CN108020661A - A kind of d-dimer immunofluorescence quantification kit and preparation method thereof - Google Patents
A kind of d-dimer immunofluorescence quantification kit and preparation method thereof Download PDFInfo
- Publication number
- CN108020661A CN108020661A CN201711176368.3A CN201711176368A CN108020661A CN 108020661 A CN108020661 A CN 108020661A CN 201711176368 A CN201711176368 A CN 201711176368A CN 108020661 A CN108020661 A CN 108020661A
- Authority
- CN
- China
- Prior art keywords
- mass concentration
- bonding pad
- dimer
- pad
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/577—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
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of D dimers immunofluorescence quantification kit and preparation method thereof.The kit pastes sample pad, bonding pad, nitrocellulose filter and water absorption pad with being overlapped successively on end liner.Bonding pad is by the immersion of bonding pad pretreatment fluid, drying.Bonding pad is sprayed with D dimer monoclonal antibody fluorescent microsphere conjugates, and the D dimer monoclonal antibody fluorescent microsphere conjugates are stored by storing liquid, diluted.By these improvement so as to accurately detect the D dimer contents in human serum sample.Meanwhile appearance effect is good in detection process, precision is high, high sensitivity, and there is very good stability, cost is low, simple operation.
Description
Technical field
The present invention relates to field of immunological detection, more particularly to a kind of d-dimer immunofluorescence quantification kit and its
Preparation method.
Background technology
D-dimer is one of special catabolite of crosslinked fibrin, is the peculiar metabolin of Secondary cases fibrinolytic.
Under certain pathology or physiological status, body blood coagulation and fibrinolytic dynamic equilibrium are destroyed, coagulation system activation, and blood coagulation tendency is continuous
Increase, causes fibrin ferment to convert fibrinogen into fibrin body, and crosslinking is formed under the action of activation factor XIII
Fibrin.Crosslinked fibrin further degrades in the fragment that the decline of fibrinolytic enzyme effect solves and produces the fragment containing γ chains,
The crosslinking of γ chains produces d-dimer.Therefore the generation or increase of d-dimer reflects blood coagulation system and fibrinolytic system in blood plasma
Activation, is the ideal indicator of unique reflection blood coagulation and fibrinolytic, has clinically been considered as internal hypercoagulative state and hyperfibrinolysis
Molecular marker.D-dimer is including disseminated intravascular coagulation (DIC), thrombotic diseases, pernicious swollen in a series of diseases
Have in the early diagnosis of knurl, hepatopathy, systemic loupus erythematosus, preeclampsia, diabetic complication etc., state of illness monitoring and guiding treatment
Significant role, while the change of d-dimer can be monitored as medicine thromboembolism treatment and the index of direction of medication usage.
Currently, it is mainly the following for the assay method of D- dimerization：Latex agglutination, latex immunoturbidimetry, enzyme
Join immunoabsorption (ELISA), colloidal gold method and fluorescence immune chromatography method.Latex agglutination can only accomplish qualitative detection, and nothing
Change of the method to d-dimer content carries out accurate characterization.The latex scattered light urbidmetry range of linearity is narrow, stability is not good enough.It is enzyme-linked to exempt from
Epidemic disease absorption method accurate quantification, sensitiveness are high, but operate stringent, complex steps, are not suitable for quick diagnosis and monitoring.Colloidal gold method
Quantify accurate, operation quickly, but heparin and blood fat etc. have result certain interference, are unsuitable for large-scale promotion use.Fluorescence is exempted from
Epidemic disease chromatographic technique, is a kind of quantitative new detection skill that immunofluorescence technique and Traditional immunochromatographic technology are combined development innovation
Art.The technology is retaining outside the advantages of colloidal gold method is easy to operate, detection is quick, portability is strong, is also strengthened by fluorescent tracing
Technology realizes the accurate quantification of testing result.
Fluorescence immune chromatography technology has following advantage compared with traditional Fast Detection Technique performance：1. high sensitivity：Should
Product is with the microsphere supported technology of functionalized nano, with reference to fluorescent marker probe, directly detection excitation fluorescence signal.2. detect model
Enclose width：Fluorescence immune chromatography technology directly excites the detection means that shines using fluorescence, and signal strength is with fluorescent microsphere quantity in straight
Line is related.The problems such as avoiding catalytic efficiency existing for enzymatic luminescence technology and amount of substrate limitation, quantification range and reactant
The specific proteins amount that reaction is participated in system is directly related.It is 3. cheap：Compared with traditional quantitative detection technique, technology tool
Have the advantages that quick, cheap.
