CN105044331B - DDi colloidal gold method detection kit - Google Patents
DDi colloidal gold method detection kit Download PDFInfo
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- CN105044331B CN105044331B CN201510292065.2A CN201510292065A CN105044331B CN 105044331 B CN105044331 B CN 105044331B CN 201510292065 A CN201510292065 A CN 201510292065A CN 105044331 B CN105044331 B CN 105044331B
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The present invention relates to field of biological detection, particularly relate to a kind of D dimer colloidal gold method detection kit and its production and use.A kind of D dimer gold-immunochromatographyreagent reagent for assay box that the present invention provides, including test card, test card includes base plate and is positioned at the sample pad, gold mark pad, nitrocellulose filter and the adsorptive pads that start to be arranged in order from sample-adding end of backplate surface, comprise the anti-D dimer monoclonal antibody 1 of colloid gold label on described gold mark pad, described nitrocellulose filter is coated with detection line and nature controlling line.D dimer colloidal gold method detection kit provided by the present invention has susceptiveness and specificity concurrently, has the advantages such as operation is fast and convenient, result is accurate, economic and practical.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of DDi colloidal gold method detection kit and preparation method thereof
And purposes.
Background technology
DDi is cross filament selective degradation product, is also the peculiar metabolite of Secondary cases fibrinolytic.DDi is respectively
Planting under pathology or physiological status, coagulation system activation causes thromboplastin to convert fibrinogen into fibrin body, and is living
Change and form crosslinked fibrin under the effect of FXIII.DDi is that crosslinked fibrin is released during fibrinolysin is explained
Put fragment further demote produce the fragment containing γ chain.Being affected by pathological factor, body blood coagulation and fibrinolytic dynamic equilibrium are suffered
Destroying, blood coagulation tendency is continuously increased, thus causes fibrin degradation product (FDP) to increase.Therefore, DDi content raises, table
There is fibrinous thrombus in bright body to be formed and fibrinolytic occurs, the most also using the change of DDi content as body Gao Ning
State and the molecular marker of hyperfibrinolysis state.
The detection of DDi is clinical, the inspection project of high specificity higher to Secondary cases fibrinolytic sensitivity, is to judge machine at present
Body hypercoagulability and discriminating constitutional and the aspect such as Secondary cases fibrinolytic, observation thromboembolism treatment effect have the index of important clinical value.
The detection of DDi has great clinical value: can be used for the early diagnosis of disseminated inravascular coagulation (DIC), deeply
Phlebothrombosis (DVT) and the examination of pulmonary infarction, the early diagnosis of acute cerebral infarction, treatment and observe the most afterwards, the treatment of hepatopathy
With diagnosis and to the curative effect of malignant tumor and observation etc. the most afterwards.
Therefore, quick, comprehensive, accurate, the specific detection of DDi is particularly important.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of DDi colloidal gold method detectable
Box and its production and use, is used for solving the problems of the prior art.
For achieving the above object and other relevant purposes, the present invention is by the following technical solutions:
A first aspect of the present invention, it is provided that a kind of DDi colloidal gold method detection kit, including test card, described test card
Including base plate and the sample pad, gold mark pad, nitrocellulose filter and the suction that start to be arranged in order from sample-adding end that are positioned at backplate surface
Water cushion, described gold mark pad comprises anti-D-dimer antibody-1 of colloid gold label, described nitrocellulose filter is coated
There are detection line and nature controlling line.
Preferably, described base plate is PVC base plate.
Preferably, on described nitrocellulose filter, detection line is positioned at side close to sample-adding end, and nature controlling line is positioned at from sample-adding end relatively
Remote side.
Preferably, described detection line is coated with anti-D-dimer antibody-2.
Preferably, described nature controlling line is coated with goat anti-mouse immunoglobulin IgG.
Described anti-D-dimer antibody-1 and anti-D-dimer antibody-2 can be identical monoclonal antibodies,
It can also be different monoclonal antibodies.
Preferably, described anti-D-dimer antibody-1 isAnti-D-Dimer antibody [DD1], described anti-D-
Dimer monoclonal antibody-2 isAnti-D-Dimer antibody [DD1], or described anti-D-dimer antibody-1
ForAnti-D-Dimer antibody [DD1], described anti-D-dimer antibody-2 isAnti-D-Dimer
Antibody [DD2].
Preferably, described sample pad use buffer process, described buffer selected from PBS, Tris-HCl buffer,
The combination of one or more in glycine buffer, borate buffer solution and citrate-phosphate salt buffer, the concentration of buffer
For 50-100mM.
