CN205538994U - Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device - Google Patents
Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device Download PDFInfo
- Publication number
- CN205538994U CN205538994U CN201620357775.9U CN201620357775U CN205538994U CN 205538994 U CN205538994 U CN 205538994U CN 201620357775 U CN201620357775 U CN 201620357775U CN 205538994 U CN205538994 U CN 205538994U
- Authority
- CN
- China
- Prior art keywords
- antibody
- antigen
- line
- microsphere
- stuck
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The utility model relates to a highly sensitive time -resolved fluorescence immunity chromatography detect reagent device, effectual to have solved the sensitivity of detection product low, the problem that differs greatly between criticizing, the technical scheme of its solution is including plastic jam, the fusion5 membrane, the cellulose nitrate membrane and the paper that absorbs water, plastic jam is the rectangle shell, be equipped with the PVC bottom plate in the plastic jam, the centre of PVC bottom plate is equipped with nitrocellulose membranes, the left and right sides of cellulose nitrate membrane respectively the overlap joint fusion5 membrane that is higher than nitrocellulose membranes with paper absorbs water, epimembranal application of sample district and the microballon district of being equipped with of fusion5, be loaded with the time -resolved fluorescence microballon of biotin mark in the microballon district, last detection line (T) and the matter accuse line (C) of being equipped with of nitrocellulose membranes, be equipped with peridium antibody or antigen on the detection line, be equipped with peridium antibody on the matter accuse line, the last application of sample hole of arranging in the application of sample district that is equipped with of plastic jam, the last observation window that is equipped with on detection line and the matter accuse line of plastic jam, the utility model has the advantages of high sensitivity, high specificity conveniently, fast.
Description
Technical field
This utility model relates to biological technical field, particularly a kind of high sensitive time resolved fluorescent immunochromatographiassay assay reagent device.
Background technology
Current immunochromatography (lateral flow immunoassay, LFIA)
Rapid detection test strip is many using gold colloidal or fluorescein as label.Based on colloidal gold-labeled method exploitation quickly detect product, there is sensitivity low, can only qualitative or sxemiquantitative, the problems such as differences between batches are bigger；Immunochromatography sensitivity based on fluorescein labelling technique is greatly improved, and also can carry out detection by quantitative, but owing to sample containing higher autofluorescent background signal, and
Stock displacement is less, detection can be produced large effect.
So this use is novel improves its sensitivity with a kind of highly sensitive detectable device.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the purpose of this utility model is just to provide a kind of high sensitive time resolved fluorescent immunochromatographiassay assay reagent device, effectively solves traditional detection product sensitivity low, can only qualitative or sxemiquantitative, the problem that differences between batches are bigger.
Its technical scheme solved is, get stuck including plastics, Fusion5 film, nitrocellulose membrane and absorbent paper, plastics get stuck for Rectangular shell, plastics are provided with PVC base plate in getting stuck, the centre of PVC base plate is provided with nitrocellulose filter, the left and right sides of nitrocellulose membrane overlaps the Fusion5 film higher than nitrocellulose filter and absorbent paper respectively, Fusion5 film is provided with sample application zone and microsphere district, microsphere district is loaded with biotin labeled time-resolved fluorescence microsphere, nitrocellulose filter is provided with detection line (T) and nature controlling line (C), detection line is provided with coated antibody or antigen, nature controlling line is provided with coated antibody, plastics get stuck and are provided with the well being placed on sample application zone, plastics get stuck and are provided with the observation window detected on line and nature controlling line.
Detection device of the present utility model uses Avidin-Biotin and Ag-Ab system to be amplified by detection signal, exempts from dilution, has easily and fast, the advantage such as high sensitivity, high specific.
Accompanying drawing explanation
Fig. 1 is this utility model cabinet axonometric chart.
Fig. 2 is that this utility model detects card schematic internal view.
Detailed description of the invention
Below in conjunction with accompanying drawing, detailed description of the invention of the present utility model is described in further detail.
nullBe given by Fig. 1 to Fig. 2，This utility model includes that plastics get stuck 1、Fusion5 film 2、Nitrocellulose membrane 3 and absorbent paper 4，Plastics get stuck 1 for Rectangular shell，Plastics get stuck and are provided with PVC base plate 5 in 1，The centre of PVC base plate 5 is provided with nitrocellulose filter 3，The left and right sides of nitrocellulose membrane 3 overlaps the Fusion5 film 2 higher than nitrocellulose filter 3 and absorbent paper 4 respectively，Fusion5 film 2 is provided with sample application zone 6 and microsphere district 7，Microsphere district 7 is loaded with biotin labeled time-resolved fluorescence microsphere 8，Nitrocellulose filter 3 is provided with detection line (T) 9 and nature controlling line (C) 10，Detection line 9 is provided with coated antibody or antigen，Nature controlling line 10 is provided with coated antibody，Plastics get stuck and 1 are provided with the well 11 being placed on sample application zone 6，Plastics get stuck 1 be provided with detection line 9 and nature controlling line 10 on observation window 12.
