CN109142336A - The kit and its detection method of spatial neighbor chemoluminescence method detection d-dimer - Google Patents
The kit and its detection method of spatial neighbor chemoluminescence method detection d-dimer Download PDFInfo
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to the kits and its detection method of a kind of spatial neighbor chemoluminescence method detection d-dimer.Kit includes: enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；Wherein, calibration object includes the calibration object and 0.1M phosphate dilution of various concentration D-Dimer antigen；The component of enzyme marker includes the D-Dimer detection antibody and 0.05M phosphate buffer of peroxidase labelling；The component of luminous marker includes the D-Dimer capture antibody and 0.05M Tris buffer of acridan label；The component of adjuvant includes shine adjuvant and citrate buffer；It triggers agent and selects 0.05M Tris buffer.Detection method provided by the invention is not necessarily to carrier, is not coated with, washing process as a kind of homogeneous chemistry luminescence technology truly, and reagent constituents are few, and high sensitivity, reproducible, easy to operate.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of examination of spatial neighbor chemoluminescence method detection d-dimer
Agent box and its detection method.
Background technique
D-dimer (D-Dimer) is the catabolite of crosslinked fibrin, formation mechenism are as follows: fibrinogen is solidifying
Under the action of hemase, polypeptide A and polypeptide B are successively sloughed from α chain and β chain, forms fibrin I and fibrin II, fiber egg
White crosslinking under the action of factor XI, plasma thromboplastin antecedent I on vascular wall, and the plasmin cleavage that is activated and generate various FDP fragments.Due to
The crosslinking of γ chain just produces 2 D segments being connected comprising γ chain, i.e., d-dimer, its molecular weight are about 180,000 dongles
, Half-life in vivo 8h.
D-dimer is that secondary fibrinolytic hyperfunction one of marker, a variety of diseases can cause in specific antimer
The raising of d-dimer.Research data shows: horizontal, the cardiovascular disease closely related with cardiovascular disease of d-dimer in blood plasma
The height of sick Plasma D-dimer Levels positive rate is followed successively by dissection of aorta, acute myocardial infarction AMI, unstable angina pectoris, steady
Sizing angina pectoris, congenital heart disease, arrhythmia cordis, show that the state of an illness gets over crisis, d-dimer ascensional range is bigger, and positive rate is got over
It is high.Wherein dissection of aorta positive rate is up to 100%, the detection of d-dimer to it with extraordinary negative desired value, therefore
It has higher application value for the exclusion diagnosis of dissection of aorta.
Deep vein thrombosis (DVT) formation is the main susceptible factor of pulmonary embolism, the blood of about 51%~71% pulmonary embolism
Bolt embolus derives from DVT.Though the discovery of DVT cannot direct Diagnosis of Pulmonary Embolism, very big prompt can be given.It is made by vein
The inspections such as shadow, ultrasonic Doppler vascular test and limbs impedance plethysmogram facilitate diagnose DVT, and at present many studies have shown that
Plasma D-dimer Levels detection is also a kind of effective means of DVT screening.Research finds that phlebography turns out to be the patient of DVT
Plasma D-Dimer levels increase, according to d-dimer > 500ng/mL be the positive, then the sensibility and specificity checked
Respectively 95% and 77%, negative predictive value 92%.Therefore, if clinically suspecting DVT patient's d-dimer testing result just
Often, then facilitate the diagnosis of exclusion VT.
In terms of anticoagulant and thromboembolism treatment, d-dimer also has certain guidance meaning, can be used as secondary increased fibrinolytic activity
Mark, observe anticoagulant heparin effect and thrombolysis.After thromboembolism treatment, the visible D-dimer level of 4~8h obviously increases
Height, this is dissolved with direct relation with a large amount of fresh thrombus, visible later to gradually decrease, and in for 24 hours~7d maintains to normal level.
This treatment of explanation at this time has no positive effect, and thrombus size variation is little, if occurring increasing phenomenon again after reducing, shows
Thrombus is formed again, and oldness thrombosis patients are not in D-dimer level rise phenomenon, illustrate exist if increasing extremely
New thrombosis.In Acute Cerebral Infarction thromboembolism treatment, with thrombolysis, in blood plasma d-dimer content sharply on
It rises, when thrombus is completely dissolved, after revascularization, content declines rapidly.Such as continue higher level not drop, prompts thrombus not complete
Fully dissolved has secondary thrombus to be formed, thus in Cerebral Infarction Treatment, especially in thromboembolism treatment, dynamic detection d-dimer contains
Amount has important clinical significance to diagnosis of cerebral infarction, observation of curative effect and prognosis.