At present, to improve the properties of this technology product, researcher is improved from many aspects, but these
Improve be mostly focused on to antigen, antibody, fluorescent grain selection and preparation on.And by existing including sample pad, bonding pad
Interior carrier part be improved reach effectively improve properties of product research it is less.
The content of the invention
The object of the present invention is to provide one kind can effectively improve including accelerated stability, appearance effect, accuracy, precision
Degree, sensitivity etc. are in d-dimer immunofluorescence quantification kit of interior properties of product and preparation method thereof.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of d-dimer immunofluorescence quantification kit, including end liner on end liner successively overlap joint be provided with sample pad,
Bonding pad, nitrocellulose filter and absorption pad, the bonding pad is in the drying and processing after the immersion of bonding pad pretreatment fluid.
Further, will be stored in storing liquid and micro- by the diluted d-dimer monoclonal antibody-fluorescence of the storing liquid
Ball conjugate is sprayed on bonding pad.
Further, the bonding pad pretreatment fluid includes the PVA of 0.2%~5.0% mass concentration, 0.1%~5.0%
Triton X-100 of mass concentration, the sucrose of 1.0%~6.0% mass concentration, 0.01%~1.5% mass concentration
Proclin300 or Sodium azide, pH 6.5~7.8.
Further, the bonding pad pretreatment fluid includes the PVA of 0.2%~3.0% mass concentration, 0.5%~3.5%
Triton X-100 of mass concentration, the sucrose of 1.0%~2.25% mass concentration, 0.03%~0.1% mass concentration
Proclin300 or Sodium azide, pH 7.0~7.8.
Further, the storing liquid include the PB of 15~50mM, the BSA of 0.5%~10% mass concentration, 0.4%~
The Tween-80 of 10% mass concentration, the glucose of 0.4%~10% mass concentration, the sweet ammonia of 1.5%~10% mass concentration
Acid, the PEG4000 of 0.8%~5% mass concentration, the PEG20000 of 1%~8% mass concentration, 0.01%~1.5% mass are dense
Proclin300 or Sodium azide, the pH 6.5~9.0 of degree.
Further, the storing liquid include the PB of 20~40mM, the BSA of 0.5%~4.8% mass concentration, 0.5%~
The Tween-80 of 6.0% mass concentration, the glucose of 0.5%~3.0% mass concentration, the sweet ammonia of 2%~7% mass concentration
Acid, the PEG4000 of 1.0%~3.0% mass concentration, the PEG20000 of 1.5%~4.0% mass concentration, 0.03%~0.1%
Proclin300 or Sodium azide, the pH 7.0~8.0 of mass concentration.
Present invention additionally comprises a kind of preparation method of d-dimer immunofluorescence quantification kit, comprise the following steps that：
The preparation of S1, sample pad：Glass fibre element film is soaked with sample pad treatment fluid, dries, takes out standby after immersion treatment
With；
The preparation of S2, bonding pad：Glass fibre element film is soaked with bonding pad pretreatment fluid, dries, is switched to after immersion treatment
Using width, bonding pad semi-finished product are made, it is spare；
S3, by polystyrene fluorescent microsphere successively add EDC and NHS, activated in activation buffer；
S4, add mouse source d-dimer monoclonal antibody, is coupled in coupling buffer with fluorescent microsphere, after add
Confining liquid, is made mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, and supernatant is abandoned after centrifugation, adds storing liquid and preserves；
S5, with storing liquid diluted mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, and drawing film instrument with metal spraying sprays
Padded in combined on the processed bonding pad of pretreatment fluid, it is spare with aluminium foil bag sealing storage after air dry oven is dried；
Sheep anti-mouse igg polyclonal antibody and mouse source d-dimer monoclonal antibody, be coated with by S6 with coating buffer respectively, with bar
Banding is individually fixed on nitrocellulose filter by the use of metal spraying stroke film instrument and is used as nature controlling line and detection line；
S7, paste with overlapping successively on end liner：Sample pad, bonding pad, nitrocellulose filter, water absorption pad, and cut into
The immunofluorescence chromatograph test strip of specific width；
The test strips cut in S7, be loaded by S8, crosses case pressing machine.
