CN204188618U - A kind of DDi immunochromatography half-quantitative detection test paper - Google Patents
A kind of DDi immunochromatography half-quantitative detection test paper Download PDFInfo
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- CN204188618U CN204188618U CN201420672665.2U CN201420672665U CN204188618U CN 204188618 U CN204188618 U CN 204188618U CN 201420672665 U CN201420672665 U CN 201420672665U CN 204188618 U CN204188618 U CN 204188618U
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Abstract
The utility model is created and is provided a kind of DDi immunochromatography half-quantitative detection test paper, comprise cellulose nitrate NC film, colloidal gold pad, sample pad and thieving paper, containing D-dimer labelled antibody and rabbit igg labelled antibody in described colloidal gold pad, described cellulose nitrate NC film is being coated with detection line successively along on liquid chromatography(LC) direction, reference line and nature controlling line, the utility model is by the setting of reference line and the accurate control to line antibody content, realize carrying out half-quantitative detection to the plasma sample of D-dimer concentration in this region of 0.1mg/L ~ 0.5mg/L.
Description
Technical field
The invention belongs to people's DDi detection field, and particularly a kind of colloidal gold immunity chromatography that adopts carries out the test paper of half-quantitative detection to people's DDi.
Background technology
DDi (D-dimer) is a kind of selective degradation product through fibrinolysin hydrolysis generation after a kind of fibrin monomer is cross-linked, to a lot of relevant to thrombosis in body and Fibrinolysis activity, the diseases such as such as myocardial infarction, phlebothrombosis, tumour, have important diagnosis, prediction and therapeutic action.
1972, the detection that first Gaffney proposes DDi can be used as " the useful instrument " of monitoring disorders of blood coagulation.By the development of four more than ten years, existing multiple method detects DDi at present, and detecting specimen types can be whole blood, also can be serum and plasma, but generally speaking, be all based on immunology principle, detectable antigens content.Main method has agglutination, enzyme linked immunosorbent assay, colloid gold immune osmosis, immunoturbidimetry etc.Agglutination belongs to qualitative experiment, and susceptibility is low, and scope, 85% ~ 90%, is referred to as medium sensitivity assay usually.Semiquantitative determination must repeatedly measure by doubling dilution, and reagent consumption is large, and result poor repeatability.Enzyme linked immunosorbent assay, the method has the advantage of susceptibility high (> 95%), specificity comparatively strong (being about 40%), belong to extremely sensitive content assaying method, be considered to the classical way that DDi detects.But this method operation steps is complicated, and take time and effort, discomfort is fit to do single sample and emergency treatment sample, can not meet the needs of the timely Treatment and diagnosis of clinical patients.Colloid gold immune osmosis, the method can coordinate detecting instrument, and detect fast, reagent is the In Aluminium Foil Packing of single sealing, plug and play, and can prevent the reagent after opening from making moist, pollute and wasting, susceptibility is higher; Its shortcoming is that specificity is not strong, operation inconvenience.Immunoturbidimetry, this method detects fast, and susceptibility is high, and reagent is liquid, plug and play, and easy to use, after uncork, the term of validity is longer; Shortcoming is that reagent generally can only coordinate specialized equipment to use, and costly, this detects to be unfavorable for Grass-roots Hospital.
Immune colloidal gold technique is that phase early 1970s Faulk and Taylor firstly appears, at first for immunoelectronmicroscopy.Colloidal gold-labeled method is using collaurum as tracer label thing or developer, in a kind of Novel immune labelling technique of antigen-antibody reaction, be used successfully to now the field such as manufacture of Electronic Speculum, flow cytometer, Western blotting, protein staining, in-vitro diagnosis preparation.Current golden labelling technique often coordinates with membrane carrier, forms specific immunoassay formats, as immunity percolation test and immunity-chromatography test etc.Immuno-chromatographic test paper strip be exactly this technology for an external important development direction of examining survey mode fast, be the novel detection technique grown up on modern monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology basis.This technical development is in recent years rapid, clinical diagnosis particularly bedside detect and arrived widespread use, as the detection of infectious disease, early pregnancy, cancer etc. in (POCT).Immune colloidal gold technique and elisa technique belong to amynologic diagnostic method, are all the specific reactions depending on antigen and antibody, but do not need special instrument and equipment, are more suitable for scene and the other detection fast of bed.
