CN101750244B - Method for separating red cells from blood sample and application - Google Patents
Method for separating red cells from blood sample and application Download PDFInfo
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- CN101750244B CN101750244B CN200910206522.6A CN200910206522A CN101750244B CN 101750244 B CN101750244 B CN 101750244B CN 200910206522 A CN200910206522 A CN 200910206522A CN 101750244 B CN101750244 B CN 101750244B
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Abstract
The invention relates to a device and a method for separating red cells from a blood sample and a device and a method for detecting analyzed substances in the blood sample. The method for separating red cells from the blood sample comprises the following steps: making the blood sample contact receptors of fibrin in a combined blood sample; and separating out the receptors of the fibrin from the blood sample. The device for detecting the analyzed substances in the blood sample comprises a carrier which comprises a detection area, and the device is characterized in that the upstream of the detection area comprises the receptors of the fibrin in the combined blood sample. The red cells in the blood sample can be effectively separated by using the devices and the methods so as to reduce the obstruction effect of the red cells on liquid flow and the interference of the red cells on a response background. The methods and the devices do not cause adverse effects on the detection results.
Description
Technical field
The invention relates to erythrocytic apparatus and method in separating whole blood sample, more specifically, is about a kind of pick-up unit, and he applies fibrin in separating whole blood and comes the method for separating red corpuscle and device to reduce the interference of red blood cell to testing result.
Background technology
Modern clinic diagnostic method normally carries out blood sample.Unfortunate, red blood cell detects and has interference effect many diagnosis.This just needs first red blood cell to be separated from blood sample, and then further sample is detected or chemically examine.On the other hand, in chromatography pick-up unit, particularly, in immunoassay device, red blood cell may stop liquid flow, and liquid flow is for reacting and be absolutely necessary on this pick-up unit.Based on this reason and some other reason, many detections are all carried out with blood plasma or serum, and this just need to isolate blood plasma or serum from whole blood.
There are many prior aries can be used on and in whole blood sample, make red blood cell and separating plasma.Centrifugal is a kind of habitual means.In addition, in patent and disclosed patented claim, also disclosed certain methods, for example United States Patent (USP) 5939331,6673629,4594327,4678757,5558834,6818180, U.S.'s publication application 2006/0029923,2004/0202783.Summary is got up, and these separated methods mainly comprise that physics and chemistry method carrys out the red blood cell in separating whole blood sample.These are expensive, complicated for making the technology of red blood cell and separating plasma many, may cause red blood cell not exclusively separated.Particularly those have been placed to for a long time time, and the whole blood that has produced blood coagulation said originally, prior art can not well be carried out separation.Especially, in chromatography immunoassay device, this blood coagulation sample can cause nonspecific combination or very high background interference, causes losing the sensitivity detecting, but also can stop up, thereby chromatography carrier hinders serum or blood plasma flows upper, and it is invalid to cause detecting.To some, need within very short time, just can draw the device of testing result especially, this shortcoming is more fatal.The apparatus and method that this just needs a kind of reagent and includes this reagent, for separating of the red blood cell in whole blood sample, and this apparatus and method do not have adverse influence to detection system.
Summary of the invention
In order to overcome the above problems, the invention provides a kind of reagent of separating red corpuscle and some utilizations of this reagent from blood sample, use this reagent separating red corpuscle from blood sample effectively.
On the one hand, the present invention relates to a kind of from blood sample a kind of method of separating red corpuscle.Concretely, comprise, allow fibrinous receptor response in blood sample sample and a kind of combination blood, remove the fibrin acceptor in blood sample.In another embodiment, the method comprises, a kind of carrier is provided, and comprises a kind of acceptor of binding fiber albumen on carrier, allows blood sample flow through this carrier.
On the other hand, the invention still further relates to a kind of pick-up unit that detects analyte in blood sample, this device comprises a carrier, comprises a surveyed area on carrier, fibrinous acceptor in surveyed area upstream process has in conjunction with blood sample.Preferably, carrier is chromatography carrier.Preferred, in the upstream of surveyed area, comprise sample region of acceptance, on sample region of acceptance, process have in conjunction with blood sample in fibrinous acceptor.
On the other hand, the present invention also provides a kind of a kind of method for detection of analyte in whole blood sample.The method is preferably applied in chromatography carrier device of the present invention.Concretely, the method comprises, a kind of chromatography carrier is provided, and comprises a sample region of acceptance on chromatography carrier, wherein, on sample region of acceptance, process have in conjunction with blood sample in the acceptor of condensating fiber albumen; Make blood sample flow through sample region of acceptance; Detect in blood sample and whether have analyte.
In above all embodiments, the acceptor of binding fiber albumen comprises fibrinous antibody, comprises the antibody of anti-erythrocyte in conjunction with erythrocytic acceptor, and antibody can be monoclonal antibody or how anti-, can be also antibody fragment; Carrier can be chromatography carrier or non-water absorptivity carrier etc.; The acceptor of binding fiber albumen can be fixed on carrier.。
Beneficial effect: utilize technology of the present invention, the effective red blood cell in the blood sample of place to go, and no matter blood sample be fresh or produced the sample of blood coagulation, increased the service time of detected sample.In addition, the pick-up unit that has used the red blood cell tripping device of acceptor that can binding fiber albumen or detected analyte in blood sample can increase the service life.