A kind of preferable preparation method, comprises the following steps：
The preparation of S1, sample pad：Glass fibre element film is soaked with sample pad treatment fluid, dries, takes out standby after immersion treatment
With；
The preparation of S2, bonding pad：Glass fibre element film is soaked with bonding pad pretreatment fluid, dries, is switched to after immersion treatment
Using width, bonding pad semi-finished product are made, it is spare；
S3, EDC and 0.5mg/mL by the 200nm polystyrene fluorescent microsphere of the 1mg successively 0.5mg/mL of 20 μ L of addition
NHS, activated in activation buffer；
S4, add 0.1mg mouse source d-dimer monoclonal antibody, is coupled in 200 μ L coupling buffers with fluorescent microsphere,
After add 20 μ L of confining liquid, be made mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, abandon supernatant after centrifugation,
Storing liquid is added to preserve；
S5, with storing liquid be diluted to 0.5mg/mL by mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, with spray
Gold is drawn film instrument and is sprayed on the combined pad processed bonding pad of pretreatment fluid, is deposited after air dry oven is dried with aluminium foil bag sealing
Put spare；
Sheep anti-mouse igg polyclonal antibody and mouse source d-dimer monoclonal antibody, be coated with by S6 with coating buffer respectively, with bar
Banding is individually fixed on nitrocellulose filter by the use of metal spraying stroke film instrument and is used as nature controlling line and detection line；
S7, paste with overlapping successively on end liner：Sample pad, bonding pad, nitrocellulose filter, water absorption pad, and cut into
The immunofluorescence chromatograph test strip of specific width；
The test strips cut in S7, be loaded by S8, crosses case pressing machine.
A kind of preferable preparation method, comprises the following steps：
The preparation of S1, sample pad：Glass fibre element film is soaked with sample pad treatment fluid, dries, takes out standby after immersion treatment
With；
The preparation of S2, bonding pad：Glass fibre element film is soaked with bonding pad pretreatment fluid, dries, is switched to after immersion treatment
Using width, bonding pad semi-finished product are made, it is spare；
S3, EDC and 0.5mg/mL by the 200nm polystyrene fluorescent microsphere of the 1mg successively 0.5mg/mL of 20 μ L of addition
NHS, activated in activation buffer；
S4, add 0.1mg mouse source d-dimer monoclonal antibody, is coupled in 200 μ L coupling buffers with fluorescent microsphere,
After add 20 μ L of confining liquid, be made mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, abandon supernatant after centrifugation,
Storing liquid is added to preserve；
S5, with storing liquid be diluted to 0.5mg/mL by mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, with spray
Gold is drawn film instrument and is sprayed on the combined pad processed bonding pad of pretreatment fluid, is deposited after air dry oven is dried with aluminium foil bag sealing
Put spare；
S6, use and wrap respectively by 1mg/mL sheep anti-mouse iggs polyclonal antibody and 1mg/mL mouse source d-dimer monoclonal antibody
It is coated with by liquid, is individually fixed in using ribbon 1.0mm by the use of metal spraying stroke film instrument on nitrocellulose filter and is used as nature controlling line and detection line；
S7, paste with overlapping successively on end liner：Sample pad, bonding pad, nitrocellulose filter, water absorption pad, and cut into
The immunofluorescence chromatograph test strip of specific width；
The test strips cut in S7, be loaded by S8, crosses case pressing machine.
Preferably, the activation buffer is MES, pH 5.5 of 75mM；The coupling buffer is PB, pH of 20mM
7.5；The confining liquid is 20%BSA；The coating buffer includes：20mM PBS, 1.2% isopropanol, 0.4% Dextran 200 00,
1.5%BSA, 0.5%Tween-80,0.03%Proclin300, pH 7.0.
The beneficial effects of the invention are as follows：Kit in the present invention can accurately detect the D- dimerization in human serum sample
Body content.Meanwhile appearance effect is good in detection process, precision is high, high sensitivity, and there is very good stability, into
This low, simple operation.