Preferably, gold mark pad of the present invention is also through pretreatment, and the pre-treatment buffer used during pretreatment is selected from sweet ammonia
Acid buffer, Tris-HCl buffer, the combination of one or more of borate buffer solution, the concentration of buffer is 10-40mM.
It is furthermore preferred that so that test kit has more preferably sensitivity and color developing effect, described buffer also includes NaCl and hat
Ether, the combination of one or more in two cyclohexyl-18-crown-s 6,15-crown ether-5, hexaoxacyclooctadecane-6-6 of the described crown ether, NaCl
Concentration in buffer is 0.1-20mg/ml, and crown ether concentration in buffer is 0.1-20mg/ml.
It is furthermore preferred that the concentration that NaCl is in buffer is 0.25-0.5mg/ml, crown ether concentration in buffer is 4-8mg/ml.
The solvent of described pre-treatment buffer is water.
Concretely comprising the following steps of described pretreatment: gold mark pad soaks in pretreatment fluid 1.5～2h, takes out and is put in 36～38 DEG C of drying.
Described pre-treatment buffer can use the various conventional pH adjusting agent in this area to carry out the regulation of pH value.
Preferably, described test kit also includes getting stuck, described in get stuck and include that back card and upper cover, described back card are provided with test card draw-in groove,
Described test card is embedded in described test card draw-in groove, described on be covered with testing window and well, the position of described testing window and institute
Matching in the position stating detection line and nature controlling line, matches with the position of described sample pad in the position of described well.
Get stuck described in it is furthermore preferred that and get stuck for plastics.
Preferably, described detection kit is for detecting the DDi in sample, and described sample is selected from people's whole blood, serum or blood
The combination of one or more in slurry.
Second aspect present invention provides the preparation method of described DDi colloidal gold method detection kit, comprises the steps:
1) with anti-D-dimer antibody-1 solution spraying gold mark pad of colloid gold label, prepare and comprise anti-DDi list
The gold mark pad of clonal antibody-1；
2) on the detection line and nature controlling line of nitrocellulose filter, spray anti-D-dimer antibody-2 respectively and sheep anti mouse is exempted from
Epidemic disease globulin IgG, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On base plate, cutting prepares Test paper card；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
Preferably, gold mark pad of the present invention is also through pretreatment, and the pre-treatment buffer used during pretreatment is selected from sweet ammonia
Acid buffer, Tris-HCl buffer, the combination of one or more of borate buffer solution, the concentration of buffer is 10-40mM.
It is furthermore preferred that so that test kit has more preferably sensitivity and color developing effect, described buffer also includes NaCl and hat
Ether, the combination of one or more in two cyclohexyl-18-crown-s 6,15-crown ether-5, hexaoxacyclooctadecane-6-6 of the described crown ether, NaCl
Concentration in buffer is 0.1-20mg/ml, and crown ether concentration in buffer is 0.1-20mg/ml.
It is furthermore preferred that the concentration that NaCl is in buffer is 0.25-0.5mg/ml, crown ether concentration in buffer is 4-8mg/ml.
The solvent of described pre-treatment buffer is water.
Concretely comprising the following steps of described pretreatment: gold mark pad soaks in pretreatment fluid 1.5～2h, takes out and is put in 36～38 DEG C of drying.
Described pre-treatment buffer can use the various conventional pH adjusting agent in this area to carry out the regulation of pH value.
Third aspect present invention provides described DDi colloidal gold method detection kit in the purposes of DDi detection field.