Accompanying drawing explanation
Fig. 1 is a specific embodiment of the present invention, in pick-up unit 10, comprise that a filtering layer 11 accepts sample and allow sample pass through this layer and a responding layer 12, in responding layer, process and have reduction reaction substrate, with hand holding handle 13, wherein on filtering layer 11, process have in conjunction with blood sample in fibrinous polyclonal antibody and in conjunction with erythrocytic polyclonal antibody.After whole blood sample 01 is applied on sample filtering layer 11, the fibrin antibody of processing on filtering layer 11 is tackled red blood cell on filtering layer together with erythrocyte antibody (EA), allow blood plasma arrive in responding layer 12 by filtering layer, if there is analyte in sample, for example blood sugar, directly can observe change color at the lower surface of responding layer 12.
As Fig. 2 has described preferred embodiment of the present invention, in pick-up unit 30, the carrier material that forms sample region of acceptance 35 is glass fibre, the material that forms surveyed area 31 is nitrocellulose filter the downstream that is positioned at sample region of acceptance, contain be labeled material region 34 between sample region of acceptance 35 and surveyed area 31.On surveyed area, comprise that being fixed with the specific bond molecular band 33 and the testing result that are fixed controls molecular band 32; The polyclonal antibody and the fibrinous polyclonal antibody that in fiberglass carrier, comprise erythrocyte surface antigen, wherein sample region of acceptance 35, interconnect can allow liquid flow to surveyed area 31 from sample region of acceptance 35 to react between marked region 34 and surveyed area 31.
Description of symbols: pick- up unit 10,30; Blood sample 01,02; Filtering layer 11; Responding layer 12; Hand holding handle 13; Sample receiving pad 35, label pad 34, surveyed area (detection line) 36, control line 32, nitrocellulose membrane 36, water accepting layer 31.
Embodiment
The structure below the utility model being related to or these technical terms using are described further.
Detect
Detect to represent chemical examination or test a kind of material or whether material exists, such as, but be not limited to this, the metabolin of chemical substance, organic compound, mineral compound, metabolism product, medicine or drug metabolite, organic organization or organic organization, nucleic acid, protein or polymkeric substance.In addition, detect the quantity that represents test substances or material.Furtherly, chemical examination also represents immune detection, chemical detection, enzyme detection etc.
Fibrin and fibrinous acceptor
Fibrinogen is a kind of clotting factor of finding the earliest.The spheroid that is elongation, is the dipolymer being formed by connecting with disulfide bond by three pairs of polypeptied chains (a pair of α chain, a pair of β chain, a pair of γ chain), and its molecular weight is about 340,000 dalton.After synthetic in liver, enter blood plasma, with dissolved form, exist.The about 0.3g of content in every 100ml human plasma.Fibrin is the insoluble protein polymer of a kind of height, is the crystalline thing as fine needle.It is the most basic variation in whole blood clotting process that fibrinogen changes fibrin into, will be through 3 links: 1. fibrinogenic hydrolysis is under thrombin action, two α chains and two every, β chains in fibrinogen molecule have a peptide bond rupture, result forms fibrin monomer, and discharge two pairs of micromolecular fibrin polypeptide (be fibrin polypeptide A and B, molecular weight adds up to and to be about 9000 dalton) simultaneously.Therefore the fibrinous molecular weight ratio fibrinogen, finally forming is less.2. the gathering of fibrin monomer.In the presence of Ca2+, some fibrin monomers aggregate into the fibrin polymer of solubility.3. under the effect that is formed on plasmase and Ca2+ of blood clot, between the α chain of different fibrin molecules, form cross linkage, make fibrin change final insoluble fibrin polymer into.Fibrin in formation is interlaced overlapping and enlist the services of haemocyte, makes the blood that is originally colloidal sol shape be transformed into gelatinous blood clot.
In our experiment, we surprisingly find, utilize the fibrin that the acceptor of binding fiber albumen is removed in blood sample can effectively isolate red blood cell.Especially, when the acceptor of this binding fiber albumen be used in conjunction with in situation in conjunction with erythrocytic acceptor, can better make the red blood cell in blood obtain separation.And no matter the erythrocytic technology of this removal is still effective equally to producing the blood sample of blood coagulation to fresh blood.
Separation method
On the one hand, the present invention relates to a kind of from whole blood sample a kind of method of separating red corpuscle.Concretely, comprise, allow fibrinous receptor response in whole blood sample and a kind of combination blood, remove the fibrin acceptor in blood sample.In specific embodiment, can as acceptor, come in conjunction with or catch fibrin monomer or the fibrin polymer in blood sample with the antibody of antifibrin, the antibody of antifibrin can be polyclonal antibody, monoclonal antibody or antibody fragment.This acceptor can also be the acceptor that synthetic or artificial synthetic other of nature can binding fiber albumen, and said combination here can be the combination of " special ", combination that also can right and wrong " special ".As long as the acceptor of this binding fiber albumen can be just passable in conjunction with the fibrin in blood sample.A fairly simple example, first allows the antibody of blood sample and antifibrin contact, and then the antibody by antifibrin in separating blood sample carrys out red blood cell in separating blood sample.In an embodiment more preferably, allow the antibody of blood sample and anti-erythrocyte contact, then allow the antibody of this blood sample and antifibrin contact, then the antibody by antifibrin in separating blood sample carrys out the red blood cell in separating sample.In another embodiment, also can allow blood sample contact with the antibody of antifibrin and the antibody of anti-erythrocyte, then by the antibody of the antifibrin in while separating sample and the antibody of anti-erythrocyte, carry out the red blood cell in separating sample simultaneously.Here said " separation " refer to by physics or chemistry mode allow directly or indirectly the antibody of antifibrin separate from blood sample.For example, can be directly next separated with a kind of antibody of antifibrin acceptor, can be also other mode, for example on some carriers, fix the antibody of some binding fiber albumen or the antibody of anti-erythrocyte, then allow whole blood sample flow through this carrier.