By measuring the content of DDi in blood plasma, the existence of activity fibrinolytic can be pointed out.To thrombosis disease as disseminated intravascular coagulation (DIC), pulmonary embolism (PE), lower limb Deep vein embolism (DVT), Acute myocardial embolism (AMI) etc. have comparatively Rapid&Early diagnosis to be worth.DDi mensuration is the screening method of PE indispensability, and < 0.5mg/L can get rid of PE.Although have some reagent manufacturers have developed kit or test paper that Monitoring lower-cut is 0.15mg/L, but all need to coordinate large-scale instrument just can detect, time-consuming, effort and expensive, all needleless is sold the test paper of the detection D-dimer of pulmonary embolism both at home and abroad at present, especially the detection for this region of mg/L, 0.1mg/L ~ 0.5 is often more difficult and loaded down with trivial details, is regarded as the gray area that D-dimer quantitatively detects.Therefore be badly in need of a kind of can the easy colloid gold test paper fast D-dimer in blood plasma being carried out to half-quantitative detection, to facilitate health care worker grasp conditions of patients promptly and accurately, reduce medical treatment cost.
Summary of the invention
The invention provides a kind of DDi immunochromatography half-quantitative detection test paper, efficiently fast and accurately semi-quantitatively detect in real time D-dimer, can have very important economy and social value.
For solving the problems of the technologies described above, the technical scheme that the invention adopts is, comprise cellulose nitrate NC film, upwards colloidal gold pad is overlapped with successively in described cellulose nitrate NC film one end, sample pad, upwards thieving paper is overlapped with at the described cellulose nitrate NC film other end, containing D-dimer labelled antibody and rabbit igg labelled antibody in described colloidal gold pad, described cellulose nitrate NC film is along liquid chromatography(LC) direction being coated with successively detection line (T line), reference line (R line) and nature controlling line (C line), described detection line is the D-dimer antibody matched with described D-dimer labelled antibody, described reference line is that goat-anti rabbit two resists, described nature controlling line is that sheep anti mouse two resists.
Wherein, described D-dimer labelled antibody is the mouse-anti people D-dimer monoclonal antibody of colloid gold label, it detection line is also mouse-anti people D-dimer monoclonal antibody with the D-dimer antibody of its pairing, the two identifies the epi-position that D-dimer is different respectively, and described rabbit igg labelled antibody is the rabbit igg antibody of colloid gold label.
Wherein, the grain size of described collaurum is 15-25nm, is preferably 20-22nm.
Wherein, after the rabbit igg labelled antibody solution of described colloidal gold pad is to be the D-dimer labelled antibody solution of 0.8-1.0 and 540nm place OD value by 540nm place OD value be 0.8-1.0 mixes with volume ratio 1: 1, form with the amount point sample of 2.5 μ l/mm.
Wherein, in described cellulose nitrate NC film, detection line evenly to be rule with 0.0008ml/cm by the D-dimer labelled antibody of 1.2mg/ml and is formed; Reference line evenly to be rule with 0.0008ml/cm by 1.2mg/ml goat-anti rabbit polyclonal antibody and is formed; Described nature controlling line evenly to be rule with 0.0008ml/cm by 2.0mg/ml sheep anti mouse polyclonal antibody and is formed.
Further, 0.4cm is spaced apart between described detection line (T line), reference line (R line) and nature controlling line (C line); Described colloidal gold pad, apart from being spaced apart 0.2-1.0cm between T line, is preferably 0.4-0.6cm.
