HBsAg concentration (nanograms/milliliter) | Average 1 (nanograms/milliliter) | Average 2 (nanograms/milliliter) | SD | Coefficient of variation CV |
| Normally | 200 | ＞16 | 18.7 | 18.475 | 18.1417 | 2.7099 | 14.94％ |
| Normally | 200 | ＞16 | 19.4 | ||||
| Normally | 200 | ＞16 | 18.6 | ||||
| Normally | 200 | ＞16 | 17.2 | ||||
| CV as a result | 4.99％ | ||||||
| Raise 10% | 200 | ＞16 | 16.7 | 16.925 | |||
| Raise 10% | 200 | ＞16 | 16.5 | ||||
| Raise 10% | 200 | ＞16 | 19.3 | ||||
| Raise 10% | 200 | ＞16 | 15.2 | ||||
| CV as a result | 10.1％ | ||||||
| Raise 20% | 200 | 16 | 18.2 | ||||
| Raise 20% | 200 | 16 | 16.7 | ||||
| Raise 20% | 200 | 16 | 15.4 | ||||
| Raise 20% | 200 | 16 | 17.3 | ||||
| CV as a result | 5.9％ | ||||||
| Raise 30% | 200 | ＜16 | 15.8 | 17.3 | |||
| Raise 30% | 200 | 15.6 | |||||
| Raise 30% | 200 | 20.8 | |||||
| Raise 30% | 200 | 17.1 | |||||
| CV as a result | 14.0％ | ||||||
| Descend 10% | 200 | ＞16 | 16.8 | 15.9 | |||
| Descend 10% | 200 | ＞16 | 16 | ||||
| Descend 10% | 200 | ＞16 | 16.2 | ||||
| Descend 10% | 200 | ＞16 | 15.6 | ||||
| CV as a result | 4.4％ | ||||||
| Descend 20% | 200 | ＞16 | 17.3 | 16.72 | |||
| Descend 20% | 200 | ＞16 | 18.6 | ||||
| Descend 20% | 200 | ＞16 | 16.1 | ||||
| Descend 20% | 200 | ＞16 | 15.9 | ||||
| CV as a result | 7.3％ | ||||||
| Descend 30% | 200 | ＞16 | 16.7 | 17.9 | |||
| Descend 30% | 200 | ＞16 | 19 | ||||
| Descend 30% | 200 | ＞16 | 18.2 | ||||
| Descend 30% | 200 | ＞16 | 17.7 | ||||
| CV as a result | 5.4％ | ||||||
| Descend 40% | 200 | ＞16 | 18.5 | 17.68 |
| Descend 40% | 200 | ＞16 | 17.6 | ||||
| Descend 40% | 200 | ＞16 | 16.4 | ||||
| Descend 40% | 200 | ＞16 | 17.4 | ||||
| CV as a result | 5.0％ | ||||||
| Whole plasm | 200 | ＞16 | 24.5 | 18.1 | |||
| Whole plasm | 200 | ＞17 | 25.6 | ||||
| Whole plasm | 200 | ＞18 | 23.7 | ||||
| Whole plasm | 200 | ＞19 | 26.8 | ||||
| CV as a result | 5.4％ | ||||||
| Annotate: SD refers to that standard deviation CV refers to the coefficient of variation | |||||||
The influence of subordinate list 8B hematid specific volume in the TSH detection by quantitative
| Sample | Port one addition (microlitre) | | Detection time (minute) | HC | LC | TC | Concentration (uIU/ml) | Average-1 (uIU/ml) | Average 2 (uIU/ml) | SD | Coefficient of variation CV% |
| Normally | 50 | 150 | 30 | 0.3679 | 0.4733 | 0.3456 | 29.66 | 28.09 | 28.5758 | 2.1258 | 7.44 |
| 0.4209 | 0.4736 | 0.3368 | 28.29 | ||||||||
| 0.4367 | 0.5451 | 0.4117 | 31.56 | ||||||||
| 0.4582 | 0.5414 | 0.3797 | 27.6 | ||||||||