D-dimer is the selective degradation product of crosslinked fibrin, is the marker of thrombosis and dissolution.It
It generates and increases and show to exist in vivo hypercoagulative state, thrombosis and secondary fibrinolytic enhancing.There is document report: the thrombus of AMI
Formation rate highest, SAP incidence of thrombus is minimum, and UAP thrombosis rate is between AMI and SAP.Therefore, d-dimer is measured
There is certain reference value for the clinical classification of coronary heart disease.
Clinically the detection of D-Dimer content mainly uses immunoturbidimetry, enzyme-catalyzed chemical luminescence method, non-enzymatic at present
Learn luminescence method.The immunoturbidimetry range of linearity is narrow, and sensitivity is low, and is affected by blood-lipoids；Enzyme-catalyzed chemical luminescence mainly wraps
Horseradish peroxidase (HRP), alkaline phosphatase (ALP) system etc. are included, common ground is the enzyme in luminescence process as marker
Substantially it is not consumed；Non- enzyme-catalyzed chemical luminescence includes acridinium ester, oxalate system etc., and common ground is marker quilt in luminescence process
Consumption.Regardless of enzymatic and non-enzyme-catalyzed chemical luminescence, it is required to solid phase carrier (such as microwell plate or magnetic particle), and after the reaction was completed
It needs to clean reaction compound, to remove unbonded free ingredient.Its is complicated for operation, and influence factor is more, to make
Unstable at testing result, repeatability is bad.
Summary of the invention
For the defects in the prior art, it is an object of that present invention to provide a kind of spatial neighbor chemoluminescence methods to detect D- bis-
The kit and its detection method of aggressiveness.The method is not necessarily to carrier, does not have as a kind of homogeneous chemistry luminescence technology truly
There are coating, washing process, reagent constituents are few, and high sensitivity, reproducible, easy to operate.
To achieve the above object, technical solution provided by the invention are as follows:
In a first aspect, the present invention provides a kind of detection kit of d-dimer, kit includes: enzyme marker, shines
Marker, adjuvant, triggering agent and calibration object；Wherein, the raw material components of enzyme marker include the D- of peroxidase labelling
Dimer detects antibody, and the raw material components of luminous marker include that the D-Dimer of 9,10- acridan label captures antibody.
Preferably, the raw material components of enzyme marker further include 0.05M phosphate buffer；It is highly preferred that preparation method packet
It includes: weighing 5mg HRP and be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH4.4, and 4 DEG C overnight；Add 20
9.5 carbonate buffer solution of μ L 0.2M pH, the addition anti-D-Dimer monoclonal antibody room temperature of 1mg, which is protected from light, immediately after is gently mixed
2h adds the 4mg/mL sodium borohydride liquid newly matched, and mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, to 0.15M pH
7.4PBS dialysis, 4 DEG C overnight；Dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Preferably, the raw material components of luminous marker further include 0.05M Tris buffer；It is highly preferred that preparation method packet
Include: the luminous substrate Acridan DMF of 500 μ L dissolves；The Acridan41.3 μ L of dissolution is drawn, it is slow that 0.05M Boratex is added
708.7 μ L of fliud flushing adds the anti-D-Dimer monoclonal antibody of 250 μ L, and overturning mixes 4~5 times, stands 30min at room temperature；
Label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, taking-up addition qs glycerin is set -20 DEG C of refrigerators and saved.
Preferably, the component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；It is highly preferred that preparation
Method includes: to weigh citric acid 1.82g, and sodium citrate 10.45g adds pure water to dissolve and is settled to 1000mL, slow in citrate
Suitable luminous adjuvant is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, 0.05M pH 8.0Tris-HCl buffer is selected in triggering agent；It is highly preferred that preparation method includes: to claim
Tris 6.06g, sodium chloride 9g are taken, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes.It is added and spits in above-mentioned solution
- 20 2mL of temperature are settled to 1000mL after mixing, and dispense after mixing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, calibration object includes the calibration object and 0.1M phosphate buffer of various concentration D-Dimer antigen；More preferably
Ground, preparation method include: to prepare calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds
Appropriate ultrapure water dissolution, 0.5~1mL of Proclin-300 after mixing, add ultrapure water to be settled to 1000mL, and as calibration object is dilute
Liquid is released, 2~8 DEG C store for future use；Prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL,
1000ng/mL, 10000ng/mL are diluted to respective concentration for d-dimer is purified with calibration object dilution, and 2~8 DEG C of storages are standby
With.