Wherein, described cellulose nitrate NC film is the porous spline structure film of aperture 8-12 μm, and described sample pad is glass fibre membrane or nonwoven fabrics, and described colloidal gold pad adopts glass fibre membrane, and described thieving paper is absorbent filter, and above material is common used material, commercially available.
The advantage that the invention has and good effect are: easy to operate, be quick on the draw, susceptibility is high, be easy to carry and preserve, being applicable to medical personnel and detecting in real time.And, the present invention is by the independence setting of reference line and the accurate control to each line antibody type and content etc., realize carrying out half-quantitative detection to D-dimer concentration at the plasma sample in this region of mg/L, 0.1mg/L ~ 0.5, there is provided solid reference for medical personnel carry out judgement to relevant disease, especially in the diagnosis of PE and prediction, there is great booster action.
Accompanying drawing explanation
Fig. 1 is the invention structural representation.
Fig. 2 is that the invention uses result to judge schematic diagram.
Wherein:
1-sample pad; 2-colloidal gold pad; 3-cellulose nitrate NC film; 4-T line; 5-R line; 6-C line; 7-thieving paper.
Embodiment
Be further described the invention below by specific embodiment, following detailed embodiment and concrete operating process are only the object setting forth the invention scheme, are not intended to limit the invention.
In following embodiment, the material of non-specified otherwise or reagent, be generally commercial commercially available product, or can obtain from disclosed approach via relevant healthcare institution.The experimental technique of unreceipted actual conditions and parameter in following embodiment, usually conveniently condition and parameter, or the condition of advising according to manufacturer and parameter.
The preparation of embodiment 1 collaurum
0.02% chlorauric acid solution heating is boiled, adds rapidly the trisodium citrate 2mL of 2%; Solution colour becomes dark blue from light blue, continues to add and occurs claret, continue to boil 10min; There is transparent claret, stop heating, continue to be stirred to room temperature, obtain the colloidal gold solution of particle diameter at about 20nm.Electronic Speculum microscopy, guarantee that the gold grain prepared makes it in the same size, even as far as possible, particle diameter at about 20nm, otherwise makes again.
Embodiment 2 is containing the preparation of the colloidal gold pad of labelled antibody
Get 1.5mL test tube, add 1mL colloidal gold solution; Add appropriate borate buffer solution wherein and pH is adjusted to 8.5; Add 10 μ g/mL D-dimer labelled antibodies, make it reach minimum protein concentration, mixing, quick oscillation 20min on shaker; Add the 10%BSA of 10 μ L, mixing, vibration 20min; 10000rpm in hydro-extractor, centrifugal 15min, absorbs supernatant gently; The collaurum precipitation suspending loose by 1mL wash buffer solution weight; The colloid gold immune compound that 540nm place OD (optical density) value is 0.8-1.0 is formulated in fix buffer;
Separately get 1.5mL test tube, add 1mL colloidal gold solution; Add appropriate borate buffer solution wherein and pH is adjusted to 9.0; Add 10 μ g/mL rabbit igg labelled antibodies, make it reach minimum protein concentration, mixing, quick oscillation 20min on shaker; Add the 10%BSA of 10 μ L, mixing, vibration 20min; 8000rpm in hydro-extractor, centrifugal 20min, absorbs supernatant gently; The collaurum precipitation suspending loose by 1mL wash buffer solution weight; The colloid gold immune compound that 540nm place OD (optical density) value is 0.8-1.0 is formulated in fix buffer;
The ratio of above-mentioned two kinds of colloid gold immune compounds according to volume ratio 1: 1 is mixed, with amount point sample on glass fibre membrane of 2.5 μ l/mm, forms colloidal gold pad, and dry under appropriate conditions, be generally 37 DEG C of dryings 12 hours.
The bag quilt of embodiment 3 cellulose nitrate NC film
Adopt spray film instrument, the D-dimer labelled antibody of 1.2mg/ml is evenly drawn with 0.0008ml/cm on cellulose nitrate NC film, forms T line; 1.2mg/ml goat-anti rabbit polyclonal antibody is evenly drawn with 0.0008ml/cm on cellulose nitrate NC film, forms R line; 2.0mg/ml sheep anti mouse polyclonal antibody is evenly drawn with 0.0008ml/cm on cellulose nitrate NC film, forms C line, and be spaced apart 0.4cm between three lines.Then drying room is put into dry under suitable temperature and humidity.