Described time-resolved fluorescence microsphere 8 surface is through biotin labeling, and particle size range is 20-500nm, is filled with lanthanide compound in fluorescent microsphere 8.
The described coated antibody on detection line 9 is the monoclonal antibody for determined antigen or multi-resistance, or is monoclonal antibody or the multi-resistance of the antigen for test antibodies, the competitive antigen that envelope antigen is determined antigen on detection line 9.
The described coated antibody on nature controlling line 10 is for Avidin traget antibody two to resist, or is the monoclonal antibody for Avidin labelled antigen or multi-resistance.
When this utility model uses,
Reaction dissolvent includes antibody or the antigenic solution of Avidin labelling, and the antibody of Avidin labelling can be the antibody of Avidin labelling or the antibody of marked by streptavidin or Avidin active subunits antibody.
Concretely comprising the following steps of the Reagent Protocol of this utility model test strip:
(1) Avidin traget antibody is used；General labeling method；
(2) biotin labeling fluorescent microsphere is used: general labeling method；
(3) blank kilocalorie is pasted
Plastic bottom board with gum uses the mode of overlap joint, first pastes nitrocellulose filter, then paste respectively at two ends, nitrocellulose filter left and right absorbent paper and
Fusion5 film.
(4) spray film
By biotin labeled fluorescent microsphere buffer a mixed diluting to finite concentration, it is sprayed onto
The fluorescent microsphere district of Fusion5 film；T line and C line buffer b dilute, and are sprayed onto T line and the C line of nitrocellulose filter respectively.
(5) be dried and cutting
The kilocalorie drying of reagent, cutting has been sprayed by above-mentioned.
(6) test strips is loaded during plastics get stuck, envelope.
(7) reactant preparation
With diluent, Avidin traget antibody is diluted to working concentration
(8) reactant subpackage
Reactant is dispensed in each hole according to usage amount, and sealer.
And biomolecule can be measured by double antibody sandwich method or competition law principle when concrete application, it is adaptable to the immunochromatography detection of all of employing double antibody sandwich method pattern or competition law pattern.
nullDouble antibody sandwich method: testing sample adds in reactant，Be combined by the characteristic of the antigen in sample with the antibody of Avidin labelling，Connect into Avidin-Antibody-antigen complex，Liquid containing Avidin-Antibody-antigen complex and the mixture of Avidin-antibody is joined the detection zone of detection card，Be combined with the time-resolved fluorescence nanometer fluorescent microspheres being marked with biotin by chromatography effect Avidin-Antibody-antigen complex and Avidin-antibody，Under capillary action，Fluorescent microsphere-biotin-avidin-Antibody-antigen complex、The mixture of fluorescent microsphere-biotin-avidin-antibody complex and biotin labeling fluorescent microsphere passes sequentially through Fusion5、Nitrocellulose filter，And with on nitrocellulose filter T line fixing another antibodies，Form fluorescent microsphere-biotin-avidin-antibody-antigen-antibody sandwich complex.
In chromatography process, in reactant, the mixture of fluorescent microsphere-biotin-avidin-antibody and biotin labeling fluorescent microsphere is continued to move along by T line and advances, the two anti-captures that fluorescent microsphere-biotin-avidin-antibody nitrocellulose filter C line position is fixed, under wash buffer, the fluorescent microsphere not reacted with Avidin continues to move to adsorptive pads position.
The fluorescent microsphere amount of T line position capture becomes positive correlation with testing sample antigen concentration, the fluorescent microsphere amount of C line position capture becomes positive negative correlation with testing sample antigen concentration, read bar instrument by time-resolved fluorescence T, C line signal is scanned, ratio according to T line signal with C line signal calculates, it is thus achieved that determined antigen concentration in testing sample.