| 0.3017 | 0.4605 | 0.2929 | 23.34 | ||||||||
| Raise 10% | 50 | 150 | 30 | 0.316 | 0.5106 | 0.3958 | 33.13 | 28.858 | |||
| 0.3918 | 0.6377 | 0.4369 | 26.49 | ||||||||
| 0.3019 | 0.4654 | 0.3739 | 35.49 | ||||||||
| 0.3851 | 0.6016 | 0.3954 | 24.66 | ||||||||
| 0.3654 | 0.5234 | 0.3428 | 24.52 | ||||||||
| Raise 20% | 50 | 150 | 30 | 0.2492 | 0.4968 | 0.3521 | 28.12 | 33.096 | |||
| 0.1325 | 0.4081 | 0.3378 | 37.64 | ||||||||
| 0.4036 | 0.4981 | 0.3921 | 34.12 | ||||||||
| 0.2374 | 0.4832 | 0.3663 | 31.77 | ||||||||
| 0.3286 | 0.4739 | 0.3714 | 33.83 | ||||||||
| Raise 30% | 50 | 150 | 30 | 0.285 | 0.5246 | 0.3487 | 25.14 | 25.976 | |||
| 0.3095 | 0.5654 | 0.3776 | 25.35 | ||||||||
| 0.387 | 0.5638 | 0.3756 | 25.24 | ||||||||
| 0.3331 | 0.5218 | 0.3613 | 26.99 | ||||||||
| 0.4314 | 0.4937 | 0.3431 | 27.16 | ||||||||
| Reduce by 10% | 50 | 150 | 30 | 0.4032 | 0.58 | 0.4178 | 28.95 | 30.118 | |||
| 0.4378 | 0.4639 | 0.3431 | 30.37 | ||||||||
| 0.4119 | 0.534 | 0.406 | 31.95 | ||||||||
| 0.3408 | 0.5217 | 0.3823 | 29.86 |
| 0.403 | 0.5726 | 0.4165 | 29.46 | ||||||||
| Reduce by 20% | 50 | 150 | 30 | 0.2859 | 0.4134 | 0.5012 | 28.8 | 27.944 | |||
| 0.3542 | 0.3538 | 0.5372 | 28.03 | ||||||||
| 0.1217 | 0.4228 | 0.5584 | 26.65 | ||||||||
| 0.3955 | 0.3661 | 0.5727 | 26.31 | ||||||||
| 0.1942 | 0.3896 | 0.4754 | 29.93 | ||||||||
| Reduce by 30% | 50 | 150 | 30 | 0.4156 | 0.531 | 0.3725 | 27.62 | 28.176 | |||
| 0.4138 | 0.5416 | 0.3874 | 28.58 | ||||||||
| 0.4028 | 0.5107 | 0.3742 | 29.85 | ||||||||
| 0.3895 | 0.5227 | 0.3686 | 27.87 | ||||||||
| 0.3955 | 0.5437 | 0.3763 | 26.96 | ||||||||
| Reduce by 40% | 50 | 150 | 30 | 0.3898 | 0.5024 | 0.3631 | 29.12 | 28.732 | |||
| 0.4023 | 0.5343 | 0.412 | 32.81 | ||||||||
| 0.369 | 0.5512 | 0.3706 | 25.64 | ||||||||
| 0.3775 | 0.4997 | 0.3581 | 28.68 | ||||||||
| 0.4182 | 0.5448 | 0.3806 | 27.41 | ||||||||
| Blood plasma | 50 | 150 | 30 | 0.4 | 0.4439 | 0.3034 | 26.39 | 26.192 | |||
| 0.361 | 0.4208 | 0.2795 | 25.12 | ||||||||
| 0.3539 | 0.4248 | 0.2846 | 25.49 | ||||||||
| 0.4089 | 0.4244 | 0.3018 | 28.29 | ||||||||
| 0.3799 | 0.448 | 0.3014 | 25.67 | ||||||||
| Annotate: HC high concentration contrast band OD value LC low concentration contrast band OD value TD calibration tape OD value " uIU/ml " refers to that " little international unit/milliliter " SD refers to that standard deviation CV% refers to the coefficient of variation | |||||||||||
Embodiment 6 hematid specific volumes are to the influence (subordinate list 9) of qualitative detection.
