CN106771145A - One kind detection D dimers spot gold diafiltration kit and quantitative detecting method - Google Patents
One kind detection D dimers spot gold diafiltration kit and quantitative detecting method Download PDFInfo
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- CN106771145A CN106771145A CN201611094832.XA CN201611094832A CN106771145A CN 106771145 A CN106771145 A CN 106771145A CN 201611094832 A CN201611094832 A CN 201611094832A CN 106771145 A CN106771145 A CN 106771145A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
Abstract
The present invention provides a kind of detection D dimers spot gold diafiltration kit and its quantitative detecting method, and kit includes pre- confining liquid, the cleaning solution that pH value is 6.2, colloid gold label D homodimeric antibodies, D dimers chromatography device and calibration card that pH value is 6.7；The pre- confining liquid includes Triton 100 and borate buffer solution；The cleaning solution includes bovine serum albumin(BSA) and borate buffer solution.It is solid phase carrier that the present invention uses nitrocellulose filter, it is solid phase with the antibody adsorbed on nitrocellulose, antigen and antibody labeling collaurum are as liquid phase with human blood, when the antibody marked by gold grain is combined with corresponding antigen ligand, gold grain just forms macroscopic punctation when antibody ligand site is largely assembled.The present invention has that detection time is short, and precision is good, and the requirement to instrument and environment is relatively low, the advantages of be easy to clinical application.
Description
Technical field
The invention belongs to field of medical examination, more particularly to a kind of detection DDi (D-Dimer) spot gold diafiltration examination
Agent box and its quantitative detecting method.
Background technology
After DDi is the crosslinking of fibrin monomer activated FXIII, then through the one kind produced by fibrinolytic enzyme hydrolysis
Selective degradation product, is a specific fibrinolytic process markup thing.DDi is fine from the crosslinking of fibrinolysin dissolving
Fibrillarin grumeleuse.
Under physiological status, body remains blood coagulation and fibrinolytic dynamic equilibrium, to ensure that fibrin is formed and clear in time
Remove, and the level of human normal D dimer is general in below 200 μ g/l, if blood coagulation in vivo and fibrinolytic system dual activation, D
Dimer levels will be raised, therefore D dimer can be as one of molecular marker of internal hypercoagulative state and hyperfibrinolysis.
What the specificity of D dimer reflected is the common pathological characteristic of this class disease for having blood coagulation and fibrinolytic process, therefore in clinic
Using quite varied.D dimer as determine fibrinolytic system Main Factors, for Clinics and Practices fibrinolytic system disease and with
The relevant disease of fibrinolytic system and thromboembolism treatment detection have great significance, therefore D dimer is DVT
(DVT), pulmonary embolism (PE), the key index of disseminated intravascular coagulation (DIC).As can be seen here, D dimer contains measurement in blood
Determining the early diagnosis to thrombotic diseases, course of disease monitoring and the Treatment monitoring to thrombolytic drug etc. all has important clinical meaning
Justice.
DDi detection technique mainly has latex agglutination, immunoturbidimetry, colloidal gold method, immunofluorescence technique, magnetic bead
Immunization etc..Latex agglutination measurement DDi is predominantly qualitative, it is impossible to the DDi concentration in enough accurate quantitative analysis samples.
The relative operation of immunoturbidimetry is relatively complicated and easily hook effect occurs in the presence of high concentration antigen, so as to influence to survey
The result of amount.The colloidal gold method testing time is more long, and precision is poor, and easily influenced by environmental conditions.Immunofluorescence and magnetic
Immune requirement of the rule to instrument of pearl is higher, is not easy to clinical expansion and uses on a large scale.
The content of the invention
Existing gold-marking immunity detection is relatively broad in qualitative detection application, and the present invention provides a kind of detection DDi
(D-Dimer) dot immunogold diafiltration kit detection method, dot immunogold filtration assay is to allow sample to be tested vertically to ooze
Filtration microporous barrier is (for example：Nitrocellulose filter), coated capture probe capture on envelope, then allow collaurum mark in the same fashion
Probe was percolated microporous barrier, with captured ligand binding after aggregation form macroscopic punctation.
It is the invention provides one kind detection DDi (D-Dimer) dot immunogold diafiltration kit, including pH value
6.7 pre- confining liquid, the cleaning solution that pH value is 6.2, colloid gold label DDi antibody, DDi chromatography device and calibration
Card；The pre- confining liquid includes Triton-100 and borate buffer solution；The cleaning solution include bovine serum albumin(BSA) and
Borate buffer solution.
Preferably, in the pre- confining liquid Triton-100 concentration be 0.6~10%, borate buffer solution it is dense
It is 0.5~10mol/L to spend；The concentration of bovine serum albumin(BSA) is 0.6~10% in the cleaning solution, borate buffer solution
Concentration is 0.5~10mol/L.
Preferably, the borate buffer solution is prepared by following raw material：Na2B4O710H2O 1.0-3.0 weight
Part, H3BO3 0.5-1.5 weight portions, the weight portions of NaCl 4, the weight portions of KCl 0.1, distilled water 800ml weight portions, ox blood are pure
The weight portion of albumen 5.