Competition law: testing sample adds in reactant, form the mixture of the antigen in sample and the antibody of Avidin labelling, liquid containing antigen Yu the mixture of the antibody of Avidin labelling is joined the detection zone of detection card, be combined with the time-resolved fluorescence nanometer fluorescent microspheres being marked with biotin by chromatography effect Avidin-antibody, under capillary action, the mixture of fluorescent microsphere-biotin-avidin-antibody-complex, free antigen and biotin labeling fluorescent microsphere passes sequentially through
Fusion5, nitrocellulose filter, in sample, free antigen and immobilized antigen competition binding fluorescent microsphere-biotin-avidin-antibody on T line, form being retained when of fluorescent microsphere-biotin-avidin-Antibody-antigen complex with the antigen being fixed on T line.
In chromatography process, move on complex and the biotin labeling fluorescent microsphere of the fluorescent microsphere-biotin-avidin-antibody-free antigen of free antigen formation in sample, the two anti-captures that fluorescent microsphere-biotin-avidin-antibody-free antigen is fixed at nitrocellulose filter C line position, under wash buffer, unreacted biotin labeling fluorescent microsphere continues to move to adsorptive pads position.
The fluorescent microsphere amount of T line position capture becomes negative correlation with the antigen concentration in testing sample, and the fluorescent microsphere of C line position capture becomes positive correlation with the antigen concentration in testing sample.Read bar instrument by time-resolved fluorescence T, C line signal is scanned, calculate according to the ratio of T line signal with C line signal, it is thus achieved that determined antigen concentration in testing sample.
In order to more express concrete preparation of the present utility model and occupation mode, this utility model provides a kind of specific embodiment to elaborate:
1. the preparation of test strips composition:
(1) preparation of biotin labeled fluorescent microsphere
With the CB of 0.01M by fluorescent microsphere 10mg/ml solution, use DMSO (dimethylformamide) configuration
Biotin-X-X-NHS (N-N-Hydroxysuccinimide modified biological element) solution is to 20mg/ml, and the amount adding 10ul Biotin-X-X-NHS according to 1mg fluorescent microsphere will
Biotin-X-X-NHS liquid joins in fluorescent microsphere solution, mix homogeneously at 4 DEG C of left overnight.Dialysis is used to remove free unreacted biotin, dialysis buffer liquid (NaCl of 0.01M PB, 0.138M, 0.0001MEDTA-Na-2H2O, pH7.4).
(2) preparation of biotin labeled fluorescent microsphere solution
With buffer (containing 7% defatted milk powder, 5% trehalose, 0.5% N, O-double trimethylsilyl acetamide (BSA), 0.1% sodium azide), by biotin labeled fluorescent microsphere solution, matter is final concentration of
0.83mg/ml。
(3) preparation of line (T line) solution is detected
With 10mM PB solution, CRP monoclonal antibody is diluted to 1mg/ml.
(4) preparation of nature controlling line (C line) solution
With 10mM PB solution, anti-mouse antibody is diluted to concentration 0.5mg/ml.
2. the preparation of reactant composition
1. with carbodlimide method by Streptavidin on another strain CRP antibody labeling.
2. with diluent (PBS containing 0.2M, 2% casein, 2.5% N, O-double trimethylsilyl acetamide (BSA), 0.1% sodium azide, 0.01%EDTA-2K), the CRP antibody of labelled streptavidin is diluted to 5 μ g/ml.
3. the preparation of test strips
(1) blank kilocalorie is pasted
According to the film compound mode of accompanying drawing 1, the plastic bottom board with gum uses the mode of overlap joint, first pastes nitrocellulose filter, then paste respectively at two ends, nitrocellulose filter left and right absorbent paper and
Fusion5 film.
(2)
Spray film
In Fig. 1, T, C line solution is sprayed in T line 7, C line 8 position respectively, and T, C line spray film liquid measure is 1 μ l/cm；In Fig. 1, biotin labeled fluorescent microsphere solution is sprayed in 6 positions, and fluorescent microsphere solution spray film amount is 4 μ l/cm.
(3) dry
Step (2) will be sprayed being stuck in greatly in constant temperature oven 37 DEG C and drying 24 hours of reagent.
(4)
Cutting and being installed
CRP kilocalorie by drying cuts into the paper slip of 4mm width, is assembled in plastic housing, forms C reactive protein detection plate.
4. prepared by reactant
The reactant diluted is joined in reactant groove, and sealer.