Brief description of the drawings
Fig. 1：D-dimer immunofluorescence prepared by the preferable bonding pad pretreatment fluid of preferable storing liquid collocation is quantitatively tried
Paper slip detects the d-dimer standard curve of System.
Fig. 2：D-dimer immunofluorescence prepared by the preferable bonding pad pretreatment fluid of preferable storing liquid collocation is quantitatively tried
The detected value of paper slip detection d-dimer recombinant antigen and the substitution curve of theoretical value.
Fig. 3：Test strips prepared by the preferable bonding pad pretreatment fluid of preferable storing liquid collocation are with compareing storing liquid collocation
Compare the fluoroscopic examination of patients serum's sample of ELISA test strip d-dimer containing various concentrations prepared by bonding pad pretreatment fluid
Line chart, a~f are the biliquid 1 of control, and g~l is preferable biliquid 2.D-dimer concentration is respectively：A, g is 260ng/mL,
B, h is 450ng/mL, c, i 970ng/mL, d, j 1860ng/mL, e, k 2470ng/mL, f, l 4220ng/mL.
Fig. 4：Patients serum pattern detection value and System of the ELISA test strip containing d-dimer prepared by control group biliquid 1
The related figure of CA7000D-dimer detected values.
Fig. 5：ELISA test strip prepared by the preferable bonding pad pretreatment fluid (biliquid 2) of preferable storing liquid collocation contains D- bis-
Patients serum's pattern detection value of aggressiveness figure related to System CA7000D-dimer detected values.
Embodiment
Embodiment 1
A kind of optimization formula of storing liquid in the present patent application：The mass concentration of PB is that the mass concentration of 20mM, BSA are
1.8%th, the mass concentration of Tween-80 is 0.5%, and the mass concentration of glucose is 0.5%, the mass concentration of glycine is
2%th, the mass concentration of PEG4000 is 1%, the mass concentration of PEG20000 is 1.5%, Proclin300 mass concentrations are
0.03%th, pH 7.8~8.0.
The preparation method of the storing liquid is：0.25g glucose is weighed, is dissolved with 45mL pure water.Add what 1mL was prepared in advance
The PB mother liquors of 100mM, vibration mix.Successively add 1g glycine, 0.5g PEG4000,0.75g PEG20000 and 0.9g
BSA, vibration mix；0.25mL Tween-80 are added with sample loading gun, are beaten repeatedly；The Proclin300 of 15 μ L is added, vibration is mixed
It is even, pH value is adjusted to 7.8~8.0 with the HCl of 1M.Last room temperature is settled to 50mL, and filtration sterilization, is made storing liquid.
The preparation method of wherein PB mother liquors is：Weigh the Na of 29g2HPO4·12H2The K of O and 2g2HPO4, it is molten to add 1L pure water
De-coordinate the PB mother liquors as 100mM.
Embodiment 2
A kind of optimization formula of bonding pad pretreatment fluid in the present patent application：The mass concentration of PVA is 2.0%, Triton
The mass concentration of X-100 is 0.8%, the mass concentration of sucrose is 1.25%, the mass concentration of Proclin300 or Sodium azide is
0.03%th, pH7.2~7.4.
The preparation method of the bonding pad pretreatment fluid is：The PVA for weighing 10g adds 480ml pure water soaked overnights, places 60
DEG C dissolve by heating.The Triton X-100 of 4mL are added with sample loading gun, are beaten repeatedly；6.25g sucrose is weighed, is put into magnetic agitation
Device dissolves；The Proclin300 of 150 μ L is added, adjusts pH value to 7.2~7.4 with the HCl of 1M, vibration mixes.Last room temperature is determined
Hold to 500mL, filtration sterilization, bonding pad pretreatment fluid is made.