The invention have the benefit that
DDi colloidal gold method detection kit provided by the present invention has high sensitivity and high specific concurrently, it is possible to quickly detect
DDi.Additionally, described detection kit has the advantages such as operation is fast and convenient, result is accurate, economic and practical.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by disclosed by this specification
Content understand other advantages and effect of the present invention easily.The present invention can also be added by the most different detailed description of the invention
To implement or application, the every details in this specification can also be based on different viewpoints and application, in the essence without departing from the present invention
Various modification or change is carried out under god.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific
Specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments,
Rather than in order to limit the scope of the invention；In description of the invention and claims, unless literary composition additionally clearly refers to
Going out, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, two end points of each numerical range with
And any one numerical value all can be selected between two end points.Unless otherwise defined, all technology used in the present invention and section are academic
The same meaning that language and those skilled in the art of the present technique are generally understood that.In addition to the concrete grammar used in embodiment, equipment, material,
According to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use and the embodiment of the present invention
Described in method, any method, equipment and the material of the similar or equivalent prior art of equipment, material realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use the art normal
Rule molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell cultivate, recombinant DNA technology and
The routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the series
METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS
IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The preparation of embodiment 1 test card of the present invention
1) using pre-treatment buffer that gold mark pad is carried out pretreatment, pre-treatment buffer is: hexaoxacyclooctadecane-6-66mg/ml sodium chloride
0.3mg/ml, the aqueous solution of glycine 2.0mg/ml, pH=7.4, concretely comprising the following steps of pretreatment: by gold mark pad at pretreatment fluid
Middle immersion 2h, takes out and is put in 37 DEG C of drying；Then with colloid gold labelAnti-D-Dimer antibody [DD1] is molten
Liquid spraying pretreated gold mark pad, prepares and is coatedThe gold mark pad of Anti-D-Dimer antibody [DD1], glue in solution
Body gold is 5:1 with the mass ratio of monoclonal antibody, and the concentration of solution is 10mg/ml, and quantity for spray is 4ul/cm；
2) on the detection line and nature controlling line of nitrocellulose filter, spray 1mg/ml's respectivelyAnti-D-Dimer antibody
[DD1] solution and goat anti-mouse immunoglobulin IgG solution, quantity for spray is 1ul/cm, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On PVC base plate, cutting prepares the Test paper card of wide 3-5mm；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
The preparation of embodiment 2 contrast agent box
The preparation of comparative example test card: using 25mM glycine buffer pretreatment gold mark pad, other reagent and experimental technique are equal
With embodiment 1.
1) 25mM glycine buffer pretreatment gold mark pad is used, then with colloid gold labelAnti-D-Dimer
Antibody [DD1] solution spraying pretreated gold mark pad, prepares and is coatedAnti-D-Dimer antibody [DD1]] gold mark
Pad, in solution, gold colloidal is 5:1 with the mass ratio of monoclonal antibody, and the concentration of solution is 10mg/ml, and quantity for spray is 4ul/cm；
2) on the detection line and nature controlling line of nitrocellulose filter, spray 1mg/ml's respectivelyAnti-D-Dimer antibody
[DD1] solution and goat anti-mouse immunoglobulin IgG solution, quantity for spray is 1ul/cm, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On PVC base plate, cutting prepares the Test paper card of wide 3-5mm；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
The sensitivity experiment of embodiment 3 detection kit
The DDi colloidal gold method detection kit that Example 1,2 prepares, is placed on test kit on level table, point
Do not detect 1ug/L, 2ug/L, 4ug/L, 10ug/L, 20ug/L, 50ug/L, 100ug/L, 200ug/L, 300ug/L,
The DDi standard substance of 400ug/L, 500ug/L, 1000ug/L, each concentration is repeated 6 times.With suction pipe pipette samples liquid
2-3 is added dropwise in sample cell, 8-15 minute sentence read result after dropping sample.
Table 1 test kit sensitivity results
Table 1, as a result, it was confirmed that DDi gold-immunochromatographyreagent reagent for assay box sensitivity≤2.0ug/L of preparing of the present invention, has the highest
Sensitivity.Detection kit to embodiment 2 carries out sensitivity test and learns further, the detection kit spirit of embodiment 2
Sensitivity is >=1000ug/L.
The repeatability of embodiment 4 detection kit and stability experiment
One, test kit batch in and batch between repeated experiment
1. experimental technique:
By with batch and the DDi gold-immunochromatographyreagent reagent for assay box of different batches detect respectively 1ug/L, 2ug/L, 4ug/L, 10ug/L,
The D-dimerization of 20ug/L, 50ug/L, 100ug/L, 200ug/L, 300ug/L, 400ug/L, 500ug/L, 1000ug/L
Body standard substance, each concentration is repeated 6 times, and observes the repeatability of test kit.
2. experimental result:
Empirical tests, DDi colloidal gold method detection kit batch in and batch between repeatability be 100%, false positive rate and false negative
Rate is 0.
Two, the stability experiment of test kit
1. experiment purpose:
DDi colloidal gold method detection kit is sealed and preserves, and deposit under 4 DEG C and room temperature (about 25 DEG C), observe
The different storage temperature impacts on stabilization of kit.
2. experimental technique:
The test kit being stored in 4 DEG C takes out weekly 4 boxes, detect respectively 1ug/L, 2ug/L, 4ug/L, 10ug/L, 20ug/L,
The DDi standard substance of 50ug/L, 100ug/L, 200ug/L, 300ug/L, 400ug/L, 500ug/L, 1000ug/L；
Every 3 days of the test kit being stored in room temperature (25 DEG C) takes out 4 boxes, detect respectively 1ug/L, 2ug/L, 4ug/L, 10ug/L,
The D-dimerization of 20ug/L, 50ug/L, 100ug/L, 200ug/L, 300ug/L, 400ug/L, 500ug/L, 1000ug/L
Body standard substance.