Purpose:
Understand in the detection of two-way horizontal effluent chromatography, hematid specific volume is to qualitative detection result's influence.
Material:
1, same routine HIV antibody positive anticoagulated whole blood, hematid specific volume is 45.3%, is distributed into 8 pipes, 1 milliliter of every pipe, centrifugal 5 minutes, changes erythrocytic specific volume by discarding or increasing blood plasma by 4,000 rev/mins.
2, through 142 filtering membranes (U.S. Ahlstrom Filtration) of the rabbit anti-human erythrocyte antibody treatment of 0.25 mg/ml.
3, HIV antibody test box is pressed accompanying drawing 5 assemblings.
The result:
For details see attached table coefficient of variation S/CO and CV% in 9.
Annotate: qualitative detection is judged testing result with signal to noise ratio (S/N ratio) (S/CO), and is positive with S/CO＞1.
From subordinate list 9 as can be seen:
Detect the whole blood and the plasma sample of same volume with anti-HIV testing cassete, the whole blood hematid specific volume+30% and-40% variation range in, the result of HIV qualitative detection does not have tangible volume dependence, and S/CO value error belongs to native system and allows difference scope between box 9.43% between sample.
Subordinate list 9 hematid specific volumes are to the influence of HIV qualitative detection
| Sample | Detection time (minute) | HC | LC | TC | S/CO | AVE1 | AVE2 | SD | CV |
| Normally | 30 | 0.2341 | 0.1272 | 0.2059 | 16.18 | 19.69 | 20.0019 | 1.8855 | 0.0943 |
| Normally | 30 | 0.2066 | 0.0941 | 0.2123 | 22.57 | ||||
| Normally | 30 | 0.2824 | 0.0942 | 0.1914 | 20.32 | ||||
| Raise 10% | 30 | 0.3702 | 0.1324 | 0.2564 | 19.36 | 19.61 | |||
| Raise 10% | 30 | 0.225 | 0.1193 | 0.2241 | 18.78 | ||||
| Raise 10% | 30 | 0.0961 | 0.1044 | 0.216 | 20.69 | ||||
| Raise 20% | 30 | 0.0503 | 0.1144 | 0.1752 | 15.32 | 17.9233 | |||
| Raise 20% | 30 | 0.0753 | 0.136 | 0.2474 | 18.19 | ||||
| Raise 20% | 30 | 0.0747 | 0.1107 | 0.2243 | 20.26 | ||||
| Raise 30% | 30 | 0.1036 | 0.1056 | 0.2463 | 23.32 | 17.3633 | |||
| Raise 30% | 30 | 0.2257 | 0.1545 | 0.2548 | 16.49 | ||||
| Raise 30% | 30 | 0.2486 | 0.1975 | 0.2425 | 12.28 | ||||
| Reduce by 10% | 30 | 0.2859 | 0.0954 | 0.2555 | 26.78 | 22.8267 | |||
| Reduce by 10% | 30 | 0.1217 | 0.12 | 0.3136 | 26.13 | ||||
| Reduce by 10% | 30 | 0.3955 | 0.1297 | 0.202 | 15.57 | ||||
| Reduce by 20% | 30 | 0.3806 | 0.1136 | 0.254 | 22.36 | 18.7033 | |||
| Reduce by 20% | 30 | 0.2666 | 0.1149 | 0.1925 | 16.75 | ||||
| Reduce by 20% | 30 | 0.3167 | 0.1075 | 0.1828 | 17 | ||||
| Reduce by 30% | 30 | 0.3931 | 0.1031 | 0.2531 | 24.55 | 20.2467 | |||
| Reduce by 30% | 30 | 0.4278 | 0.1615 | 0.2365 | 14.64 | ||||
| Reduce by 30% | 30 | 0.1701 | 0.1014 | 0.2185 | 21.55 | ||||
| Reduce by 40% | 30 | 0.2957 | 0.0805 | 0.2067 | 25.67 | 21.3167 | |||
| Reduce by 40% | 30 | 0.3816 | 0.1069 | 0.188 | 17.59 | ||||
| Reduce by 40% | 30 | 0.3317 | 0.0905 | 0.1873 | 20.69 | ||||
| Blood plasma | 15 | 0.3661 | 0.0844 | 0.1688 | 20 | 22.3367 | |||
| Blood plasma | 15 | 0.2843 | 0.0741 | 0.1689 | 22.79 | ||||
| Annotate: HC refers to that high concentration contrast band OD value LC refers to that low concentration contrast band OD value TD refers to that calibration tape OD value S/CO refers to that signal to noise ratio AVE refers to that mean value SD refers to that standard deviation CV% refers to the coefficient of variation | |||||||||
Embodiment 7:
Whole blood application of sample volume is to quantitative or qualitative detection result's influence (subordinate list 10A, 10B)
Purpose:
Understand of the influence of whole blood application of sample volume to two-way horizontal sidestream immune chromatography testing result.