Embodiment 3
The present embodiment provides a kind of specific preparation method of d-dimer immunofluorescence quantitative test paper bar is as follows：
(1) preparation of storing liquid：The mass concentration of PB be the mass concentration of 20mM, BSA be 1.8%, the matter of Tween-80
It is 0.5% to measure concentration, and the mass concentration of glucose is 0.5%, the mass concentration of glycine is 2%, the mass concentration of PEG4000
Mass concentration for 1%, PEG20000 is 1.5%, Proclin300 mass concentrations are 0.03%, pH 7.8~8.0, by above-mentioned
Formula prepares filtration sterilization after mixing, and storing liquid is made；
(2) preparation of sample pad：Glass fibre element film 10min is soaked with sample pad treatment fluid, it is wet to be placed in 37 DEG C of drying room
Degree 30%, dries 5h, and sample pad is made, spare；
(3) preparation of bonding pad：Glass fibre element film 10min is soaked with bonding pad treatment fluid, after immersion treatment, is placed in dry
Dry 37 DEG C of humidity 30%, dry 5h, are switched to paper knife using width (1cm*30cm), and bonding pad semi-finished product are made, spare；
(4) the 200nm polystyrene fluorescent microsphere of 1mg is successively added to the EDC and 0.5mg/mL of the 0.5mg/mL of 20 μ L
NHS, in 37 DEG C of activation buffer activation 1hr；
(5) 0.1mg mouse source d-dimer monoclonal antibody is added, is coupled in 200 μ L coupling buffers with fluorescent microsphere,
After add 20 μ L of confining liquid, be made mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, 19000rpm freeze from
After heart 15min, supernatant discarding, 200 μ L storing liquids of addition, the mouse source d-dimer monoclonal antibody that obtained concentration is 5mg/mL-
Fluorescent microsphere conjugate, 2~8 DEG C of preservations；
(6) mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate is diluted to 0.5mg/mL with storing liquid, passed through
Metal spraying is drawn film instrument and is sprayed on the combined pad processed bonding pad for the treatment of fluid, and 7h is dried with air dry oven.Aluminium foil bag is close
Be sealed and placed in 20-25 DEG C, store under conditions of humidity about 30% it is spare；
(7) by 1mg/mL sheep anti-mouse iggs polyclonal antibody and 1mg/mL mouse source d-dimer monoclonal antibody respectively with bag
It is coated with by liquid, is individually fixed in using ribbon 1.0mm by the use of metal spraying stroke film instrument on nitrocellulose filter and is used as nature controlling line and detection line；
(8) paste with being overlapped successively on PVC bottom plates：Sample pad, bonding pad, nitrocellulose filter, water absorption pad, and shear
Into the fluorescence immune chromatography test paper bar of width 4.1mm；
(9) test strips cut in (8) are loaded, cross case pressing machine, it is spare.
In above-mentioned preparation process, used portion of reagent formula is as follows：
Sample pad treatment fluid：100mM PBS, 1%Triton, 0.75%BSA, 0.5%PVA, 0.9%NaCl, 0.3% Portugal
Glycan, 0.05%Proclin300, pH 7.8；
Bonding pad treatment fluid：0.8%Triton X-100,2.0%PVA, 1.25% sucrose, 0.03%Proclin300,
PH 7.2~7.4；
Activation buffer is 75mM MES, pH 5.5；
Coupling buffer：20mM PB, pH 7.5；
Confining liquid：20%BSA；
Coating buffer：20mM PBS, 1.2% isopropanol, 0.4% Dextran 200 00,1.5%BSA, 0.5%Tween-80,
0.03%Proclin300, pH 7.0.
Embodiment 4
Control storing liquid is prepared, specific formula is：The mass concentration of PB be the mass concentration of 50mM, BSA be 1%,
The mass concentration of Tween-80 is 1%, and the mass concentration of glucose is 1%, the mass concentration of glycine is 0.5%, PEG4000
Mass concentration be 2%, Proclin300 mass concentrations be 0.3%, pH 7.0~7.5.
Control bonding pad pretreatment fluid is prepared, specific formula is：The mass concentration of PVA is 0.5%, Triton X-100's
Mass concentration is 0.5%, the mass concentration of Proclin300 or Sodium azide is 0.3%, pH 7.0~7.2.