Preferably, the colloid gold label DDi antibody is prepared as follows：
The gold chloride that mass fraction is 3% is first made into A liquid with distilled water by volume 1: 86；It is 3% lemon by mass fraction
Lemon acid trisodium, mass fraction are 2.8% tannic acid, mass fraction is 40mM K2CO3With distilled water by volume 6: 0.05~0.98:
0.05~0.98: 16.04~18.99 are made into B liquid；The A liquid is heated to 62~69 DEG C again, B liquid is heated to 55~58 DEG C, stirs
A liquid is mixed, B liquid is rapidly joined, continues to stir 3 minutes, boiling was heated in 5~9 minutes, be made collaurum liquid；
Using 0.5M K2CO3It is 7.0~7.5 to adjust the collaurum liquid pH value；
By DDi antibody stoste at 2 DEG C with 10000~13000r/min centrifugations 40 minutes, draw supernatant
Liquid；The antibody is loaded into bag filter, after distilled water dialysis desalination, bottling freezing obtains DDi antibody；
The DDi antibody is slowly added to be coated with collaurum liquid, the DDi antibody and collaurum
The mass volume ratio of liquid is 0.31~0.39mg：After 110ml, stirring 25 minutes, sequentially add 20~30% sodium azides and 1~
10% bovine serum albumin(BSA), the sodium azide and bovine serum albumin(BSA) are 500 μ l with the volume ratio of the collaurum liquid：100μl：
100ml, at 4.1~5.0 DEG C after stirring evenly, is centrifuged 20 minutes with 7000r/min speed, removes supernatant, and sediment is glue
Body gold mark DDi colloid.
Preferably, the trisodium citrate, tannic acid, K2C03It is 6 with the volume ratio of distilled water：1.2：1.2：19；The glue
The colloid gold particle of a diameter of 7.8 ＜ D ＜ 9.8nm is formed in body gold liquid.A diameter of 6~the 16nm's of colloid gold particle, should
When collaurum liquid color is by black → blue → purple → red.
Preferably, the reaction box of a diameter of 3.5cm is provided with the DDi chromatography device, the reaction box includes box
Bottom and lid two parts, the lid top surface center are provided with an a diameter of 0.8cm in upper surface, a diameter of 0.4cm of lower surface
Small sircle hole；The full absorbent material of pad in the reaction box, below small sircle hole and above absorbent material between place one 1.5 ×
The nitrocellulose filter of the coating DDi antibody of 1.5cm.
Preferably, the nitrocellulose filter of the coating DDi antibody is prepared according to following steps：
By the DDi antibody stoste at 2 DEG C with 10000~13000r/min centrifugations 40 minutes, in absorption
Clear liquid；The antibody is loaded into bag filter, after distilled water dialysis desalination, freezing of being bottled after ultrafiltration concentration obtains D- bis-
Dimer antibody；
Above-mentioned DDi antibody is diluted with borate buffer solution, the coating buffer of 1.0mg/ml is obtained；
The coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking by every diaphragm 0.025ml
The nitrocellulose filter for being coated with DDi antibody is made after being dried in case.
Preferably, the calibration card detects the corresponding detection signal of color by DDi gradient mass concentration solution
Value.According to the signal value that Test paper shows, compared with calibration card signal value, you can quantitatively judge its mass concentration.
Preferably, the gradient mass concentration be respectively 0.1,0.3,1,2.5,5,10mg/L, and the gradient quality is dense
The signal value for spending detection is corresponding with calibration card.
It is a kind of to detect the quantitative detecting method that DDi dot immunogold is percolated kit, comprise the following steps：
Collection 5.1~10ml of blood sample, room temperature places 15min-2h, with 4000r/min centrifugation 20min, draws supernatant
Blood sample mixed well is obtained sample to be tested by liquid；
Pre- confining liquid is accurately drawn using pipettor, is put on nitrocellulose face in reaction box circular hole；
After after the infiltration of pre- confining liquid, detection sample is accurately drawn with pipettor, put the nitrocellulose in reaction box circular hole
On face；
After sample to be detected penetrates into, then colloid gold label DDi antibody is added dropwise to reaction box face；The collaurum
Mark DDi antibody is 1 with the volume ratio of the detection sample：1.5；
After after the infiltration of DDi antibody labeling collaurum, then to cleaning solution is added dropwise on reaction box face；The cleaning solution
Volume ratio with the detection sample is 1：1.5；
Observing response box face, after cleaning solution fully penetrates into face, detection DDi chromatography device signal value, by instrument
Device combines calibration card information and calculates detection sample DDi concentration, can quantitatively judge its mass concentration.
DDi (D-Dimer) spot gold diafiltration kit has detection time short, and precision is good, to instrument and environment
Requirement it is relatively low, the advantages of be easy to clinical application.
The invention provides one kind detection DDi dot immunogold diafiltration kit and its quantitative detecting method, including
Cleaning solution that pre- confining liquid that pH value is 6.7, pH value are 6.2, colloid gold label DDi antibody, DDi chromatography device and
Calibration card；The pre- confining liquid includes Triton-100 and borate buffer solution；The cleaning solution includes bovine serum albumin
White and borate buffer solution.Dot immunogold filtration assay of the present invention based on affinity chromatography principle, using cellulose nitrate
Plain film is solid phase carrier, is solid phase with the antibody adsorbed on nitrocellulose, with antigen in human blood and antibody labeling colloid
Gold is liquid phase, and simultaneously using antibody labeling collaurum as probe and indicator, and a kind of new immune inspection set up
Survey method.