Material:
1, through pretreated 142 filtering membrane of rabbit anti-human erythrocyte antibody (Ahlstrom Filtration, the U.S.) of 0.25 mg/ml
2, HIV antibody positive anticoagulated whole blood (hematid specific volume is 45.3%)
3, the positive anticoagulated whole blood (hematid specific volume is 44%) of HBsAg
4, assemble HIV antibody test box by Fig. 5
5, assemble the HBsAg testing cassete by Fig. 6
The result:
1, subordinate list 10A, for different whole blood application of sample volumes to HIV antibody qualitative detection result
2, subordinate list 10B, for different whole blood application of sample volumes to HBsAg detection by quantitative result
Annotate: 1, the HIV qualitative detection is with signal to noise ratio (S/N ratio) (S/CO) judged result, and is positive with S/CO＞1.
2, the HBsAg detection by quantitative is with concentration unit judged result, i.e. mg/ml.
Conclusion:
1, is applicable to that it is 40 microlitres that minimum whole blood that HIV detects adds kith and kin's volume.Whole blood application of sample amount is when 40～70 μ l, and the coefficient of variation of testing result (CV) is worth in 5～20% scopes.
2, be applicable to that the best whole blood application of sample volume that HBsAg detects is 200～250 microlitres.Detection time is when being 30 minutes, the coefficient of variation of testing result (CV)＜10%.
The HIV testing result of the different application of sample volume of table 10A whole blood sample relatively
| Sample | Application of sample amount (microlitre) | Cell has or not filtration | Blood plasma migration velocity (mm/min) | The sample surplus | Background | High concentration contrast band (Dr) | Low concentration contrast band (Dr) | Calibration tape (Dr) | S/CO | The S/CO average | CV | Detection time (minute) |
| The positive whole blood of HIV | 30 | Do not have | ＜16 | Do not have | Totally | 0.4752 | 0.226 | 0 | 0 | 0 | --- | 20 |
| 30 | Do not have | ＜16 | Do not have | Totally | 0.4154 | 0.2432 | 0 | 0 | ||||
| 30 | Do not have | ＜16 | Do not have | Totally | 0.4163 | 0.2317 | 0 | 0 | ||||
| 40 | Do not have | ＜16 | Do not have | Totally | 0.2936 | 0.1979 | 0.2043 | 10.3223 | 9.0592 | 0.1290 | 20 | |
| 40 | Do not have | ＜16 | Do not have | Totally | 0.2697 | 0.1958 | 0.1731 | 8.8396 | ||||
| 40 | Do not have | ＜16 | Do not have | Totally | 0.2994 | 0.1986 | 0.1592 | 8.0156 | ||||
| 40 | Do not have | ＜16 | Do not have | Totally | 0.371 | 0.225 | 0.2507 | 9.1408 | 9.5644 | 0.0886 | 30 | |
| 40 | Do not have | ＜16 | Do not have | Totally | 0.1701 | 0.1983 | 0.209 | 10.5396 | ||||
| 40 | Do not have | ＜16 | Do not have | Totally | 0.3206 | 0.1955 | 0.1762 | 9.0128 | ||||
| 50 | Do not have | ≤16 | Less | Totally | 0.2037 | 0.1697 | 0.2164 | 12.7513 | 12.0134 | 0.0650 | 30 | |
| 50 | Do not have | ≤16 | Less | Totally | 0.2572 | 0.1876 | 0.2271 | 12.1034 | ||||
| 50 | Do not have | ≤16 | Less | Totally | 0.0603 | 0.1729 | 0.1934 | 11.1856 | ||||
| 60 | Do not have | ＞16 | More | Totally | 0.3271 | 0.2474 | 0.2005 | 8.105 | 9.8273 | 0.2042 | 50 | |
| 60 | Do not have | ＞16 | More | Totally | 0.3456 | 0.1984 | 0.2387 | 12.0302 | ||||