The CRP antigen standard being separately added into variable concentrations in reagent trough (takes six different concentration mixings, concentration is respectively 0,2.5,10,25,50,100 μ g/mL, each concentration sets 5 repetitions), the most in order the reagent in reagent trough is added Mobile phone card, after film chromatographs 10 minutes, instrument reads C, T line signal.
Time-resolved fluorescence (Time-resolved Fluorescence, TRF) it is compared with common fluorescent, there is stock displacement big, the features such as fluorescence lifetime length, can effectively avoid the background fluorescence in sample, and the impact of the veiling glare such as exciting light, therefore compare common fluorescent and there is higher sensitivity and capacity of resisting disturbance.
Avidin-Biotin system has Cascaded amplification effect, strengthens fluorescence signal, reduces the interference of background fluorescence background, and sensitivity is higher, and testing result is more accurate.
During spray film, the biotin labeled fluorescent microsphere of unified use is sprayed onto on Fusion5 film, different products all uses biotin labeled fluorescent microsphere to spray film, can be mass-produced, for different products, i.e. decrease the batch of different product, make product more stable, need again to debug different labelling microsphere spray films with conventional different product compared with reduce cost savings raw material and energy consumption.
Reactant is liquid single part dress, only need to be added thereto by sample to be tested, mix gently, then according to prescribed dose is added to detect in card sample aperture, simple and easy to operate.
Reactant contains anticoagulant, whole blood is added thereto and is diluted solidifying, improve the hemofiltration effect of Fusion5 film, when reducing whole blood, the detection of reagent is affected by hemocyte, and additionally traditional quantitative immuno-chromatographic test paper strip major part project needs diluted sample to avoid the occurrence of " HOOK " phenomenon before detection, liquid reactant also instead of the effect of diluent, only need to adjust sample loading alternative just can reach the dilution effect of different product, decreases dilution step, convenient, workable.
Time-resolved fluoroimmunoassay chromatograph test strip sensitivity high energy of the present utility model detects, it is possible to the sample that detectable concentration is extremely low, can detect whole blood, serum, blood plasma, urine specimen or other body fluid sample, 300mg/dL simultaneously
Hemoglobin, 700mg/dL triglyceride, 15mg/dL
Bilirubin on the detection of this test strips without impact.
Time-resolved fluoroimmunoassay chromatography detectable device is by double antibody sandwich method or competition law principle, utilize time-resolved fluorescence microsphere as immune marker, determined antigen or antibody are carried out detection by quantitative quickly and accurately, detection method of the present utility model uses Avidin-Biotin and Ag-Ab system to be amplified by detection signal, exempt from dilution, have easily and fast, the advantage such as high sensitivity, high specific.
Claims (4)
- null1. a high sensitive time resolved fluorescent immunochromatographiassay assay reagent device，(1) is got stuck including plastics、Fusion5 film (2)、Nitrocellulose membrane (3) and absorbent paper (4)，It is characterized in that，Plastics get stuck (1) be Rectangular shell，Plastics get stuck and are provided with PVC base plate (5) in (1)，The centre of PVC base plate (5) is provided with nitrocellulose filter (3)，The left and right sides of nitrocellulose membrane (3) overlaps the Fusion5 film (2) higher than nitrocellulose filter (3) and absorbent paper (4) respectively，Fusion5 film (2) is provided with sample application zone (6) and microsphere district (7)，Microsphere district (7) is loaded with biotin labeled time-resolved fluorescence microsphere (8)，Nitrocellulose filter (3) is provided with detection line (T) (9) and nature controlling line (C) (10)，Detection line (9) is provided with coated antibody or antigen，Nature controlling line (10) is provided with coated antibody，Plastics get stuck (1) be provided with the well (11) being placed on sample application zone (6)，Plastics get stuck (1) be provided with detection line (9) and nature controlling line (10) on observation window (12).
- A kind of high sensitive time resolved fluorescent immunochromatographiassay assay reagent device the most according to claim 1, it is characterized in that, described time-resolved fluorescence microsphere (8) surface is through biotin labeling, particle size range is 20-500nm, and fluorescent microsphere is filled with lanthanide compound in (8).
- A kind of high sensitive time resolved fluorescent immunochromatographiassay assay reagent device the most according to claim 1, it is characterized in that, the described coated antibody on detection line (9) is the monoclonal antibody for determined antigen or multi-resistance, or it is monoclonal antibody or the multi-resistance of the antigen for test antibodies, the competitive antigen that envelope antigen is determined antigen in detection line (9).