Kit of the present invention detects the content of d-dimer (D-Dimer) in human plasma using double antibody sandwich method.By sample
Originally, the anti-D-Dimer monoclonal antibody of horseradish peroxidase (HRP) label, acridan (Acridan) mark
Anti- D-Dimer monoclonal antibody one reacts, and antigen-antibody sandwich complex is formed, so that horseradish peroxidase and 9,10-
Acridan (Acridan) is spatially able to close to each other, the luminous adjuvant of addition and triggering agent, generation flash type chemistry hair
Light；And unbonded free HRP labelled antibody and Acridan labelled antibody does not shine then.D-Dimer content in sample is got over
The luminous value (RLU) of height, measurement is bigger.So luminous value is positively correlated with concentration of specimens within the scope of a certain concentration, pass through
The calibration object and its luminous value of known concentration draw working curve, can calculate D- in sample according to the luminous value of sample
The content of Dimer.
Second aspect, detection kit provided by the invention answering in spatial neighbor chemoluminescence method detection d-dimer
With.
The third aspect, above-mentioned detection kit provided by the invention is when spatial neighbor chemoluminescence method detects d-dimer
Method, comprising steps of S1: be separately added into 25 μ L standard items, 25 μ L peroxidase labellings D-Dimer detection antibody and 25
μ L luminous marker is to reaction tube；S2: 15min is reacted in 37 DEG C of insulating boxs；S3: 5 μ L adjuvants are separately added into reaction tube, shake
Mixing is swung, 1~2min is stood；It is separately added into triggering 75 μ L of agent later, concussion is mixed, detected immediately, reads signal value；S4: right
The concentration and luminous value of calibration object carry out tetra- parameter fitting of logistic, calculate concentration of specimens by sample luminous value.
Technical solution provided by the invention, have it is following the utility model has the advantages that
(1) it is needed with traditional chemiluminescence using microwell plate or magnetic particle as carrier coated antibody or antigen phase
Than, carrier is not necessarily in kit provided by the invention and detection method, is not coated with, washing process, be it is a kind of truly
Homogeneous chemistry luminescence technology.
(2) traditional enzyme-catalyzed chemical luminescence technology is in the test analyte successively detection antibody with capture antibody and enzyme label
It must be cleaned after combining 2~3 times, to remove the unstable non-specific substance of be not associated with or combination；And the present invention is
After the combination of test analyte and two specific antibodies, the interference that shines is eliminated by adjuvant effect, triggering agent is added and generates
Luminous signal, whole process is without washing.
(3) traditional enzyme-catalyzed chemical luminescence technology needs substrate for enzymatic activity, 5~10 minutes or so detection luminous values；And this
Invention is flash type chemiluminescence, generates luminous signal immediately after triggering agent is added.
(4) reagent constituents provided by the invention are few, and production process is simple, and production cost reduces, and are easy to amplify production；And
Detection process is convenient, it is easy to accomplish full-automatic.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described.Implement below
Example is only used for clearly illustrating technical solution of the present invention, therefore is intended only as example, and cannot be used as a limitation and limit this hair
Bright protection scope.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantitative test in following embodiment,
Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.
The present invention provides a kind of detection kit of d-dimer, and kit includes: enzyme marker, luminous marker, auxiliary
Auxiliary agent, triggering agent and calibration object；Wherein, the component of enzyme marker include peroxidase labelling D-Dimer detection antibody and
0.05M phosphate buffer；The component of luminous marker include acridan label D-Dimer capture antibody and
0.05M Tris buffer；The component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；Agent is triggered to select
0.05M pH 8.0Tris-HCl buffer；Calibration object includes the calibration object and 0.1M calibration object of various concentration D-Dimer antigen
Dilution.
One, the preparation of calibration object
(1) it prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, 0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, and 2~8 DEG C
It stores for future use.