| 60 | Do not have | ＞16 | More | Totally | 0.1926 | 0.246 | 0.2299 | 9.3467 | ||||
| 70 | Do not have | ＞16 | More | Totally | 0.1296 | 0.1983 | 0.1913 | 9.649 | 9.6556 | 0.0840 | 70 | |
| 70 | Do not have | ＞16 | More | Totally | 0.1387 | 0.245 | 0.2168 | 8.8477 | ||||
| 70 | Do not have | ＞16 | More | Totally | 0.1119 | 0.2401 | 0.2514 | 10.4702 |
The HBsAg testing result of the different application of sample volume of subordinate list 10B whole blood sample relatively
| Whole blood (microlitre) | Red blood cell spills (being/deny) | Blood plasma migration rate (mm/min) | Testing result | ||||||||
| Detected in 20 minutes | Detected in 30 minutes | Detected in 40 minutes | |||||||||
| The remaining red blood cell in application of sample place | HBsAg (nanograms/milliliter) | CV％ | The remaining red blood cell in application of sample place | HBsAg (nanograms/milliliter) | CV％ | The remaining red blood cell in application of sample place | HBsAg (nanograms/milliliter) | CV％ | |||
| 150 | Do not have | >16 | Few | 3.2 | 42％ | Do not have | 3.94 | 42％ | Do not have | 4.1 | 46％ |
| 200 | Do not have | >16 | Many slightly | 3.267 | 9％ | Few | 3.467 | 3％ | Do not have | 3.267 | 19％ |
| 250 | Do not have | >16 | Many | 2.633 | 6％ | Many slightly | 2.7 | 6％ | Few | 2.7 | 4％ |
| 300 | Do not have | >16 | Many | 2.767 | 6％ | Many | 2.833 | 9％ | Many | 3.133 | 10％ |
| 350 | -------- | -------- | -------- | -------- | -------- | ||||||
| Blood plasma 150 (microlitre) | 30 minutes | CV％ | |||||||||
| 3.36 | 5％ | ||||||||||
| Annotate: CV refers to the coefficient of variation | |||||||||||
The reference catalogue:
Reference-1 raw material supplier inventory
1, NC Nitroncellulose film (water wettability nitration fiber, Co., Ltd among the Millipore) in order to bag the be hunted down district and the reagent of control zone, provides the place of biochemical reaction.
2, chemical coupling pad (glass fibre, hydrophobicity, Co., Ltd among the Millipore) is used to wrap the reagent by the collaurum coupling.
3, damping fluid absorption pad (water wettability composite fibre, Cytosep, U.S. AhlstromFiltration company) absorbs damping fluid or sample.
4, sample pad (water wettability composite fibre, Cytosep, U.S. Ahlstrom Filtration company) absorbs sample.
5, liquid absorption pad (water wettability composite fibre, U.S. Whatman company), the liquid of absorption chromatography.
6, supporting pad (hydrophobicity plastic film, U.S. G﹠amp; L company), the solid support structure of test-strips.
7, whole blood filtering membrane (hydrophobic glass fiber or water wettability composite fibre, U.S. Ahlstrom Filtration company), the red blood cell of separating whole blood.
8, rabbit polyclonal anti-human erythrocyte antibody (Chinese Rui Taitesi) is used for pre-service whole blood filtering membrane and chemical coupling pad.
9, mouse monoclonal anti-human erythrocyte antibody (EA) (Chinese Xiamen ripple give birth to) is used for pre-service whole blood filtering membrane and liquid collecting pad.
10, plastic testing box (biology is contained by China), the framework as the ReLIA immunochromatography provides immunoreactive place.