Carrier, the tripping device that contains carrier
In another embodiment, the method comprises, a kind of carrier is provided, and comprises a kind of acceptor of binding fiber albumen on carrier, allows blood sample flow through this carrier.In addition, the invention still further relates to erythrocytic device in a kind of separating whole blood sample, this device comprises a carrier, wherein on carrier, processes and has binding fiber protein receptor.This carrier can, by water absorptivity, make again " chromatography carrier " or non-water-absorbing material form or form.
Non-absorptive material includes, but not limited to plastics, glass, pottery and other metal materials.For example, can first allow the acceptor of binding fiber albumen, antibody for example, be fixed on frosting, then allow blood sample contact with the acceptor on frosting, fibrin in blood is just fixed on the receptor capture of frosting like this, thereby causes the fibrin in blood separated from blood sample, and red blood cell is also separated like this.One preferred embodiment in, a kind of so non-absorptive material is provided, wherein in conjunction with the antibody of erythrocytic antibody and binding fiber albumen, be fixed simultaneously and process on these materials, allow blood sample flow through non-absorptive material surface.
On the other hand, this carrier can be also chromatography carrier, and said chromatography carrier refers to any applicable poriness, absorbent material or capillary materials here.This material itself has certain receptivity, and liquid can flow upper by capillary action, for example some filter paper, glass fibre element, polyester film, nylon membrane, nitrocellulose filter, cellulose acetate, natural material (for example cotton) fabric and synthesis material (nylon) fabric; And porous gel etc.In a preferential embodiment, when supporting the carrier of liquid flow with these, can first the acceptor of binding fiber albumen be fixed and be processed on these carriers, it can not be dissolved in liquid because of flowing of liquid.When allowing blood sample be applied on these carriers, the fibrin molecule in blood sample is collected on carrier by its receptors bind, and along with the continuation of blood sample is flowed, the fibrin in sample comes with regard to separated.Another preferred embodiment in, one or more on these carriers, can also have been processed in conjunction with erythrocytic acceptor, these acceptors can be assembled the red blood cell in blood sample, these erythrocytic acceptors can be processed at the upstream of fibrin acceptor, for example fixing processing on carrier.Process the acceptor of binding fiber albumen and the position on carrier can be positioned at same position with fibrin acceptor in conjunction with erythrocytic acceptor, can also lay respectively at up and down or front and back, or other position; When blood sample flows through carrier, blood sample can contact with erythrocytic acceptor with fibrinous acceptor simultaneously simultaneously; Also can be that blood sample first contacts with fibrinous acceptor and then contacts with erythrocytic acceptor, or first contact with erythrocytic acceptor and then contact with fibrinous acceptor.How these acceptors to be processed and allow it be fixed on carrier is the common practise of this area.And, it should be appreciated by those skilled in the art, carrier can be that homogenous material forms or (for example consists of more than one material, can be formed by different materials different positions, district or floor), as long as the state that the material of these multilayers is in contact with one another in liquid stream, thereby liquid can be passed through at these storerooms.For example, this carrier is accepted can allow liquid sample vertically pass through after sample, and as shown in Figure 1, or level is by carrier, as shown in Figure 2, can be also other form.
This can be separated or remove erythrocytic reason and may be by fibrin in separating blood sample, fibrinous acceptor, the antibody of antifibrin for example, by binding fiber protein molecular, allow single fibrin molecular aggregates, form one and assemble net, interlaced and the network of this net is lived the red blood cell in sample, thus the red blood cell in separating sample indirectly of the acceptor by separating fibrin.Another preferred embodiment in, when having in conjunction with erythrocytic acceptor, the antibody of erythrocyte surface antigen for example, in situation about existing, ER can make single erythrocyte aggregation form " the little group of red blood cell " together, in the network-like fabric that has fibrin molecule to form, can live these " the little groups of red blood cell " and be not easy by blood sample, dissolved or take away by network, so better the red blood cell in separating blood sample simultaneously.In an embodiment more preferably, the acceptor of this binding fiber albumen is polyclonal antibody and is fixed processing on chromatography carrier, in addition, on chromatography carrier, also fixing processing has the polyclonal antibody in conjunction with erythrocyte surface antigen, when blood sample being applied on this chromatography carrier, the erythrocyte antibody (EA) of processing on carrier in conjunction with or the red blood cell of catching in sample form " the little group of red blood cell ", fibrin antibody combination on carrier simultaneously or the fibrin of catching in sample form fibrin molecular network, this protein molecular network can be caught these than single erythrocyte volume larger " the little group of red blood cell " thereby can continue to flow forward by more effective obstruction red blood cell, can effectively from blood sample, isolate red blood cell like this.The method of fibrinous acceptor separating red corpuscle on carrier is processed in this utilization can, by allowing acceptor directly be fixed on carrier and to be achieved, also can carry out separating fibrin indirectly.For example, the antibody of Biciromab (being called for short fibrin two anti-) is processed and is fixed on carrier, first allow the antibody hybrid reaction of blood sample and antifibrin, then this mixed solution being applied to process has on two anti-carriers, this two antiantibody is caught fibrin antibody, thereby indirectly catches fibrin.As a same reason, also the antibody of anti erythrocyte antibody (be called for short red blood cell two anti-) can be processed and is fixed on and on carrier, indirectly catch the red blood cell in blood sample.In a preferred mode, the antibody of the antibody of antifibrin and anti-erythrocyte is directly fixed and processed on carrier.