Prepared using the preferred storing liquid collocation embodiment 2 of the preparation of embodiment 1 (following simple preferably in combination with pad pretreatment fluid
Claim biliquid 2) carry out stability and precision with compareing storing liquid collocation control bonding pad pretreatment fluid (hereinafter referred to as biliquid 1)
Contrast, implementation and result are as follows：
With biliquid 1 and biliquid 2, spray film is carried out to the fluorescent microsphere of same process mark d-dimer (D-dimer) monoclonal antibody
It is dry, 40 test strips are assembled into respectively, put aluminium foil bag, drier sealing.Place 4 DEG C of refrigerator for one bag and preserve 7 days (control group),
37 DEG C of incubator is placed for other one bag to accelerate 7 days.After complete 7 days, strip is taken out, it is 50ng/mL and 500ng/ to be separately added into concentration
The D-dimer recombinant antigens detection of mL.Accelerate preserved 1 year equivalent to 4 DEG C within 7 days according to theoretical 37 DEG C, the antigen inspection after simulating 1 year
Survey situation, as a result as shown in table 1 below, table 2.
The Contrast on effect of test strips test 50ng/mL D-dimer recombinant antigens made from 1 two kinds of biliquids of table
The Contrast on effect of test strips test 500ng/mL D-dimer recombinant antigens made from 2 two kinds of biliquids of table
Being drawn from table 1,2, preferable biliquid 2 only declines within 5% after 37 DEG C accelerate seven days, and precision is preferable,
5% or so；And compare biliquid 1 37 DEG C accelerate seven days after, decline 18% or so, precision is relatively poor.
Embodiment 5
D-dimer immunofluorescence quantitative test paper bar prepared by embodiment 2 is established into standard curve and is detected, is implemented
Method is as follows：
(1) standard curve is established：It is height value calibration product mixed diluting to 200ng/ with Sysmex D dimer concentration
Seven concentration of mL, 500ng/mL, 900ng/mL, 1800ng/mL, 3600ng/mL, 6000ng/mL, 8000ng/mL.With preparing
Test strips be loaded 80 μ L on immunofluorescence analysis to detect, each concentration, which is repeated 3 times, to be averaged.It is with diluted concentration (Xi)
Independent variable, obtains equation of linear regression, as shown in Figure 1 with testing result measured value (Yi) for dependent variable；
(2) with antigenic dilution to recombinant antigen d-dimer doubling dilution to 250ng/mL, 500ng/mL, 1000ng/
Six concentration of mL, 2000ng/mL, 4000ng/mL, 8000ng/mL.Antigen diluent formula of liquid is：20mM PBS, 0.5%BSA,
0.5%Tween-20, pH 7.2.T/C peaks face will be tried to achieve and substitute into Y in y=0.001x+0.050, try to achieve X to be surveyed antigen value.
As shown in Figure 2, it can be seen that ELISA test strip value and the small (R of theoretical value deviation prepared by embodiment 22=0.997),
The accuracy rate of detection is high.
Embodiment 6
With biliquid 1 and biliquid 2 (with embodiment 4), make bonding pad semi-finished product and to same process mark D-dimer monoclonal antibodies
Fluorescent microsphere carry out spray film drying, assemble test strips, be respectively 260ng/mL, 450ng/mL with concentration, 970ng/mL,
Patients serum's sample pair of the Sysmex CA7000 assignment containing D-dimer of 1860ng/mL, 2470ng/mL, 4220ng/mL
Two kinds of test strips sample-adding detections, are then read out test strips window information with supporting detecting instrument.Pass through Instruments Laser pair
The fluorescent microsphere of window is excited, then the transmitting fluorescence intensity (making ordinate) of 300 positions (making abscissa) of acquisition window,
300 points are connected together to form line chart in order using fluorescence analysis software.
As shown in figure 3, line chart can reflect race film situation of the fluorescent microsphere on NC films, there are two peaks, left side T in figure
Peak (corresponding naked eyes regard strip as detection line), the right are C peaks (corresponding naked eyes regard strip as nature controlling line).Use the entirety of biliquid 2
Appearance effect is preferable, and it is preferable to illustrate that biliquid 2 is discharged into NC film effects to antibody-fluorescent microsphere conjugate from bonding pad.Using double
Not high, the baseline out-of-flatness that then goes out peak-to-peak signal of liquid 1, leading portion lift more apparent, influence calculating of the software to peak area, and then influence
The accuracy of readings.Biliquid 2 can also improve the sensitivity of the project, and the sample of same concentration, T peaks are substantially than biliquid 1
Will height.