11, other filtering membrane: CytoSep Grade 1660,1661,1662 and 1663 (water wettability is mixed natural and the synthetic fiber, U.S. Ahlstrom Filtration company); Cellulose Grade111 (100% textile paper, water wettability, U.S. Ahlstrom Filtration company).
The pretreated filter membrane technological process of reference-2 anti erythrocyte antibody
The Tween-20 solution of A, preparation 10%;
B, preparation final concentration are rabbit (mouse) the anti-human erythrocyte antibody-solutions of 0.25～5 mg/ml:
Damping fluid: trishydroxymethylaminomethane 50 mMs (final concentration)
EDTANa
24.1 mM (final concentration)
PH 8.5±1
Tween-20 0.8% (final concentration)
The solution-treated filtering membrane that contains anti erythrocyte antibody that C, usefulness have been prepared
1, a filtering membrane is put into pallet.Measure 52 milliliters of anti-erythrocyte Treatment Solution, pour pallet into.Make solution evenly infiltrate whole filtering membrane.
2, roll at the film upper reaches for several times with roller, to remove steam bubble.
3, repeating step 1 and 2 is until the whole film of solution uniformly penetrating.
4, film is placed on the gauze, blows over night with electric fan under the room temperature condition.
5, the half-dried film of processing was put into the exsiccator drying at least 5 hours.
6, the whole blood filtering membrane of handling well is put into the aluminium foil bag that contains drying agent, the sealing room temperature preservation.
Reference-3 filtering membrane physicochemical property
| Filtering membrane | Chemical characteristic | Hydrophobicity/water wettability | Pore diameter distribution (micron) | Flow rate of liquid (ml/min) | Capillary attraction (mm/min) | |
| #111 (whole blood filtering membrane) | | Hydrophilic | 1 | 130 | 51 | |
| #141 (whole blood filtering membrane) | Glass fibre | Hydrophilic | 3 | 350 | 79 | |
| #142 (whole blood filtering membrane) | Glass fibre | Hydrophilic | 5 | 300 | 55 | |
| #1660 (whole blood filtering membrane) | Synthon | Hydrophilic | 3 | 100 | 52 | |
| #1661 (whole blood filtering membrane) | Synthon | Hydrophilic | 5 | 260 | 41 | |
| #1662 (whole blood filtering membrane) | Synthon | Hydrophilic | 3 | 35 | 46 | |
| #1663 (whole blood filtering membrane) | | Hydrophilic | 2 | 35 | 46 | |
| The NC Nitroncellulose film * | NC Nitroncellulose | Hydrophilic | Change 5～20 | Change | 200～270 | |
| The chemical coupling pad | Glass fibre | Hydrophobic | 42 | 250 | 46 |
Remarks:
1, the aperture of NC Nitroncellulose film is a uneven distribution, but the aperture of other filtering membrane is a conformance to standard, be referred to as " pore diameter distribution ".
2, the flow rate of liquid of NC Nitroncellulose film represents that with XX second/milliliter/square centimeter other is all represented with ml/min.
3, the capillary attraction of NC Nitroncellulose film is different with the liquid of different product, with centimetre representing that flow rate of liquid depends on the aperture to a great extent, pore diameter distribution, the thickness of film in XX second/4.These factors have determined the porous proterties (air that includes certain volume in the film) of NC Nitroncellulose film, and in the middle of the detection of all films, the NC Nitroncellulose film has maximum capillary attraction.
About the flow velocity of film and the further detailed theoretical description of rate of capillary flow, see also " immuno-chromatographic test paper strip Minute Ride " (Millipore), P.7.
Claims (25)
1, a kind of quantitative quick horizontal lateral flow methods, platform with biochemical reaction, enforcement is made various projects to be checked to being not limited to human liquid biological sample to be checked, it is characterized in that intercepting cell contained in the liquid sample with filtration, obtains finishing the required liquid volume of its biochemical reaction; And by this platform structure, form effective capillary chromatography pressure reduction, make filtered liquid and be dissolved in wherein material to be checked on this platform through capillary chromatography effect directed flow; And according to material to be checked in the biochemical reaction and contrast material produced can be detected optical density judge testing result.