Pick-up unit
On the other hand, the invention still further relates to a kind of pick-up unit that detects analyte in whole blood sample, this device comprises a carrier, comprises a surveyed area on carrier, fibrinous acceptor in surveyed area upstream process has in conjunction with blood sample.Preferably, carrier is chromatography carrier; Preferred, on this carrier, also comprise a sample region of acceptance that is positioned at surveyed area upstream; On sample region of acceptance, process have in conjunction with blood sample in fibrinous acceptor.Any detection system may be used to object of the present invention.Chromatography immune detection system preferably, including, but not limited to horizontal flow system, vertical liquid fluid system and test bar.To some known detection systems be described below.
In general, at least arranging a sample region of acceptance and a surveyed area on chromatography carrier reagent strip, on surveyed area, comprise some special moleculars or chemical substance, by them, can detect in sample, whether have analyte or quantity.When detected sample liquid is applied to sample region of acceptance, by capillary action, it will move along chromatography carrier, and pass through the effect of association reaction (as immunological response) at surveyed area, the markd material that is labeled of being with on chromatography carrier is presetted in accumulation, this summation (is for example directly proportional to existence or the content of analyte in sample, double antibody sandwich method) or inverse ratio (competition law), this by measuring existence or the content with mark substance, just can detect in sample liquid, whether exist or content how many.Certainly, it on surveyed area, can be also the color reaction of the low thing of pure chemistry character, redox reaction etc. for example, by the depth of color on surveyed area, measure that analyte has or not or quantity, utilize the pick-up unit of this principle to common are blood sugar test reagent strip etc., for example, described in United States Patent (USP) 6818180.In specific embodiment, sample region of acceptance be labeled material in identical position, preferably in the upstream that is labeled material (direction that causes liquid flow by capillary action is called to " upstream ", and contrary direction is called " downstream ").When allowing sample liquids (suspection contains analyte) contact with sample region of acceptance, sample liquid is by capillary action, along chromatography carrier together with analyte flow further downstream together.Conventionally analyte is a kind of compound, and it is incorporated into the detection molecules being fixed on surveyed area in a particular manner.For example, analyte is a kind of antigen, and the first antibody that the material being labeled is this antigen is fixed on the molecule of surveyed area for another antibody molecule of this antigen.Utilize these principles to detect in sample, whether having the apparatus and method of analyte is prior art, is not emphasis of the present invention.The present invention can comprise serum and plasma for any blood sample, and still preferential is with containing erythrocytic whole blood sample.
If wish to detect with desirable sensitivity, before preferably required analyte in blood sample being detected, red blood cell is removed.Therefore, according to pick-up unit of the present invention, allow the acceptor of binding fiber albumen be fixed processing on chromatography carrier, preferably process on sample region of acceptance.Why be preferred, once be because blood sample is applied on sample region of acceptance, this processing can effectively be removed the red blood cell in blood sample, and reduce serum or blood plasma afterwards along the least interference of carrier flow, thereby make serum or blood plasma flowing velocity afterwards unaffected, reduced in addition background interference.As Fig. 2 has described preferred embodiment of the present invention, in pick-up unit 30, the carrier material that forms sample region of acceptance 35 is glass fibre, the material that forms surveyed area 31 is nitrocellulose filter the downstream that is positioned at sample region of acceptance, contain be labeled material region 34 between sample region of acceptance 35 and surveyed area 31.On surveyed area, comprise that being fixed with the specific bond molecular band 33 and the testing result that are fixed controls molecular band 32; The polyclonal antibody and the fibrinous polyclonal antibody that in fiberglass carrier, comprise erythrocyte surface antigen, wherein sample region of acceptance 35, interconnect can allow liquid flow to surveyed area 31 from sample region of acceptance 35 to react between marked region 34 and surveyed area 31.
Fig. 1 has described another preferred embodiment of the present invention, in pick-up unit 10, comprise that a filtering layer 11 accepts sample and allow sample pass through this layer and responding layer 12, in responding layer, process and have reduction reaction substrate, with hand holding handle 13, wherein on filtering layer, process and have in conjunction with fibrinous polyclonal antibody in blood sample with in conjunction with erythrocytic polyclonal antibody.After whole blood sample 01 is applied on sample receiving layer 11, the fibrin antibody of processing on filtering layer 11 is tackled red blood cell on filtering layer together with erythrocyte antibody (EA), allow blood plasma arrive in responding layer 12 by filtering layer, if there is analyte in sample, for example blood sugar, directly can observe change color at the lower surface of responding layer 12.About the present invention relates to remove the application of red blood cell method in blood sample, not only limit to and the enumerating of above embodiment, can also be applied in other any detection systems and go, for example can be applied in the pick-up unit that United States Patent (USP) 6818180 Fig. 1 describe and go, concretely, can be processed to the separated whole blood sample applying by hole 21 on the top layer 5 of porous membrane 1.The those skilled in the art in this field are appreciated that, method described in the invention or device in can with the method for other separating red corpuscle in prior art, structure or reagent are combined with, for example, be combined with the total described such structure of United States Patent (USP) 6818180 and reagent.