Compared with prior art, the negatively charged positive charge with DDi antibody protein molecule in the surface of colloid gold particle
Group firm combination because electrostatic interaction is formed, while gold grain has the characteristic of high electron density again, when what is marked by gold grain
Antibody is combined with corresponding antigen ligand, and gold grain just forms macroscopic red when antibody ligand site is largely assembled
Spot.DDi antibody spot gold diafiltration kit of the invention has detection time short, and precision is good, to instrument and environment
Requirement it is relatively low, the advantages of be easy to clinical application.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described with reference to embodiment, but
It should be appreciated that these descriptions are simply to further illustrate the features and advantages of the present invention, rather than to the claims in the present invention
Limitation.
The embodiment of the invention discloses one kind detection DDi (D-Dimer) dot immunogold diafiltration kit, including
Cleaning solution that pre- confining liquid that pH value is 6.7, pH value are 6.2, colloid gold label DDi antibody, DDi chromatography device and
Calibration card；Wherein, the concentration of Triton-100 is the concentration of 0.6~10%, borate buffer solution in the pre- confining liquid
It is 0.5~10mol/L；The concentration of bovine serum albumin(BSA) (BSA) is 0.6~10%%, borate buffer solution in the cleaning solution
The concentration of solution is 0.5~10mol/L.
Preferably, the borate buffer solution is by Na2B4O710H2O 1.0-3.0 weight portions, H3BO3
0.5-1.5 weight portions, the weight portions of NaCl 4, the weight portions of KCl 0.1, distilled water 800ml weight portions, the weight of bovine serum albumin(BSA) 5
Part.
Preferably, the colloid gold label DDi antibody is prepared from according to the following steps：
(1) preparation of collaurum is respectively 3% gold chloride, 3% trisodium citrate, 2.8% tannic acid, 40mM with mass fraction
K2CO3And distilled water is raw material, and 3% gold chloride and distilled water first are made into A liquid by volume 1: 86；By 3% trisodium citrate,
2.8% tannic acid, 40mMK2CO36: 0.05~0.98: 0.05~0.98: 16.04~18.99 B is made into distilled water by volume
Liquid；The A liquid is heated to 62~69 DEG C again, B liquid is heated to 55~58 DEG C, stirs A liquid, rapidly join B liquid, continue to stir 3
Minute, boiling was heated in 5~9 minutes, collaurum liquid is made, when the collaurum liquid color is by black → blue → purple → red,
It is made the colloid gold particle of a diameter of 8~12nm；
(2) using 0.5M K2CO3The collaurum liquid pH value is adjusted for standby after 7.0~7.5；
(3) preparation of DDi antibody with dialysis, by antibody at 2 DEG C with 10000~13000r/min centrifugations
40 minutes, Aspirate supernatant；The antigen is loaded into bag filter, after distilled water dialysis desalination, bottling freezing is standby；
(4) DDi antibody labeling collaurum, it is slow to add by DDi 0.31~0.39mg of antigen in step (3)
It is coated with the 110ml collaurum liquid for entering step (2) preparation, after stirring 15 minutes, is sequentially added 20~30% sodium azide
500 μ l, 1~10% μ l of bovine serum albumin(BSA) 100, at 4.1~5.0 DEG C after stirring evenly, so that 20 points to be centrifuged under 7000r/min speed
Clock, remove supernatant, DDi antibody labeling collaurum.
Preferably, collaurum A formula of liquid is prepared ibid, the component for preparing collaurum B liquid is 3% citric acid
Trisodium 6.0ml, 2.8% tannic acid 1.2ml, 40mM K2C031.2ml and distilled water 19ml；The colloid gold particle a diameter of 7.8 being made
＜ D ＜ 9.8nm；Collaurum liquid pH value is adjusted to 6.8；Collaurum liquid is 100ml with the envelope-bulk to weight ratio of DDi antigen:
0.30mg。
Preferably, the DDi chromatography device is provided with reaction box, and the plastic caddy of a diameter of 3.5cm divides
Cassette bottom and lid two parts, it is the small sircle hole of 0.4cm, box for 0.8cm, lower diameter that there is a upper diameter in lid top surface center
The interior full absorbent material of pad, below small sircle hole and above absorbent material between place 1.5 × 1.5cm be coated with D- dimerization
The nitrocellulose filter of body antibody, it is anabolic reaction box to close tight lid.Most of sample to be tested and label probe from spot can be avoided
Non-relevant areas beyond point are lost in, and influence detection sensitivity, can cause that the technology is widely used in clinical examination.
Preferably, the nitrocellulose filter of the coating DDi antibody is prepared from according to the following steps：
(1) purifying of antibody, the purifying of DDi coated antibody and dialysis, by antibody stoste at 2 DEG C with 10000~
13000r/min centrifugations 40 minutes, Aspirate supernatant is DDi antibody；The antibody is loaded into bag filter, double steamings are used
After water dialysis desalination, freezing of being bottled after ultrafiltration concentration is standby；
(2) above-mentioned antibody is diluted with borate buffer solution, obtains the coating buffer of 1.0mg/ml；
(3) coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking by every diaphragm 0.025ml
The nitrocellulose filter for being coated with DDi antibody is made after being dried in case.