3. experimental result:
Empirical tests, paper box can preserve at 4 DEG C 24 months, at room temperature can preserve 18 months；Within the preservable time limit,
Test kit all can reach the detection sensitivity of 2.0ug/L.
In sum, detection kit provided by the present invention has good anti-interference and specificity, and has good spirit
Quick property, negative background is lower, effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any it is familiar with this skill
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage of art.Therefore, such as
All that in art, tool usually intellectual is completed under without departing from disclosed spirit and technological thought etc.
Effect is modified or changes, and must be contained by the claim of the present invention.
Claims (8)
1. a DDi colloidal gold method detection kit, including test card, test card includes base plate and is positioned at backplate surface
Start sample pad, gold mark pad, nitrocellulose filter and the adsorptive pads being arranged in order from sample-adding end, described gold mark pad comprises glue
Anti-D-dimer antibody-1 of body gold labelling, described nitrocellulose filter is coated with detection line and nature controlling line；Described
Gold mark pad uses buffer to process, and described buffer is selected from glycine buffer, Tris-HCl buffer, borate buffer solution
The combination of one or more, the concentration of buffer is 10～40mM；Described buffer also includes NaCl and crown ether, institute
Stating the crown ether combination of one or more in two cyclohexyl-18-crown-s 6,15-crown ether-5, hexaoxacyclooctadecane-6-6, NaCl is slow
Rushing the concentration in liquid is 0.1-20mg/ml, and crown ether concentration in buffer is 0.1-20mg/ml.
2. colloidal gold method detection kit as claimed in claim 1, it is characterised in that on described nitrocellulose filter, detect line position
In side close to sample-adding end, nature controlling line is positioned at from sample-adding end side farther out.
3. colloidal gold method detection kit as claimed in claim 1, it is characterised in that be coated with anti-D-dimerization on described detection line
Body monoclonal antibody-2.
4. colloidal gold method detection kit as claimed in claim 1, it is characterised in that be coated with sheep anti mouse immune globulin on nature controlling line
White IgG.
5. colloidal gold method detection kit as claimed in claim 1, it is characterised in that described sample pad uses buffer to process, institute
State buffer selected from PBS, Tris-HCl buffer, glycine buffer, borate buffer solution and citric acid-phosphorus
The combination of one or more in phthalate buffer, the concentration of buffer is 50～100mM.
6. colloidal gold method detection kit as claimed in claim 1, it is characterised in that also include getting stuck, described in get stuck and include back card
And upper cover, described back card is provided with test card draw-in groove, and described test card is embedded in described test card draw-in groove, described on be covered with survey
Trying window and well, match with the position of described detection line and nature controlling line in the position of described testing window, the position of described well
Put the position with described sample pad to match.
7. colloidal gold method detection kit as claimed in claim 1, it is characterised in that described detection kit is used for detecting in sample
DDi, the combination of one or more in people's whole blood, serum or blood plasma of the described sample.
8., according to the preparation method of the gold-immunochromatographyreagent reagent for assay box described in claim 1～7 any claim, specifically include following step
Rapid:
1) with anti-D-dimer antibody-1 solution spraying of colloid gold label pretreated gold mark pad, prepare comprise anti-
The gold mark pad of DDi monoclonal antibody-1；
2) on the detection line and nature controlling line of nitrocellulose filter, spray anti-D-dimer antibody-2 respectively and sheep anti mouse is exempted from
Epidemic disease globulin IgG, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On base plate, cutting prepares Test paper card；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
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| CN106405078B (en) * | 2016-08-31 | 2018-06-26 | 中山市创艺生化工程有限公司 | A kind of d-dimer immunofluorescence quantitative test paper item and preparation method thereof |
| CN110007094B (en) * | 2019-04-29 | 2022-04-05 | 江苏众红生物工程创药研究院有限公司 | Human D-dimer quantitative detection card and clinical application thereof |
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| JP4578570B1 (en) * | 2010-01-08 | 2010-11-10 | 田中貴金属工業株式会社 | Reagent composition for immunochromatography |
| CN102010472B (en) * | 2010-10-22 | 2012-09-05 | 上海贝西生物科技有限公司 | Anti-D-dimer monoclonal antibody and application thereof |
| US9194859B2 (en) * | 2011-12-23 | 2015-11-24 | Abbott Point Of Care Inc. | Reader devices for optical and electrochemical test devices |
| CN204188618U (en) * | 2014-11-12 | 2015-03-04 | 正元盛邦(天津)生物科技有限公司 | A kind of DDi immunochromatography half-quantitative detection test paper |
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