Detection method
On the other hand, the present invention also provides a kind of a kind of method for detection of analyte in whole blood sample.The method is preferably applied in chromatography carrier device of the present invention.Concretely, the method comprises, a kind of carrier is provided, and comprises a sample region of acceptance on carrier, wherein, on sample region of acceptance, process have in conjunction with blood sample in fibrinous acceptor; Make blood sample flow through sample region of acceptance; Detect in blood sample and whether have analyte.Preferably, on sample region of acceptance, also process and have in conjunction with erythrocytic antibody.Preferably, in the downstream of sample region of acceptance, also comprise a surveyed area, this surveyed area comprises a specific bond molecule that is fixed and processes, and allows the sample flow that flows through sample region of acceptance cross surveyed area.Carrier can be chromatography carrier or non-water absorptivity carrier.
The following examples will further illustrate the present invention, in any case but should not be construed as limitation of the scope of the invention.
Experiment
experiment 1, utilizes Biciromab to come separating blood sample red blood cell detecting CTI (myocardium calcium egg
application in vain)
This experiment, the how anti-three rich polygala root biotechnology Ltds that buy in Beijing that comprise antifibrin used in following other experiments, No. 26, South Road, Northeast Wang, Haidian District, Beijing City, address, lot number is G11135796J162; The monoclonal antibody Mai Yu Genclonn company of the anti-erythrocyte using, address is No. 198 ，Gu Dang Economy Gardens of Zhejiang Province, China Tianmushan Road, Hangzhou, lot number is ME060822-1-0419; The anti-erythrocyte using how anti-bought the company in Genclonn, and address is No. 198 ，Gu Dang Economy Gardens of Tianmushan Road, Hangzhou, and lot number is Q05920-1113..Except the place additionally marking, can manufacture CTI by the described method of prior art and detect reagent strip.This experiment illustrates how to prepare and to assemble reagent strip with reference to Fig. 2.
1. the configuration of the antibody of antifibrin and process sample receiving pad 35
Sample receiving pad is glass fibre, first processes Tris buffer solution in the above, and glass fibre is done to following several different processing.
1), only on glass fibre, process antifibrin how anti-(100, each processes 20).The polyclonal antibody of antifibrin proportionally 1: 50,1: 100,1: 200,1: 300, dilution in 1: 400, and process equably on glass fibre, be respectively and process A, B, C, D, E.
2), on glass fibre, process anti-erythrocyte and fibrinous many anti-(50, each processes 10) simultaneously.The A in (1), B, C, D, E is divided into 2 groups, and 10 every group, then process therein the polyclonal antibody of anti-erythrocyte on each glass fibre of one group, the dilution ratio of anti erythrocyte antibody is 1: 10, is respectively G=1: 50 (how anti-antifibrin is)+1: 10 (how anti-anti-erythrocyte is); H=1: 100 (how anti-antifibrin is)+1: 10 (how anti-anti-erythrocyte is), I=1: 200 (how anti-antifibrin is)+1: 10 (how anti-anti-erythrocyte is), J=1: 300 (how anti-antifibrin is)+1: 10 (how anti-anti-erythrocyte is), K=1: 400 (how anti-antifibrin is)+1: 10 (how anti-anti-erythrocyte is).
3), how anti-the red blood cell of only processing 1: 10 on glass fibre is, is treated to F (10).
The last glass fibre that drying and processing is good in 37 ℃ of baking ovens is standby.
2. the preparation 34 of label pad
With conventional collaurum latex, mean grain size is 20-40nm sodium rice. the monoclonal antibody of the anti-CTI of mark rabbit and mouse-anti human IgG be how anti-, and be sprayed at uniformly on polyester film with machine, and under the baking oven of 37 ℃ the good label pad of drying and processing.
3. the preparation 36 of nitrocellulose membrane
First the microsyringe of being controlled by a microprocessor is the how anti-(1.3mg/ml of sheep anti-mouse igg, as testing result control line 32) process to nitrocellulose filter, and then mouse-anti CTI monoclonal antibody (concentration is 4.0mg/ml) is processed to nitrocellulose filter as detection line 33, as analyte calmodulin binding domain CaM.Discharge rate is all 1.1 μ l/cm.After completing, nitrocellulose filter fixes antibody reagent 45 ℃ dry (2 hours) immediately.
4. the assembling of reagent strip
The style assembling of describing according to Fig. 2 detects the reagent strip of CTI, allows glass fibre as absorption of sample pad 35 and is superimposed upon in label pad 34, allows label pad be superimposed upon on nitrocellulose membrane 36 simultaneously; And absorbent filter 31 is superimposed upon composition reagent strip as shown in Figure 2 on nitrocellulose membrane.
5. the preparation of blood sample
Prepare 20 whole blood samples (be stored in 2-8 ℃, acquisition time all surpasses 7 days)
6. testing process
In the sample receiving pad of each processing, drip the whole blood sample of 100ul, and time of occurring of record controls lines (C line), the speed of control line time of occurrence can illustrate liquid mobile speed on reagent strip.According to the standard setting in advance, within 5 minutes, to occur that control line is qualified, in 10 minutes, needed detection, blood sample need to flow through nitrocellulose membrane and then arrives absorbent filter; Otherwise be substandard product.Select F and J to process to carry out sensitivity and specific detection.By negative sample, be configured to positive sample, allow the concentration of CT1 to be detected be 0.5ng/ml, record testing result.Specific detection, detects these reagent strips by 20 of the negative sample of standard.In addition, if there is red background, be also judged to substandard product.