Preferably, the calibration blocks for DDi gradient mass concentration solution institute detected signal value is corresponding.
According to the signal value that Test paper shows, compared with calibration card signal value, you can quantitatively judge its mass concentration.
Preferably, the gradient mass concentration be respectively 0.1,0.3,1,2.5,5,10mg/L, and the gradient
The signal of mass concentration detection is corresponding with calibration card.
Further, the present invention also provides a kind of determining for detection DDi (D-Dimer) spot gold diafiltration kit
Quantity measuring method, operates according to the following steps：
(1) collection of human plasma to be checked：5.1~10ml of blood sample is gathered with the vacuum test tube of sodium citrate anti-freezing, is mixed,
Put room temperature and place 15min-2h, with 4000r/min centrifugation 20min, Aspirate supernatant is sample to be tested, freeze standby；
(2) pre- confining liquid sample-adding：Pre- confining liquid, point nitrocellulose filter in reaction box circular hole are accurately drawn with pipettor
On face；
(3) sample application is detected：After pre- confining liquid is fully penetrated into, detection sample is accurately drawn with pipettor, put anti-
Answer in box circular hole on nitrocellulose face；
(4) DDi antibody labeling collaurum is added dropwise：After sample to be detected penetrates into, then D- bis- is added dropwise toward reaction box face
Dimer antibody, the colloid gold label DDi antibody is 1 with the volume ratio of the detection sample：1.5；
(5) after after the infiltration of DDi antibody labeling collaurum, then toward cleaning solution is added dropwise on reaction box face；The washing
Liquid is 1 with the volume ratio of the detection sample：1.5；
(6) detect and record result：Observing response box face, after cleaning solution fully penetrates into face, detects DDi
Chromatography device signal value, combines calibration card information and calculates detection sample DDi concentration, you can quantitatively judge its matter by instrument
Amount concentration.
From above scheme as can be seen that the present invention is simple and convenient, the color that shows of device is chromatographed according to DDi, it is and corresponding
Batch targeted message, you can the mass concentration of quantitative material to be detected.
For a further understanding of the present invention, the technical scheme that the present invention is provided is carried out specifically with reference to embodiment
Bright, protection scope of the present invention is not limited by the following examples.
The raw material and chemical reagent that the embodiment of the present invention is used are purchased in market.
Embodiment 1
One kind detection DDi (D-Dimer) dot immunogold diafiltration kit, including the pre- closing that pH value is 6.7
Liquid, pH value are 6.2 cleaning solution, colloid gold label DDi antibody, DDi chromatography device and calibration card；Wherein, it is described
The concentration of Triton-100 is that the concentration of 1.5%, borate buffer solution is 1.5mol/L in pre- confining liquid；The cleaning solution
The concentration of middle bovine serum albumin(BSA) (BSA) is that the concentration of 1.5%, borate buffer solution is 1.5mol/L.The borate
Buffer soln is by the weight portions of Na2B4O710H2O 2.0, the weight portions of H3BO3 1.0, the weight portions of NaCl 4.0, the weights of KCl 0.1
Amount part, distilled water 800ml weight portions, the weight portion of bovine serum albumin(BSA) 5.
The DDi chromatography device is provided with reaction box, and the plastic caddy of a diameter of 3.5cm is divided to cassette bottom and lid two
Point, it is the full water-absorption material of pad in the small sircle hole of 0.4cm, box for 0.8cm, lower diameter that there is a upper diameter in lid top surface center
Material, below small sircle hole and above absorbent material between place the nitric acid for being coated with DDi antibody of 1.5 × 1.5cm
Cellulose membrane.The nitrocellulose filter of the coating DDi antibody is prepared from according to the following steps：
(1) purifying of antibody, the purifying of DDi coated antibody and dialysis, by antibody stoste at 2 DEG C with 11000r/
Min centrifugations 40 minutes, Aspirate supernatant is DDi antibody；The antibody is loaded into bag filter, is dialysed with distilled water
After desalination, freezing of being bottled after ultrafiltration concentration is standby；
(2) above-mentioned antibody is diluted with borate buffer solution, obtains the coating buffer of 1.0mg/ml；
(3) coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking by every diaphragm 0.025ml
The nitrocellulose filter for being coated with DDi antibody is made after being dried in case.
The colloid gold label DDi antibody is prepared from according to the following steps：
(1) preparation of collaurum is respectively 3% gold chloride, 3% trisodium citrate, 2.8% tannic acid, 40mM with mass fraction
K2CO3And distilled water is raw material, and 3% gold chloride and distilled water first are made into A liquid by volume 1: 86；By 3% trisodium citrate,
2.8% tannic acid, 40mMK2CO36: 0.05~0.98: 0.05~0.98: 16.04~18.99 B is made into distilled water by volume
Liquid；The A liquid is heated to 64 DEG C again, B liquid is heated to 56.5 DEG C, stirs A liquid, rapidly join B liquid, continue to stir 3 minutes,
Boiling is heated in 7 minutes, collaurum liquid is made, when the collaurum liquid color is by black → blue → purple → red, is made a diameter of
The colloid gold particle of 10nm；
(2) using 0.4M K2CO3The collaurum liquid pH value is adjusted for standby after 7.2；
(3) preparation of DDi antibody and dialysis, by antibody at 2 DEG C with 11000r/min centrifugations 40 minutes,
Aspirate supernatant；The antigen is loaded into bag filter, after distilled water dialysis desalination, bottling freezing is standby；
(4) DDi antibody labeling collaurum, by step (3) DDi antigen 0.35mg, is slowly added to step (2)
It is coated with the 110ml collaurum liquid of preparation, after stirring 15 minutes, is sequentially added the 25% μ l of sodium azide 500,5% ox
The μ l of seralbumin 100, at 4.5 DEG C after stirring evenly, with 7000r/min speed be centrifuged 20 minutes, remove supernatant, D- bis-
Dimer antibody marks collaurum.