2, quantitative quick horizontal lateral flow methods according to claim 1, the platform that it is characterized in that described biochemical reaction is made up of test-strips and testing cassete shell, and the structure of test-strips has two ends and to guarantee effective cell filtration, capillary chromatography and biochemical reaction; Guarantee application of sample result's the observation and the fixing of test-strips physical arrangement of liquid biological sample with the testing cassete shell; Its biological sample can be added in an one or both ends of application of sample mouth in two ends on the testing cassete shell.
3, quantitative quick horizontal lateral flow methods according to claim 2, the cell filtration structure that it is characterized in that test-strips is made up of the filtering membrane in the different apertures of single or multiple lift, and the filtering membrane aperture of cells contacting is less than cell dia to be intercepted in its direct and sample.
4, quantitative quick horizontal lateral flow methods according to claim 1 and 2 is characterized in that the cell filtration structure of described test-strips has been used double faced adhesive tape, impels filtered solution to be advantage towards preset direction and flows.
5, quantitative quick horizontal lateral flow methods according to claim 1 and 2 is characterized in that being that the cell filtration structure of described test-strips and capillary chromatography pressure reduction require to be consistent, to guarantee the flow rate of liquid in effective liquid filtration and the chromatography process.
6, quantitative quick horizontal lateral flow methods according to claim 5, it is characterized in that described cell filtration structure, require consistent with capillary chromatography pressure reduction, by membrane material with different apertures, capillary attraction, set up cell filtration---chromatograph test strip, make celliferous liquid state biological sample to be checked VOLUME LOSS in filtration minimum, and after by physical filtering, liquid is being collected initial point place capillary chromatography pressure reduction less than biochemical reaction zone, thereby makes the liquid after the filtration be subjected to capillary attraction to drive directed moving on the test chromatography strip.
7, quantitative quick horizontal lateral flow methods according to claim 6, it is characterized in that described cell filtration structure, the available pretreated filter membrane of chemical reagent that makes cell aggregation to be filtered makes thing to be filtered increase because of the gathering volume, and improves the barriering effect of filtering membrane pair cell.
8, quantitative quick horizontal lateral flow methods according to claim 7, it is characterized in that the described chemical reagent of cell aggregation to be filtered that makes adopts anti erythrocyte antibody or vegetable protein agglutinin, anti erythrocyte antibody can be monoclonal antibody or polyclonal antibody at the antigenic determinant that the erythrocyte membrane surface exposes.
9, quantitative quick horizontal lateral flow methods according to claim 8 is characterized in that described is to comprise with surfactant Tween-20 handling film to increase the absorption of antibody to filtering membrane with the pretreated filter membrane of anti erythrocyte antibody.
10, quantitative quick horizontal lateral flow methods according to claim 1 and 2 is characterized in that the capillary chromatography pressure reduction of described test-strips comes from the material and the package assembly thereof of assembling test bar; According to the biochemistry detecting item requirement, but celliferous liquid biological sample application of sample is in the one or both ends of appointing of testing cassete; The different sample loading mode that add have different cell filtration-chromatographies-biochemical reaction structure.
11, quantitative quick horizontal lateral flow methods according to claim 10 is characterized in that the capillary chromatography pressure reduction of test-strips comes from the material and the package assembly thereof of assembling test bar; According to the biochemistry detecting item requirement, but celliferous liquid biological sample application of sample is in the one or both ends of appointing of testing cassete; The different sample loading mode that add have different cell filtration-chromatographies-biochemical reaction structure.
12, quantitative quick horizontal lateral flow methods according to claim 1 is characterized in that liquid biological sample to be checked is celliferous whole blood, but is not limited to whole blood sample; Celliferous liquid biological sample to be checked can contain anti-coagulants or not contain anti-coagulants, and it is required that the liquid volume that obtains behind the filtration through the reaction platform satisfies biochemical reaction.
13, quantitative quick horizontal lateral flow methods according to claim 1 and 2 is characterized in that its biochemical reaction process has utilized the background in the two-way horizontal effluent reduction biochemical reaction; And the relative optical density value RI analyzing and testing result of the ratio of the optical density value that is produced with thing to be checked in the biochemical reaction and tester, and improve the sensitivity that detects, it is poor to reduce between box, makes testing result be non-volume dependence relatively.