Experimental result
Table 1, the timing that C line occurs
Attention: in the result of table 1, at F, H, although the time that I occurs with control line in the numeral of underscore in processing is all less than 5 minutes, they did not have to complete reaction in 10 minutes, were judged as substandard product yet.
Table 2: disqualification rate
Table 3: sensitivity ("+" represents positive findings, and "-" represents negative findings)
Table 4: specific detection ("+" represents positive findings, and "-" represents negative findings)
7. analyze and conclusion
By experimental result above, can be found out, utilize the antibody of the antifibrin red blood cell in can separating blood sample, greatly reduce the probability that blood blocks chromatography carrier, improve the efficiency of blood separation, and do not bring adverse influence to detecting itself.In addition, if be combined with the antibody of anti-erythrocyte, act on more obvious.
experiment 2, (prostate-specific is anti-detecting PSA to utilize Biciromab to carry out separating blood sample red blood cell
former) in application
With reference to accompanying drawing 2, reagent strip how to assemble this experiment is described equally.Compare with experiment 1, structure does not have too large difference, and erythrocyte antibody (EA) used is identical with source with fibrinous antibody lot number.The analyte that just detects is different and in sample receiving pad, do different processing.
1. the configuration of the antibody of antifibrin and process sample receiving pad
Sample receiving pad is glass fibre, first processes Tris buffer solution in the above, and glass fibre is done to following several different processing.
1), process 1.At glass fibre, process the polyclonal antibody of antifibrin and anti-erythrocyte.The antibody of antifibrin proportionally 1: 300, dilution, and process on glass fibre equably.
2), process 2.On glass fibre, process the polyclonal antibody (buy the company in Genclonn, used in 1 with experiment is same lot number) of anti-erythrocyte.The dilution ratio of red blood cell polyclonal antibody is that 1: 10 last glass fibre that drying and processing is good in 37 ℃ of baking ovens is standby.
3), control treatment CK.The monoclonal antibody (buy the company in Genclonn, lot number is: used in 1 with experiment is same lot number) of processing anti-erythrocyte on glass fibre, dilution ratio is 1: 75.
2. the preparation 34 of label pad
With conventional collaurum latex, particle diameter is 20-40nm sodium rice. how anti-the monoclonal antibody body of the anti-PSA of mark rabbit and mouse-anti human IgG be is sprayed on polyester film uniformly with machine, and under the baking oven of 37 ℃ the good label pad of drying and processing.
3. the preparation 36 of nitrocellulose membrane
First the microsyringe of being controlled by a microprocessor sheep anti-mouse igg, how process to nitrocellulose filter by anti-(1.3mg/ml, as testing result control line 32), and then mouse-anti
pSA monoclonal antibody(concentration is 4.0mg/ml) processes nitrocellulose filter as detection line 31, as analyte calmodulin binding domain CaM.Discharge rate is all 1.1 μ l/cm.After completing, nitrocellulose filter fixes antibody reagent 45 ℃ dry (2 hours) immediately.
4. the assembling of reagent strip
The style assembling of describing according to Fig. 2 detects the reagent strip of PSA, allows glass fibre superpose as absorption of sample pad and with label pad, allows label pad and nitrocellulose membrane and absorbent filter stack form reagent strip as shown in Figure 2 simultaneously.
5. the preparation of blood sample
Prepare 20 7 whole blood samples (be stored in 2-8 ℃, acquisition time was over 7 days).
6. testing process
The whole blood phosphate buffer solution that drips the whole blood sample of 40ul and drip 40ul in the sample receiving pad of each processing help to run plate, and time of occurring of record controls lines (C line), the speed of control line time of occurrence can illustrate liquid mobile speed on reagent strip.According to standard, within 3 minutes, to occur that control line is qualified, in 5 minutes, to complete detection, blood sample need to flow through nitrocellulose membrane and then arrives absorbent filter, otherwise is substandard product.Select contrast and process 1 and carry out sensitivity and specific detection.By negative sample, be configured to positive sample, allow the concentration of PSA to be detected be 2ng/ml, 4ng/ml, 10ng/ml, 20ng/ml, records testing result.Specific detection, detects these reagent strips by 20 of the negative sample of standard.
7. testing result
Table 1: contrast and process the time comparison (second) that 2 control line occurs
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Qualification rate | |
| CK | - ＊ | - | 72 | 60 | - | - | - | - | - | - | 20％ |
| Process 2 | - | 163 | 74 | 134 | - | 136 | - | - | 124 | - | 50％ |
Table 2: process 1 and process the time comparison (second) that 2 control line occurs
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | Averaging time | |
| Process 2 | 110 | - | 170 | 90 | 129 | 100 | 100 | 92 | 67 | 81 | 116 | 148 | 149 | 87 | 124 |
| Process 1 | 103 | 80 | 120 | 96 | 63 | 82 | 84 | 122 | 67 | 64 | 105 | 140 | 116 | 84 | 94 |
Note: "-" is illustrated in sample in 3 minutes can not arrive control line,
*be illustrated in 5 minutes and still can not complete detection.