The calibration card is respectively 0.1 for DDi gradient mass concentration, 0.3,1,2.5,5,10mg/L, and the ladder
The signal of degree mass concentration detection is corresponding with calibration card.
Further, the present invention also provides a kind of determining for detection DDi (D-Dimer) spot gold diafiltration kit
Quantity measuring method, operates according to the following steps：
(1) collection of human plasma to be checked：Blood sample 7ml is gathered with the vacuum test tube of sodium citrate anti-freezing, is mixed, put room temperature
1h is placed, with 4000r/min centrifugation 20min, Aspirate supernatant is sample to be tested, freezed standby；
(2) pre- confining liquid sample-adding：Pre- confining liquid, point nitrocellulose filter in reaction box circular hole are accurately drawn with pipettor
On face；
(3) sample application is detected：After pre- confining liquid is fully penetrated into, detection sample is accurately drawn with pipettor, put anti-
Answer in box circular hole on nitrocellulose face；
(4) DDi antibody labeling collaurum is added dropwise：After sample to be detected penetrates into, then D- bis- is added dropwise toward reaction box face
Dimer antibody, the colloid gold label DDi antibody is 1 with the volume ratio of the detection sample：1.5；
(5) after after the infiltration of DDi antibody labeling collaurum, then toward cleaning solution is added dropwise on reaction box face；The washing
Liquid is 1 with the volume ratio of the detection sample：1.5；
(6) detect and record result：Observing response box face, after cleaning solution fully penetrates into face, detects DDi
Chromatography device signal value, combines calibration card information and calculates detection sample DDi concentration, you can quantitatively judge its matter by instrument
Amount concentration.
Embodiment 2
One kind detection DDi (D-Dimer) dot immunogold diafiltration kit, including the pre- closing that pH value is 6.7
Liquid, pH value are 6.2 cleaning solution, colloid gold label DDi antibody, DDi chromatography device and calibration card；Wherein, it is described
The concentration of Triton-100 is that the concentration of 0.6%, borate buffer solution is 0.5mol/L in pre- confining liquid；The cleaning solution
The concentration of middle bovine serum albumin(BSA) (BSA) is that the concentration of 0.6%, borate buffer solution is 0.5mol/L.The borate
Buffer soln is by the weight portions of Na2B4O710H2O 1.0, the weight portions of H3BO3 0.5, the weight portions of NaCl 4.0, the weights of KCl 0.1
Amount part, distilled water 800ml weight portions, the weight portion of bovine serum albumin(BSA) 5.
The DDi chromatography device is provided with reaction box, and the plastic caddy of a diameter of 3.5cm is divided to cassette bottom and lid two
Point, it is the full water-absorption material of pad in the small sircle hole of 0.4cm, box for 0.8cm, lower diameter that there is a upper diameter in lid top surface center
Material, below small sircle hole and above absorbent material between place the nitric acid for being coated with DDi antibody of 1.5 × 1.5cm
Cellulose membrane.The nitrocellulose filter of the coating DDi antibody is prepared from according to the following steps：
(1) purifying of antibody, the purifying of DDi coated antibody and dialysis, by antibody stoste at 2 DEG C with 10000r/
Min centrifugations 40 minutes, Aspirate supernatant is DDi antibody；The antibody is loaded into bag filter, is dialysed with distilled water
After desalination, freezing of being bottled after ultrafiltration concentration is standby；
(2) above-mentioned antibody is diluted with borate buffer solution, obtains the coating buffer of 1.0mg/ml；
(3) coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking by every diaphragm 0.025ml
The nitrocellulose filter for being coated with DDi antibody is made after being dried in case.
The colloid gold label DDi antibody is prepared from according to the following steps：
(1) preparation of collaurum is respectively 3% gold chloride, 3% trisodium citrate, 2.8% tannic acid, 40mM with mass fraction
K2CO3And distilled water is raw material, and 3% gold chloride and distilled water first are made into A liquid by volume 1: 86；By 3% trisodium citrate,
2.8% tannic acid, 40mMK2CO36: 0.05~0.98: 0.05~0.98: 16.04~18.99 B is made into distilled water by volume
Liquid；The A liquid is heated to 62 DEG C again, B liquid is heated to 55 DEG C, stirs A liquid, rapidly join B liquid, continue to stir 3 minutes, 5
Boiling is heated in minute, collaurum liquid is made, when the collaurum liquid color is by black → blue → purple → red, is made a diameter of
The colloid gold particle of 8nm；
(2) using 0.4M K2CO3The collaurum liquid pH value is adjusted for standby after 7.0；
(3) preparation of DDi antibody and dialysis, by antibody at 2 DEG C with 10000r/min centrifugations 40 minutes,
Aspirate supernatant；The antigen is loaded into bag filter, after distilled water dialysis desalination, bottling freezing is standby；
(4) DDi antibody labeling collaurum, by step (3) DDi antigen 0.31mg, is slowly added to step (2)
It is coated with the 110ml collaurum liquid of preparation, after stirring 15 minutes, is sequentially added the 20% μ l of sodium azide 500,1% ox
The μ l of seralbumin 100, at 4.1 DEG C after stirring evenly, with 7000r/min speed be centrifuged 20 minutes, remove supernatant, D- bis-
Dimer antibody marks collaurum.