(2) prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL,
10000ng/mL is diluted to respective concentration for d-dimer is purified with calibration object dilution, and 2~8 DEG C store for future use.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4,
4 DEG C overnight.
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, the anti-D-Dimer monoclonal antibody of 1mg is added immediately after
Room temperature, which is protected from light, is gently mixed 2h, adds the 4mg/mL sodium borohydride liquid newly matched, and mixes, and in 4 DEG C of placement 2h, above-mentioned liquid is packed into and is dialysed
It in bag, dialyses to 0.15M pH 7.4PBS, 4 DEG C overnight.
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved.
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, adds 250 μ L
Anti- D-Dimer monoclonal antibody, overturning mix 4~5 times, stand 30min at room temperature.
(3) label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, taken out addition qs glycerin and set -20 DEG C
Refrigerator saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled
200mL。
D- bis- is detected in spatial neighbor chemoluminescence method using d-dimer detection kit the present invention also provides a kind of
Method when aggressiveness, comprising steps of
S1: 25 μ L standard items, the D-Dimer detection antibody of 25 μ L peroxidase labellings and 25 μ L are separately added into and shines and marks
Remember object to chemiluminescence reaction pipe.
S2: 15min is reacted in 37 DEG C of insulating boxs.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, calculate sample by sample luminous value
This concentration.
Technical solution provided by the invention is described further combined with specific embodiments below.
Embodiment one
The present embodiment provides a kind of detection kits of d-dimer, comprising: enzyme marker, luminous marker, adjuvant,
Trigger agent and calibration object；Wherein, calibration object includes the calibration object and 0.1M phosphoric acid of no less than 6 various concentration D-Dimer antigen
Salt buffer；Enzyme marker includes the D-Dimer detection antibody and 0.05M phosphate buffer of peroxidase labelling；It shines
Marker includes the D-Dimer capture antibody and 0.05M Tris buffer of acridan label；The group subpackage of adjuvant
Include 6.0 citrate buffer of luminous adjuvant and pH；It triggers agent and selects 0.05M pH 8.0Tris-HCl buffer.
One, the preparation of calibration object
(1) it prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, Proclin-300 0.8mL after mixing, add ultrapure water to be settled to 1000mL, obtain calibration object dilution, and 4 DEG C of storages are standby
With.
(2) prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL,
10000ng/mL is diluted to respective concentration for d-dimer is purified with calibration object dilution, and 4 DEG C store for future use.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4,
4 DEG C overnight；
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, the anti-D-Dimer monoclonal antibody of 1mg is added immediately after
Room temperature, which is protected from light, is gently mixed 2h, adds the 4mg/mL sodium borohydride liquid newly matched, and mixes, and in 4 DEG C of placement 2h, above-mentioned liquid is packed into and is dialysed
It in bag, dialyses to 0.15M pH 7.4PBS, 4 DEG C overnight；
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved；
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, adds 250 μ L
Anti- D-Dimer monoclonal antibody, overturning mix 5 times, stand 30min at room temperature；
(3) label reaction tube is placed on shaking table, is mixed at 4 DEG C overnight, taken out addition qs glycerin and set -20 DEG C of refrigerators
It saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Embodiment two
D- dimerization is detected in spatial neighbor chemoluminescence method using d-dimer detection kit the present embodiment provides a kind of
Method when body, comprising steps of
S1: 25 μ L standard items, the D-Dimer detection antibody of 25 μ L peroxidase labellings and 25 μ L are separately added into and shines and marks
Remember object to chemiluminescence reaction pipe.
S2: 15min is reacted in 37 DEG C of insulating boxs.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, calculate sample by sample luminous value
This concentration.
The present invention detects d-dimer using spatial neighbor chemoluminescence method, and sensitivity can reach 10ng/mL；D-dimer
It is 86.3% to acute cardiovascular disease specificity, is especially 100% (23/ to diagnosis of aorta dissection positive rate
23)；To the sensibility of diagnosis of acute pulmonary embolism up to 90% or more.
Certainly, the case where being enumerated in addition to embodiment one and embodiment two, other conditions and parameter in preparation process etc.
It is possible.
Applicant has found after creative work: detection method provided by the invention is as a kind of truly equal
Phase chemiluminescence is not necessarily to carrier, is not coated with, washing process, and reagent constituents are few, and high sensitivity, reproducible, behaviour
Make simple.