The assembling of embodiment 4 Test paper
In the hothouse that condition is suitable, the cellulose nitrate NC film that the bag that the colloidal gold pad obtain embodiment 2 and embodiment 3 obtain is done and sample pad and thieving paper are cut into suitable size, carry out bonding according to the structure shown in Fig. 1, control colloidal gold pad apart from being spaced apart 0.4-0.6cm between T line, this interval can by controlling bag by the scribing position of NC film, adjustment cutting size and bonding portion size realize, the bonding portion of colloidal gold pad and NC film is not less than 1/7 of colloidal gold pad, the bonding portion of colloidal gold pad and sample pad is 1/7 ~ 1/2 of colloidal gold pad, the bonding portion of thieving paper and NC film is not less than 1/10 of thieving paper.Above-mentioned mutually bonding each several part can in advance or bonding after be fixed on a loading plate, described loading plate can be plastic plate.Suitable size is cut to after fixedly completing.Described Test paper and then can also load one and moulds in box, and on plastic casing, counter sample pad and cellulose nitrate NC film dashed part arrange opening, are convenient for carrying and preserve.
Embodiment 5 Cleaning Principle and method
The principle of double-antibody sandwich colour developing in immunochromatographic assays is make use of in the invention, when containing certain density D-dimer in test plasma, after being added sample pad, mouse source D-dimer monoclonal antibody in liquid dissolves colloidal gold pad in sample, and the mouse source D-dimer monoclonal antibody generation specific binding of D-dimer and this colloid gold label, form Ag-Ab-colloidal gold composite, and continue to move to detection line by capillary action.When sample arrives detection line, above-mentioned Ag-Ab-colloidal gold composite and the D-dimer antibody being coated on the pairing on detection line further combined with, form antibody-antigen-antibody-collaurum double-antibody sandwich compound, and assemble colour developing on detection line.Along with capillary further forward, when arriving nature controlling line, because in colloidal gold pad, D-dimer monoclonal antibody is mouse, therefore no matter in test plasma whether containing D-dimer, all be fixed in the sheep anti mouse two of nature controlling line anti-catch and develop the color, namely this colour developing ensure that whole reaction system is correct.Simultaneously, the invention is in order to semiquantitative object, the rabbit IgG antibody of colloid gold label is also added in colloidal gold pad, and arrange between detection line and nature controlling line can with the anti-reference line of goat-anti rabbit two of its generation specific reaction, no matter in test plasma with or without D-dimer, capital is driving rabbit igg labelled antibody to spring up forward through colloidal gold pad, and there is chromogenic reaction in reference line position, and its colour developing level is set to be equivalent to the colour developing level of D-dimer concentration when 0.5mg/L, like this, just can very easily by observing the concentration information obtaining D-dimer in test plasma intuitively.Due to rabbit igg antibody there is easy acquisition, stationary phase is long, specificity good, susceptibility advantages of higher, the colour developing level under certain concentration can be played consistently when there is specific binding, and interference can not be produced to other specific reactions, therefore become the optimal selection of the invention as colour developing reference substance.
During detection, the plasma sample extracted is balanced to room temperature, sample pad instills tested sample, after 5min, observes the colour developing situation of T line, R line, C line.C line develops the color, and it is identical that T line and R line go out line color, shows that in sample, D-dimer equals 0.5mg/L (see accompanying drawing 2a); C line develops the color, and T line goes out line color and is weaker than R line and goes out line color, shows the D-dimer level (see accompanying drawing 2b) between 0.1-0.5mg/L in sample; C line develops the color, and T line goes out line color and is better than R line and goes out line color, shows that the D-dimer in sample is greater than 0.5mg/L (see accompanying drawing 2c); C line, R line develop the color, and T line does not develop the color, and shows that the D-dimer level in sample is less than 0.1mg/L (see accompanying drawing 2d); C line or R line do not develop the color (see accompanying drawing 2e, 2f, 2g), show the failure of an experiment, need retest.