The calibration card is respectively 0.1 for DDi gradient mass concentration, 0.3,1,2.5,5,10mg/L, and the ladder
The signal of degree mass concentration detection is corresponding with calibration card.
Further, the present invention also provides a kind of determining for detection DDi (D-Dimer) spot gold diafiltration kit
Quantity measuring method, operates according to the following steps：
(1) collection of human plasma to be checked：Blood sample 5.1ml is gathered with the vacuum test tube of sodium citrate anti-freezing, is mixed, put room
Temperature places 15min, and with 4000r/min centrifugation 20min, Aspirate supernatant is sample to be tested, freezes standby；
(2) pre- confining liquid sample-adding：Pre- confining liquid, point nitrocellulose filter in reaction box circular hole are accurately drawn with pipettor
On face；
(3) sample application is detected：After pre- confining liquid is fully penetrated into, detection sample is accurately drawn with pipettor, put anti-
Answer in box circular hole on nitrocellulose face；
(4) DDi antibody labeling collaurum is added dropwise：After sample to be detected penetrates into, then D- bis- is added dropwise toward reaction box face
Dimer antibody, the colloid gold label DDi antibody is 1 with the volume ratio of the detection sample：1.5；
(5) after after the infiltration of DDi antibody labeling collaurum, then toward cleaning solution is added dropwise on reaction box face；The washing
Liquid is 1 with the volume ratio of the detection sample：1.5；
(6) detect and record result：Observing response box face, after cleaning solution fully penetrates into face, detects DDi
Chromatography device signal value, combines calibration card information and calculates detection sample DDi concentration, you can quantitatively judge its matter by instrument
Amount concentration.
Embodiment 3
One kind detection DDi (D-Dimer) dot immunogold diafiltration kit, including the pre- closing that pH value is 6.7
Liquid, pH value are 6.2 cleaning solution, colloid gold label DDi antibody, DDi chromatography device and calibration card；Wherein, it is described
The concentration of Triton-100 is that the concentration of 10%, borate buffer solution is 10mol/L in pre- confining liquid；In the cleaning solution
The concentration of bovine serum albumin(BSA) (BSA) is that the concentration of 10%, borate buffer solution is 10mol/L.The boric acid salt buffer
Liquor is by the weight portions of Na2B4O710H2O 3.0, the weight portions of H3BO3 1.5, the weight portions of NaCl 4.0, the weight of KCl 0.1
Part, distilled water 800ml weight portions, the weight portion of bovine serum albumin(BSA) 5.
The DDi chromatography device is provided with reaction box, and the plastic caddy of a diameter of 3.5cm is divided to cassette bottom and lid two
Point, it is the full water-absorption material of pad in the small sircle hole of 0.4cm, box for 0.8cm, lower diameter that there is a upper diameter in lid top surface center
Material, below small sircle hole and above absorbent material between place the nitric acid for being coated with DDi antibody of 1.5 × 1.5cm
Cellulose membrane.The nitrocellulose filter of the coating DDi antibody is prepared from according to the following steps：
(1) purifying of antibody, the purifying of DDi coated antibody and dialysis, by antibody stoste at 2 DEG C with 13000r/
Min centrifugations 40 minutes, Aspirate supernatant is DDi antibody；The antibody is loaded into bag filter, is dialysed with distilled water
After desalination, freezing of being bottled after ultrafiltration concentration is standby；
(2) above-mentioned antibody is diluted with borate buffer solution, obtains the coating buffer of 1.0mg/ml；
(3) coating buffer is dropped on the nitrocellulose filter for cutting, diaphragm is placed in baking by every diaphragm 0.025ml
The nitrocellulose filter for being coated with DDi antibody is made after being dried in case.
The colloid gold label DDi antibody is prepared from according to the following steps：
(1) preparation of collaurum is respectively 3% gold chloride, 3% trisodium citrate, 2.8% tannic acid, 40mM with mass fraction
K2CO3And distilled water is raw material, and 3% gold chloride and distilled water first are made into A liquid by volume 1: 86；By 3% trisodium citrate,
2.8% tannic acid, 40mMK2CO36: 0.05~0.98: 0.05~0.98: 16.04~18.99 B is made into distilled water by volume
Liquid；The A liquid is heated to 69 DEG C again, B liquid is heated to 58 DEG C, stirs A liquid, rapidly join B liquid, continue to stir 3 minutes, 9
Boiling is heated in minute, collaurum liquid is made, when the collaurum liquid color is by black → blue → purple → red, is made a diameter of
The colloid gold particle of 12nm；
(2) using 0.4M K2CO3The collaurum liquid pH value is adjusted for standby after 7.5；
(3) preparation of DDi antibody and dialysis, by antibody at 2 DEG C with 133000r/min centrifugations 40 minutes,
Aspirate supernatant；The antigen is loaded into bag filter, after distilled water dialysis desalination, bottling freezing is standby；
(4) DDi antibody labeling collaurum, by step (3) DDi antigen 0.39mg, is slowly added to step (2)
It is coated with the 110ml collaurum liquid of preparation, after stirring 15 minutes, is sequentially added the 30% μ l of sodium azide 500,10% ox
The μ l of seralbumin 100, at 5.0 DEG C after stirring evenly, with 7000r/min speed be centrifuged 20 minutes, remove supernatant, D- bis-
Dimer antibody marks collaurum.