It should be noted that unless otherwise indicated, technical term or scientific term used in this application should be this hair
The ordinary meaning that bright one of ordinary skill in the art are understood.Unless specifically stated otherwise, it otherwise illustrates in these embodiments
Component and opposite step, numerical expression and the numerical value of step are not limit the scope of the invention.It is illustrated and described herein
In all examples, unless otherwise prescribed, any occurrence should be construed as merely illustratively, not as limitation, because
This, other examples of exemplary embodiment can have different values.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover in protection scope of the present invention.
Claims (8)
1. a kind of detection kit of d-dimer, which is characterized in that the kit includes:
Enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；
Wherein, the raw material components of the enzyme marker include the D-Dimer detection antibody of peroxidase labelling, the luminous mark
The raw material components of note object include the D-Dimer capture antibody of acridan label.
2. the detection kit of d-dimer according to claim 1, it is characterised in that:
The raw material components of the enzyme marker further include 0.05M phosphate buffer；
Preferably, preparation method includes:
It weighs 5mg HRP to be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4, and 4 DEG C overnight；
20 μ L 0.2M pH, 9.5 carbonate buffer solution is added, the anti-D-Dimer monoclonal antibody room temperature of 1mg is added immediately after and keeps away
Light is gently mixed 2h, adds the 4mg/mL sodium borohydride liquid newly matched, and mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter,
It dialyses to 0.15M pH 7.4PBS, 4 DEG C overnight；
Dialysate is taken out, adds qs glycerin to be placed on -20 DEG C of refrigerators and saves.
3. the detection kit of d-dimer according to claim 1, it is characterised in that:
The raw material components of the luminous marker further include 0.05M Tris buffer；
Preferably, preparation method includes:
The luminous substrate Acridan DMF of 500 μ L is dissolved；
The 41.3 μ L of Acridan of dissolution is drawn, 708.7 μ L of 0.05M sodium borate buffer liquid is added, adds the 250 anti-D- of μ L
Dimer monoclonal antibody, overturning mix 4~5 times, are stored at room temperature 30min；
Label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, addition qs glycerin is taken out and is placed on -20 DEG C of refrigerators
It saves.
4. the detection kit of d-dimer according to claim 1, it is characterised in that:
The component of the adjuvant includes the citrate buffer of luminous adjuvant and pH 6.0；
Preferably, preparation method includes:
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, in citrate buffer
It is middle that the adjuvant that shines is added, it is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
5. the detection kit of d-dimer according to claim 1, it is characterised in that:
0.05M pH 8.0Tris-HCl buffer is selected in the triggering agent；
Preferably, preparation method includes:
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added and spits later
- 20 2mL of temperature, are settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 200mL.
6. the detection kit of d-dimer according to claim 1, it is characterised in that:
The calibration object includes the calibration object and 0.1M calibration object dilution of various concentration D-Dimer antigen；
Preferably, preparation method includes:
It prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, NaH2PO4·2H2O 3.0g, adds ultrapure water to dissolve,
0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, 2~8 DEG C of storages
It is spare；
Prepare calibration object: the concentration of calibration object be 0,10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL, 10000ng/mL,
It is diluted to respective concentration by d-dimer is purified with the calibration object dilution, 2~8 DEG C store for future use.
7. the described in any item detection kits of claim 1~6 are in spatial neighbor chemoluminescence method detection d-dimer
Using.
8. method of the described in any item kits of claim 1~6 when spatial neighbor chemoluminescence method detects d-dimer,
Comprising steps of
S1: the D-Dimer detection antibody and 25 μ L luminous markers of 25 μ L standard items, 25 μ L peroxidase labellings are separately added into
To chemiluminescence reaction pipe；
S2: 15min is reacted in 37 DEG C of insulating boxs；
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；It is separately added into touching later
75 μ L of agent is sent out, concussion is mixed, detected immediately, reads signal value；
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, and it is dense to calculate sample by sample luminous value
Degree.
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| CN202110794241.8A CN113514448A (en) | 2018-09-25 | 2018-09-25 | Application of kit for detecting D-dimer by using space proximity chemiluminescence method |
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| CN113376378A (en) * | 2020-02-25 | 2021-09-10 | 上海奥普生物医药股份有限公司 | D-dimer detection kit, preparation method and application |
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| CN107525933A (en) * | 2017-07-21 | 2017-12-29 | 王贤俊 | The measure kit and its application process of a kind of D dimers |
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