Claims (6)
1. a DDi immunochromatography half-quantitative detection test paper, comprise cellulose nitrate NC film, upwards colloidal gold pad is overlapped with successively in described cellulose nitrate NC film one end, sample pad, upwards thieving paper is overlapped with at the described cellulose nitrate NC film other end, containing D-dimer labelled antibody and rabbit igg labelled antibody in described colloidal gold pad, described cellulose nitrate NC film is being coated with detection line successively along on liquid chromatography(LC) direction, reference line and nature controlling line, described detection line is the D-dimer antibody matched with described D-dimer labelled antibody, described reference line is that goat-anti rabbit two resists, described nature controlling line is that sheep anti mouse two resists.
2. a kind of DDi immunochromatography half-quantitative detection test paper according to claim 1, it is characterized in that: described D-dimer labelled antibody is the mouse-anti people D-dimer monoclonal antibody of colloid gold label, it detection line is also mouse-anti people D-dimer monoclonal antibody with the D-dimer antibody of its pairing, the two identifies the epi-position that D-dimer is different respectively, and described rabbit igg labelled antibody is the rabbit igg antibody of colloid gold label.
3. a kind of DDi immunochromatography half-quantitative detection test paper according to claim 2, is characterized in that: the grain size of described collaurum is 15-25nm.
4. a kind of DDi immunochromatography half-quantitative detection test paper according to claim 1, it is characterized in that: after the rabbit igg labelled antibody solution of described colloidal gold pad is to be the D-dimer labelled antibody solution of 0.8-1.0 and 540nm place OD value by 540nm place OD value be 0.8-1.0 mixes with volume ratio 1: 1, form with the amount point sample of 2.5 μ l/mm.
5. a kind of DDi immunochromatography half-quantitative detection test paper according to claim 1, is characterized in that: in described cellulose nitrate NC film, and detection line evenly to be rule with 0.0008ml/cm by the D-dimer labelled antibody of 1.2mg/ml and formed; Reference line evenly to be rule with 0.0008ml/cm by 1.2mg/ml goat-anti rabbit polyclonal antibody and is formed; Described nature controlling line evenly to be rule with 0.0008ml/cm by 2.0mg/ml sheep anti mouse polyclonal antibody and is formed.
6. a kind of DDi immunochromatography half-quantitative detection test paper according to claim 1, is characterized in that: be spaced apart 0.4cm between described detection line, reference line and nature controlling line; Described colloidal gold pad is apart from being spaced apart 0.2-1.0cm between detection line.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104865382A (en) * | 2015-06-01 | 2015-08-26 | 上海凯创生物技术有限公司 | Detecting kit for detecting D-dimer according to near-infrared fluorometric method |
| CN105044331A (en) * | 2015-06-01 | 2015-11-11 | 上海凯创生物技术有限公司 | Detection kit with D-dimer colloidal gold method |
| CN106950373A (en) * | 2017-03-29 | 2017-07-14 | 杭州博拓生物科技股份有限公司 | A kind of half-quantitative detection kits of CA15 3 and preparation technology |
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2014
- 2014-11-12 CN CN201420672665.2U patent/CN204188618U/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104865382A (en) * | 2015-06-01 | 2015-08-26 | 上海凯创生物技术有限公司 | Detecting kit for detecting D-dimer according to near-infrared fluorometric method |
| CN105044331A (en) * | 2015-06-01 | 2015-11-11 | 上海凯创生物技术有限公司 | Detection kit with D-dimer colloidal gold method |
| CN106950373A (en) * | 2017-03-29 | 2017-07-14 | 杭州博拓生物科技股份有限公司 | A kind of half-quantitative detection kits of CA15 3 and preparation technology |
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