The calibration card is respectively 0.1 for DDi gradient mass concentration, 0.3,1,2.5,5,10mg/L, and the ladder
The signal of degree mass concentration detection is corresponding with calibration card.
Further, the present invention also provides a kind of determining for detection DDi (D-Dimer) spot gold diafiltration kit
Quantity measuring method, operates according to the following steps：
(1) collection of human plasma to be checked：Blood sample 10ml is gathered with the vacuum test tube of sodium citrate anti-freezing, is mixed, put room
Temperature places 2h, and with 4000r/min centrifugation 20min, Aspirate supernatant is sample to be tested, freezes standby；
(2) pre- confining liquid sample-adding：Pre- confining liquid, point nitrocellulose filter in reaction box circular hole are accurately drawn with pipettor
On face；
(3) sample application is detected：After pre- confining liquid is fully penetrated into, detection sample is accurately drawn with pipettor, put anti-
Answer in box circular hole on nitrocellulose face；
(4) DDi antibody labeling collaurum is added dropwise：After sample to be detected penetrates into, then D- bis- is added dropwise toward reaction box face
Dimer antibody, the colloid gold label DDi antibody is 1 with the volume ratio of the detection sample：1.5；
(5) after after the infiltration of DDi antibody labeling collaurum, then toward cleaning solution is added dropwise on reaction box face；The washing
Liquid is 1 with the volume ratio of the detection sample：1.5；
(6) detect and record result：Observing response box face, after cleaning solution fully penetrates into face, detects DDi
Chromatography device signal value, combines calibration card information and calculates detection sample DDi concentration, you can quantitatively judge its matter by instrument
Amount concentration.
Embodiment 4
Embodiment 4 is with the difference of embodiment 3, embodiment 1 and embodiment 2, in colloid gold label DDi antibody
Preparation process in, prepare collaurum A formula of liquid ibid, the component for preparing collaurum B liquid is 3% citric acid three with content
Sodium 6.0ml, 2.8% tannic acid 0.05ml, 40mM K2C030.05ml and distilled water 16.04ml；The colloid gold particle being made is a diameter of
8.1 ＜ D ＜ 9.3nm；Collaurum liquid pH value is adjusted to 6.7；Collaurum liquid is 100ml with the envelope-bulk to weight ratio of DDi antibody:
0.30mg。
Embodiment of above and embodiment are both needed to the usage amount of appropriateness regulation temperature and distilled water as the case may be, this theory
It is bright to be only intended to help and understand the method for the present invention and its core concept.It should be pointed out that for the ordinary skill of the art
For personnel, under the premise without departing from the principles of the invention, some improvement and modification can also be carried out to the present invention, these improvement
Also fallen into the protection domain of the claims in the present invention with modification.
The foregoing description of the disclosed embodiments, enables professional and technical personnel in the field to realize or uses the present invention.
Various modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The scope most wide for causing.
Claims (10)
1. a kind of detection DDi dot immunogold is percolated kit, it is characterised in that including：
Cleaning solution, colloid gold label DDi antibody, DDi chromatography that pre- confining liquid that pH value is 6.7, pH value are 6.2
Device and calibration block；The pre- confining liquid includes Triton-100 and borate buffer solution；The cleaning solution includes cow's serum
Albumin and borate buffer solution.
2. detection DDi dot immunogold according to claim 1 is percolated kit, it is characterised in that the pre- envelope
It is 0.6~10% to close the concentration of Triton-100 in liquid, and the concentration of borate buffer solution is 0.5~10mol/L；It is described to wash
It is 0.6~10% to wash the concentration of bovine serum albumin(BSA) in liquid, and the concentration of borate buffer solution is 0.5~10mol/L.
3. detection DDi dot immunogold according to claim 1 is percolated kit, it is characterised in that the boric acid
Salt buffer solution is prepared by following raw material：Na2B4O710H2O 1.0-3.0 weight portions, H3BO3 0.5-1.5 weight portions,
The weight portions of NaCl 4, the weight portions of KCl 0.1, distilled water 800ml weight portions, the weight portion of bovine serum albumin(BSA) 5.