Table 3: process 1 and contrast remolding sensitivity
" G " represents the color of T line/detection line, and numeral is below larger, represents that color is darker.
Table 4: process 1 and contrast specificity comparison
| ' negative ' specimens | Clinical samples | |
| Contrast CK | G1 100％ | G1 100％ |
| Process 1 | G1 100％ | G1 100％ |
Conclusion
By experimental result above, can be found out, utilize the antibody of the antifibrin red blood cell in can separating blood sample, greatly reduce the probability that blood blocks chromatography carrier, improve the efficiency of blood separation, and do not bring adverse influence to detecting itself.In addition, if be combined with the antibody of anti-erythrocyte, act on more obvious.
experiment 3, accelerated stability experiment.
With the processing F in experiment 1 and J, do accelerated stability experiment, be used for checking the longest effective time of product.Method: the sample of processing F and J is placed at 55 ℃ and is processed, and carry out sensitivity according to the listed number of days of following table 1, specificity is carried out with 10 products at every turn, in the sample receiving pad of each processing, drip the whole blood sample of 100ul, and the time of record controls lines (C line) appearance.
Table 1, stability test number of days and detection time the table of comparisons
| Temperature | Day | Day | Day | Day | Day | Day | Day |
| 0 ＊ | 7 | 14 | 21 | 30 | 35 | 40 | |
| 55℃ | X | X | X | X | X | X | X |
" X " is illustrated in to need to test in the situation of 55 ℃ and verifies.
Experimental result
0 day
Table 2-1 processes the result of F
| NO. | Negative sample | 0.5ng/ml | 5ng/ml | The time that C line occurs |
| 1 | - | 3+ | 5.5+ | 4’15” |
| 2 | - | 3+ | 5.5+ | 3’45” |
| 3 | - | 3+ | 6+ | 4’15” |
| 4 | - | 3.5+ | 5.5+ | 3’15” |
| 5 | - | 3+ | 6+ | 3’10” |
| 6 | - | 3+ | 5.5+ | 3’35” |
| 7 | - | 3.5+ | 6+ | 4’35” |
| 8 | - | 3+ | 5.5+ | 2’40” |
| 9 | - | 3+ | 6+ | 4’35” |
| 10 | - | 3+ | 6+ | 4’15” |
Table 2-2 processes the result of J
| NO. | Negative sample | 0.5ng/ml | 5ng/ml | The time that C line occurs |
| 1 | - | 3+ | 5.5+ | 3’25” |
| 2 | - | 3.5+ | 5.5+ | 3’15” |
| 3 | - | 3+ | 6+ | 4’ |
| 4 | - | 3.5+ | 5.5+ | 2’45” |
| 5 | - | 3+ | 5.5+ | 3’10” |
| 6 | - | 3+ | 5.5+ | 3’05” |
| 7 | - | 3.5+ | 6+ | 3’45” |
| 8 | - | 3+ | 5.5+ | 2’30” |
| 9 | - | 3+ | 6+ | 3’45” |
| 10 | - | 3+ | 6+ | 3’20” |
Table 3 experimental result of the 7th day
| F | J | |
| Negative sample | -，-，- | -，-，- |
| 0.5ng/ml | 3+，3+，3.5+ | 3+，3+，3+ |
| 5ng/ml | 6+，6+，6+ | 6+，6+，6+ |
| The time that C line occurs | 4’20”，3’35”，4’50” | 3’35”，2’45”，3’20” |
Table 4, the experimental result of the 14th day:
| F | J | |
| Negative sample | -，-，，-， | -，-，-， |
| 0.5ng/ml | 3+，3.5+，3.5+ | 3+，3+，3.5+ |
| 5ng/ml | 6.5+，6+，6+ | 6+，6+，6+ |
| The time that C line occurs | 4’50”，4’25”，4’45” | 3’30”，3’15”，3’30” |
Table 5, the experimental result of the 21st day
| F | J | |
| Negative sample | -，-，，-， | -，-，-， |
| 0.5ng/ml | 3+，3.5+，3.5+ | 3+，3+，3.5+ |
| 5ng/ml | 6.5+，6+，6+ | 6+，6+，6+ |
| The time that C line occurs | 5’20”，4’45”，4’10” | 3’40”，3’10”，3’35” |
Table 6, the experimental result of the 30th day:
| F | J | |
| Negative sample | -，-，，-， | -，-，-， |
| 0.5ng/ml | 3+，3.5+，3.5+ | 3+，3+，3.5+ |
| 5ng/ml | 6.5+，6+，6+ | 6+，6+，6+ |
| The time that C line occurs | 6’20”，4’35”，4’25” | 3’50”，3’25”，3’45” |
Table 7, the experimental result of the 35th day::
| F | J | |
| Negative | -，-，，-， | -，-，-， |
| 0.5ng/ml | 3+，3.5+，3.5+ | 3+，3+，3.5+ |
| 5ng/ml | 6.5+，6+，6+ | 6+，6+，6+ |
| The time that C line occurs | 5’10”，5’15”，4’50” | 2’55”，2’35”，2’50” |
Table 8, the experimental result of the 40th day:
| F | J | |
| Negative sample | -，-，，-， | -，-，-， |
| 0.5ng/ml | 3+，3.5+，3.5+ | 3+，3+，3.5+ |
| 5ng/ml | 6.5+，6+，6+ | 6+，6+，6+ |
| The time that C line occurs | 5’10”，4’25”， 5’50” | 3’15”，2’55”，3’35” |
Note: "-" represents negative findings; Numeral adds "+" and represents that result is positive, and numeral is larger, represents that the color of detection lines is darker.