- A kind of high sensitive time resolved fluorescent immunochromatographiassay assay reagent device the most according to claim 1, it is characterized in that, the described coated antibody on nature controlling line (10) is for Avidin traget antibody two to resist, or is the monoclonal antibody for Avidin labelled antigen or multi-resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620357775.9U CN205538994U (en) | 2016-04-26 | 2016-04-26 | Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620357775.9U CN205538994U (en) | 2016-04-26 | 2016-04-26 | Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN205538994U true CN205538994U (en) | 2016-08-31 |
Family
ID=56797572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201620357775.9U Active CN205538994U (en) | 2016-04-26 | 2016-04-26 | Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN205538994U (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109374903A (en) * | 2018-10-08 | 2019-02-22 | 杭州康知生物科技有限公司 | A kind of hypersensitive IL-6 fluorescence immune chromatography assay kit and measuring method |
| CN110346575A (en) * | 2018-04-04 | 2019-10-18 | 南京东纳生物科技有限公司 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
| CN113495156A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Enzyme conjugate diluent, AMH quantitative detection kit and use method thereof |
-
2016
- 2016-04-26 CN CN201620357775.9U patent/CN205538994U/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346575A (en) * | 2018-04-04 | 2019-10-18 | 南京东纳生物科技有限公司 | A kind of homocysteine fluorescence immune chromatography detection kit and its detection method |
| CN109374903A (en) * | 2018-10-08 | 2019-02-22 | 杭州康知生物科技有限公司 | A kind of hypersensitive IL-6 fluorescence immune chromatography assay kit and measuring method |
| CN113495156A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Enzyme conjugate diluent, AMH quantitative detection kit and use method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xu et al. | Development of lateral flow immunoassay system based on superparamagnetic nanobeads as labels for rapid quantitative detection of cardiac troponin I | |
| KR101540608B1 (en) | Assay strip having variable control line, and diagnosis kit using the same | |
| CA2669667C (en) | Saturation assay | |
| JP6741013B2 (en) | Immunochromatographic test strip | |
| Lin et al. | Washing-free centrifugal microchip fluorescence immunoassay for rapid and point-of-care detection of protein | |
| CN104090248A (en) | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay | |
| WO2009083975A2 (en) | Small molecules and protein analysis devices based on molecular imprinted polymers | |
| CN107132359B (en) | Pepsinogen Cgene and Pepsinogen II detection method and its kit | |
| JP2024074809A (en) | System, device, and method that amplify a signal of lateral flow assay | |
| KR102586991B1 (en) | Immunochromatography device | |
| CN205538994U (en) | Highly sensitive time -resolved fluorescence immunity chromatography detect reagent device | |
| CN105785041A (en) | Test strip for quantitatively detecting calprotectin, preparation method thereof and determining method for calprotectin concentration | |
| CN107328938A (en) | Propepsin and helicobacter pylori antibody detection method and its kit | |
| US20210164974A1 (en) | Chromatographic strip comprising multiple test lines, diagnostic kit comprising same, and qualitative, semi-quantitative or quantitative analysis method comprising multiple competitive reaction measurement steps | |
| JPWO2017094825A1 (en) | Immunochromatographic test piece | |
| US8110403B2 (en) | Immunoassay method | |
| JP5831448B2 (en) | Test instrument for measuring an analyte in a sample and method for measuring an analyte using the same | |
| WO2014051141A1 (en) | Additive for measuring diluted sample in non-dilution-type immunochromatographic method reagent | |
| US20140011190A1 (en) | Method for performing a rapid test | |
| JP7533226B2 (en) | Immunochromatographic test piece and measurement method using the same | |
| JP2001272405A (en) | Examination kit | |
| KR20150111391A (en) | Immunochromatography Analysis Device | |
| JP2010025887A (en) | Simple and easy measurement method | |
| JP2003262637A (en) | Inspection piece and inspection method | |
| WO2024062265A1 (en) | Membrane based chemiluminescence immunochromatography assay and its use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder |
Address after: Yuanhui district business road and South Ring Road of Luohe city of Henan Province in the northwest corner of 462000 Patentee after: HENAN MAINCARE BIOLOGICAL TECHNOLOGY CO., LTD. Address before: Sha Li Luohe City Industrial Agglomeration in Henan province Xiangjiang District 462000 road and five road intersection Patentee before: HENAN MAINCARE BIOLOGICAL TECHNOLOGY CO., LTD. |
|
| CP02 | Change in the address of a patent holder |