4. detection DDi dot immunogold according to claim 1 is percolated kit, it is characterised in that the colloid
Gold mark DDi antibody is prepared as follows：
The gold chloride that mass fraction is 3% is first made into A liquid with distilled water by volume 1: 86；It is 3% citric acid by mass fraction
Trisodium, mass fraction are 2.8% tannic acid, mass fraction is 50mM K2CO3With distilled water by volume 6: 0.05~0.98: 0.05
~0.98: 16.04~18.99 are made into B liquid；The A liquid is heated to 62~69 DEG C again, B liquid is heated to 55~58 DEG C, stirs A
Liquid, rapidly joins B liquid, continues to stir 3 minutes, and boiling was heated in 5~9 minutes, is made collaurum liquid；
Using 0.5M K2CO3It is 7.0~7.5 to adjust the collaurum liquid pH value；
By DDi antibody stoste at 2 DEG C with 10000~13000r/min centrifugations 40 minutes, Aspirate supernatant；Will
The antibody loads bag filter, and after distilled water dialysis desalination, bottling freezing obtains DDi antibody；
The DDi antibody is slowly added to be coated with collaurum liquid, the DDi antibody and collaurum liquid
Mass volume ratio is 0.31~0.39mg：110ml, after stirring 25 minutes, sequentially adds 20~30% sodium azides and 1~10% N
Seralbumin, the sodium azide and bovine serum albumin(BSA) are 500 μ l with the volume ratio of the collaurum liquid：100μl：100ml,
At 4.1~5.0 DEG C after stirring evenly, it is centrifuged 20 minutes with 7000r/min speed, removes supernatant, sediment is collaurum mark
Note DDi colloid.
5. detection DDi dot immunogold according to claim 4 is percolated kit, it is characterised in that the lemon
Sour trisodium, tannic acid, K2C03It is 6 with the volume ratio of distilled water：1.2：1.2：19；A diameter of 7.8 ＜ is formed in the collaurum liquid
The colloid gold particle of D ＜ 9.8nm.
6. detection DDi dot immunogold according to claim 1 is percolated kit, it is characterised in that the D- bis-
The reaction box of a diameter of 3.5cm is provided with aggressiveness chromatography device, the reaction box includes cassette bottom and lid two parts, the lid
Top surface center is provided with an a diameter of 0.8cm in upper surface, the small sircle hole of a diameter of 0.4cm of lower surface；Pad in the reaction box
Full absorbent material, below small sircle hole and above absorbent material between place a coating DDi antibody of 1.5 × 1.5cm
Nitrocellulose filter.
7. detection DDi dot immunogold according to claim 6 is percolated kit, it is characterised in that the coating
The nitrocellulose filter of DDi antibody is prepared according to following steps：
By the DDi antibody stoste at 2 DEG C with 10000~13000r/min centrifugations 40 minutes, draw supernatant
Liquid；The antibody is loaded into bag filter, after distilled water dialysis desalination, freezing of being bottled after ultrafiltration concentration obtains D- dimerization
Body antibody；
Above-mentioned DDi antibody is diluted with borate buffer solution, 1.0mg/ml is obtained for coating buffer；
The coating buffer is dropped on the nitrocellulose filter for cutting, be placed in diaphragm in baking oven by every diaphragm 0.025ml
The nitrocellulose filter for being coated with DDi antibody is made after drying.
8. detection DDi dot immunogold according to claim 1 is percolated kit, it is characterised in that the calibration
Block and the corresponding detected signal value of color is detected by DDi gradient mass concentration solution.
9. detection DDi dot immunogold according to claim 8 is percolated kit, it is characterised in that the gradient
Mass concentration is respectively 0.1,0.3,1,2.5,5,10mg/L.
10. it is a kind of to detect the quantitative detecting method that DDi dot immunogold is percolated kit, it is characterised in that including following
Step：
Collection 5.1~10ml of blood sample, room temperature places 15min-2h, and with 4000r/min centrifugation 20min, Aspirate supernatant will
Blood sample mixed well, obtains sample to be tested；
Pre- confining liquid is accurately drawn using pipettor, is put on nitrocellulose face in reaction box circular hole；
After after the infiltration of pre- confining liquid, detection sample is accurately drawn with pipettor, put the nitrocellulose face in reaction box circular hole
On；
After sample to be detected penetrates into, then colloid gold label DDi antibody is added dropwise to reaction box face；The colloid gold label
DDi antibody is 1 with the volume ratio of the detection sample：1.5；
After after the infiltration of DDi antibody labeling collaurum, then to cleaning solution is added dropwise on reaction box face；The cleaning solution and institute
The volume ratio for stating detection sample is 1：1.5；
Observing response box face, after cleaning solution fully penetrates into face, detection DDi chromatography device signal value, by instrument knot
Close calibration card information and calculate detection sample DDi concentration, its mass concentration can be quantitatively judged.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2016107073623 | 2016-08-23 | ||
| CN201610707362 | 2016-08-23 | ||
| CN2016109110260 | 2016-10-18 | ||
| CN201610911026 | 2016-10-18 |
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| CN201611094832.XA Pending CN106771145A (en) | 2016-08-23 | 2016-12-02 | One kind detection D dimers spot gold diafiltration kit and quantitative detecting method |
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Cited By (3)
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| CN112578113A (en) * | 2020-09-22 | 2021-03-30 | 苏州市药品检验检测研究中心 | Abietic acid colloidal gold detection card and manufacturing method thereof |
| CN112578112A (en) * | 2020-09-22 | 2021-03-30 | 苏州市药品检验检测研究中心 | 808scarlet colloidal gold detection card and preparation method thereof |
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