Embodiment 7
With biliquid 1 and biliquid 2 (with embodiment 4), make bonding pad semi-finished product and to same process D-dimer monoclonal antibodies mark
Fluorescent microsphere carries out spray film drying, 33 test strips is assembled respectively, with patient's blood of the antigen containing D-dimer of 33 various concentrations
Final proof sheet is at the same time to two kinds of test strips sample-adding detections.Test strips window information is read out with supporting detecting instrument, uses fluorescence
Analysis software calculates T peak areas and C peak areas, while full-automatic with the CA7000 under blood clotting aspect authorized carriers Sysmex
The end value of coagulo meter detection.
The clinical diagnosis accuracy of ELISA test strip result prepared by 2 kinds of biliquids is evaluated with Sysmex CA7000.Such as figure
4th, shown in 5, ordinate is T peak areas/C peak areas, abscissa Sysmex CA7000 end values, test strips prepared by biliquid 2
Testing result and the relatively identical (R of CA7000 Instrumental results of Sysmex company2=0.982), and the serum of biliquid 1 is linear
Related poor (R2=0.925), repeatability is also bad.This makes fluorescent microsphere releasing effect more preferable with foregoing biliquid 2, and software calculates
Area is more accurate related.
The above description is merely a specific embodiment, but protection scope of the present invention is not limited thereto, any
Belong to those skilled in the art the invention discloses technical scope in, the change or replacement that can readily occur in, all should
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be subject to scope of the claims.
Claims (10)
1. a kind of d-dimer immunofluorescence quantification kit, including end liner, overlap joint is provided with sample pad, knot successively on end liner
Close pad, nitrocellulose filter and absorption pad, it is characterised in that the bonding pad is by the immersion of bonding pad pretreatment fluid, drying.
2. d-dimer immunofluorescence quantification kit according to claim 1, it is characterised in that the bonding pad is sprayed with
D-dimer monoclonal antibody-fluorescent microsphere conjugate, the d-dimer monoclonal antibody-fluorescent microsphere conjugate is by storing
Liquid storage, dilution.
3. d-dimer immunofluorescence quantification kit according to claim 1 or 2, it is characterised in that the bonding pad
Pretreatment fluid include the PVA of 0.2%~5.0% mass concentration, 0.1%~5.0% mass concentration Triton X-100,
The sucrose of 1.0%~6.0% mass concentration, the Proclin300 of 0.01%~1.5% mass concentration or Sodium azide, pH 6.5~
7.8。
4. d-dimer immunofluorescence quantification kit according to claim 3, it is characterised in that the bonding pad is located in advance
Manage liquid include the PVA of 0.2%~3.0% mass concentration, Triton X-100 of 0.5%~3.5% mass concentration, 1.0%~
The sucrose of 2.25% mass concentration, the Proclin300 of 0.03%~0.1% mass concentration or Sodium azide, pH 7.0~7.8.
5. d-dimer immunofluorescence quantification kit according to claim 1 or 2, it is characterised in that the storing liquid
PB comprising 15~50mM, the BSA of 0.5%~10% mass concentration, the Tween-80 of 0.4%~10% mass concentration, 0.4%
The glucose of~10% mass concentration, the glycine of 1.5%~10% mass concentration, 0.8%~5% mass concentration
PEG4000, the PEG20000 of 1%~8% mass concentration, the Proclin300 of 0.01%~1.5% mass concentration or Sodium azide,
PH 6.5~9.0.
6. d-dimer immunofluorescence quantification kit according to claim 5, it is characterised in that the storing liquid includes
The PB of 20~40mM, the BSA of 0.5%~4.8% mass concentration, the Tween-80 of 0.5%~6.0% mass concentration, 0.5%~
The glucose of 3.0% mass concentration, the glycine of 2%~7% mass concentration, 1.0%~3.0% mass concentration PEG4000,
The PEG20000 of 1.5%~4.0% mass concentration, the Proclin300 of 0.03%~0.1% mass concentration or Sodium azide, pH
7.0~8.0.