14, quantitative quick horizontal lateral flow methods according to claim 13; it is characterized in that described biochemical reaction utilizes the material of the optical density that colloid gold particle or silver-colored particle or fluorescent dye or other generation can detect by the light reflection principle thing that serves as a mark, make the ratio of the optical density that the result of biochemical reaction can produce by thing to be checked and tester determined.
15, quantitative quick horizontal lateral flow methods according to claim 1, it is characterized in that its biochemical reaction is based on immunology principle, but be not limited to the detection of the big intermolecular marriage relation of immunology principle, be used for detecting human, but be not limited to the big molecule in people's fluid-like state sample, as antigen, antibody, polysaccharide, nucleic acid, polypeptide, pathogenic microorganism etc.
16, a kind of quantitative quick horizontal side streaming system, platform with biochemical reaction, enforcement is made various projects to be checked to being not limited to human liquid biological sample to be checked, the platform that it is characterized in that biochemical reaction is made up of test-strips and testing cassete shell, and the structure of test-strips has two ends and to guarantee effective cell filtration, capillary chromatography and biochemical reaction; Biological sample can be added in an one or both ends of application of sample mouth in two ends on the shell.
17, quantitative quick horizontal side streaming system according to claim 16 is characterized in that cell filtration-chromatography-biochemical reaction structure, and is partly or entirely overlapping according to the order of sequence between each group; Its lap is 0.1～20 millimeter.
18, quantitative quick horizontal side streaming system according to claim 17 is characterized in that described cell filtration-chromatography-biochemical reaction structure, and the cell filtration film is hydrophobic glass fibre but does not limit the what glass fibre, or hydrophilic composite fibre.
19, quantitative quick horizontal side streaming system according to claim 18, it is characterized in that its liquid biological sample liquid collection membrane after filtering is the hydrophobic glass fiber but does not limit the what glass fibre, or hydrophilic composite fibre, and has the aperture that is not less than the cell filtration film.
20, quantitative quick horizontal side streaming system according to claim 19 is characterized in that its liquid biological sample liquid collection membrane aperture after filtering and ratio＞1 in filtering membrane aperture.
21, quantitative quick horizontal side streaming system according to claim 20, it is characterized in that label that biochemical reaction is required such as collaurum by pre-bag by and dry on hydrophobic glass fibre but do not limit the what glass fibre, be positioned at an end of chromatography strip; Collaurum is lower than the reaction zone of biochemical reaction at the chromatography pressure reduction of present position on the chromatography strip when discharging in that collaurum is dissolved, so collaurum is can coating antigen point mobile to the biochemical reaction zone orientation.
22, quantitative quick horizontal side streaming system according to claim 21, it is characterized in that biochemical reaction zone is by hydrophilic NC Nitroncellulose film but do not limit what NC Nitroncellulose film to form, and have in first time of two-way horizontal flow measurement during liquid flow, the highest capillary attraction in this structure makes liquid move to its reaction zone is directed from sample application zone or liquid collection region.
23, quantitative quick horizontal side streaming system according to claim 22, it is characterized in that the other end at chromatography strip, the position that corresponds to chemical coupling thing pad is equipped with hydrophilic suction pad, its effect is soaked at the NC Nitroncellulose film, after the water-intake capacity decline collaurum dissolving, provide with its height water-intake capacity and to impel liquid to move required capillary chromatography pressure reduction to suction pad end is directed from the collaurum end.
24, quantitative quick horizontal side streaming system according to claim 23 is characterized in that its cell filtration film directly contacts with the NC Nitroncellulose film but do not contact with the pad that absorbs water when celliferous liquid biological sample application of sample during at suction pad one end; When celliferous liquid biological sample application of sample during at collaurum one end, its cell filtration film contacts with whole with liquid collection membrane part, but does not directly contact with chemical coupling thing pad; When containing the liquid biological sample of cell application of sample be in two ends application of sample mouth at the same time or separately, the combination of its cell filtration structure during for application of sample respectively.
25, quantitative quick horizontal side streaming system according to claim 24 is characterized in that its liquid biological sample liquid collection membrane after filtering makes and both can be used for immune indirect method with a kind of test strips structure and detect, and can be used for immune sandwich method again and detects.
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