Conclusion
From above experimental result, can obviously find out, when having added the antibody of antifibrin in sample receiving pad when, the effective storage life of product can reach 2 years, has reduced the time of sample arrival C line simultaneously.
Claims (23)
1. a method for separating red corpuscle from blood sample, comprising: allow the antibody of blood sample and antifibrin contact, then the antibody by antifibrin in separating blood sample carrys out red blood cell in separating blood sample.
2. method according to claim 1, is characterized in that: the method also comprises allows blood sample contact with erythrocytic acceptor in a kind of combination blood sample.
3. method according to claim 2, is characterized in that: the antibody of described antifibrin and being fixed on a carrier of supporting liquid flow in conjunction with erythrocytic acceptor.
4. method according to claim 1, is characterized in that: the antibody of described antifibrin comprises fibrinous monoclonal or polyclonal antibody or antibody fragment.
5. method according to claim 3, is characterized in that: described carrier is chromatography carrier.
6. the method for a separating red corpuscle from blood sample, comprise: a kind of chromatography carrier is provided, allow blood sample flow through from this carrier, wherein, on this chromatography carrier, process and have in conjunction with fibrinous antibody in blood sample, the antibody by antifibrin in separating blood sample carrys out red blood cell in separating blood sample.
7. method according to claim 6, is characterized in that: on this carrier, also comprise in conjunction with erythrocytic acceptor.
8. method according to claim 6, described antibody comprises monoclonal, polyclonal antibody or antibody fragment.
9. erythrocytic device in a separating blood sample, comprise: a carrier, on this carrier, fix a kind of antibody of antifibrin, allow blood sample contact with the antibody that is fixed on the antifibrin on carrier, then the antibody by antifibrin in separating blood sample carrys out red blood cell in separating blood sample.
10. device according to claim 9, is characterized in that, described carrier is chromatography carrier, fixes the erythrocytic acceptor of a kind of combination on this chromatography carrier.
11. devices according to claim 9, is characterized in that: the antibody of described antifibrin comprises fibrinous monoclonal, polyclonal antibody or antibody fragment.
12. devices according to claim 10, is characterized in that: the erythrocytic acceptor of described combination comprises erythrocytic monoclonal, polyclonal antibody or antibody fragment.
13. 1 kinds of pick-up units that detect analyte in blood sample, comprise: a carrier, this carrier comprises a surveyed area, it is characterized in that: in the upstream of surveyed area, comprise fibrinous antibody in a kind of combination blood sample, allow the antibody of blood sample and antifibrin contact, then the antibody by antifibrin in separating blood sample carrys out red blood cell in separating blood sample.
14. devices according to claim 13, is characterized in that: this carrier is chromatography carrier.
15. devices according to claim 14, is characterized in that: described device also comprises a sample region of acceptance that is positioned at surveyed area upstream; The antibody of described antifibrin is positioned on sample region of acceptance.
16. devices according to claim 15, is characterized in that: on sample region of acceptance, also comprise the erythrocytic acceptor of a kind of combination.
17. devices according to claim 13, is characterized in that: described antibody comprises monoclonal, polyclonal antibody or antibody fragment.
18. devices according to claim 15, is characterized in that: in downstream, sample region of acceptance, also comprise the material being labeled, this material being labeled can flow to surveyed area with liquid.
19. according to the device one of claim 13-18 Suo Shu, it is characterized in that: a kind of specific bond molecule is fixed on surveyed area.
20. 1 kinds of methods that detect analyte in blood sample, comprising: a kind of chromatography carrier is provided, allows blood sample flow through from chromatography carrier; Measure the content of analyte in blood sample, wherein on this carrier, fix fibrinous antibody in a kind of combination blood sample, this antibody contacts with blood sample, and the antibody by antifibrin in separating blood sample carrys out red blood cell in separating blood sample.
21. methods according to claim 20, is characterized in that, on described carrier, also comprise in conjunction with erythrocytic acceptor.
22. methods according to claim 21, is characterized in that, described acceptor comprises antibody or antibody fragment.
23. methods according to claim 22, is characterized in that, also comprise the surveyed area of analyte in a detection sample in the downstream of this chromatography carrier.
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| US10953101B2 (en) | 2014-02-21 | 2021-03-23 | École Polytechnique Fédérale De Lausanne (Epfl) | Glycotargeting therapeutics |
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| CN106442079B (en) * | 2016-08-31 | 2020-04-14 | 杭州博拓生物科技股份有限公司 | Blood filtration sample pad and preparation method thereof |
| CN106546729B (en) * | 2016-10-18 | 2020-04-24 | 上海凯璟生物科技有限公司 | Novel process method for removing serum matrix effect in dry immunofluorescence quantitative detection |
| CN106840828B (en) * | 2017-03-29 | 2020-02-14 | 天津中新科炬生物制药股份有限公司 | Method and device for quickly separating plasma from trace whole blood |
| WO2018232176A1 (en) | 2017-06-16 | 2018-12-20 | The University Of Chicago | Compositions and methods for inducing immune tolerance |
| CN114414306A (en) * | 2022-01-13 | 2022-04-29 | 杭州安旭生物科技股份有限公司 | Sample collection device and sample collection method |
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