7. a kind of preparation method of d-dimer immunofluorescence quantification kit as claimed in claim 1, includes the following steps：
The preparation of S1, sample pad：Glass fibre element film is soaked with sample pad treatment fluid, dries, takes out spare after immersion treatment；
The preparation of S2, bonding pad：Glass fibre element film is soaked with bonding pad pretreatment fluid, is dried after immersion treatment, is switched to use
Width, is made bonding pad semi-finished product, spare；
S3, by polystyrene fluorescent microsphere successively add EDC and NHS, activated in activation buffer；
S4, add mouse source d-dimer monoclonal antibody, is coupled in coupling buffer with fluorescent microsphere, after add closing
Liquid, is made mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, and supernatant is abandoned after centrifugation, adds storing liquid and preserves；
S5, with storing liquid diluted mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, with metal spraying draw film instrument be sprayed on through
It is spare with aluminium foil bag sealing storage after air dry oven is dried on the processed bonding pad of bonding pad pretreatment fluid；
Sheep anti-mouse igg polyclonal antibody and mouse source d-dimer monoclonal antibody, be coated with by S6 with coating buffer respectively, with ribbon
It is individually fixed in by the use of metal spraying stroke film instrument on nitrocellulose filter and is used as nature controlling line and detection line；
S7, paste with overlapping successively on end liner：Sample pad, bonding pad, nitrocellulose filter, water absorption pad, and cut into specific
The immunofluorescence chromatograph test strip of width；
The test strips cut in S7, be loaded by S8, crosses case pressing machine.
8. preparation method according to claim 7, it is characterised in that comprise the following steps：
The preparation of S1, sample pad：Glass fibre element film is soaked with sample pad treatment fluid, dries, takes out spare after immersion treatment；
The preparation of S2, bonding pad：Glass fibre element film is soaked with bonding pad pretreatment fluid, is dried after immersion treatment, is switched to use
Width, is made bonding pad semi-finished product, spare；
S3, the 200nm polystyrene fluorescent microsphere of 1mg successively added 20 μ L 0.5mg/mL EDC and 0.5mg/mL
NHS, activates in activation buffer；
S4, add 0.1mg mouse source d-dimer monoclonal antibody, is coupled, finishes with fluorescent microsphere in 200 μ L coupling buffers
20 μ L of confining liquid are added afterwards, mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate is made, supernatant is abandoned after centrifugation, are added
Storing liquid preserves；
S5, with storing liquid be diluted to 0.5mg/mL by mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, is drawn with metal spraying
Film instrument is sprayed on the combined pad processed bonding pad of pretreatment fluid, standby with aluminium foil bag sealing storage after air dry oven is dried
With；
Sheep anti-mouse igg polyclonal antibody and mouse source d-dimer monoclonal antibody, be coated with by S6 with coating buffer respectively, with ribbon
It is individually fixed in by the use of metal spraying stroke film instrument on nitrocellulose filter and is used as nature controlling line and detection line；
S7, paste with overlapping successively on end liner：Sample pad, bonding pad, nitrocellulose filter, water absorption pad, and cut into specific
The immunofluorescence chromatograph test strip of width；
The test strips cut in S7, be loaded by S8, crosses case pressing machine.
9. preparation method according to claim 8, it is characterised in that comprise the following steps：
The preparation of S1, sample pad：Glass fibre element film is soaked with sample pad treatment fluid, dries, takes out spare after immersion treatment；
The preparation of S2, bonding pad：Glass fibre element film is soaked with bonding pad pretreatment fluid, is dried after immersion treatment, is switched to use
Width, is made bonding pad semi-finished product, spare；
S3, the 200nm polystyrene fluorescent microsphere of 1mg successively added 20 μ L 0.5mg/mL EDC and 0.5mg/mL
NHS, activates in activation buffer；
S4, add 0.1mg mouse source d-dimer monoclonal antibody, is coupled, finishes with fluorescent microsphere in 200 μ L coupling buffers
20 μ L of confining liquid are added afterwards, mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate is made, supernatant is abandoned after centrifugation, are added
Storing liquid preserves；
S5, with storing liquid be diluted to 0.5mg/mL by mouse source d-dimer monoclonal antibody-fluorescent microsphere conjugate, is drawn with metal spraying
Film instrument is sprayed on the combined pad processed bonding pad of pretreatment fluid, standby with aluminium foil bag sealing storage after air dry oven is dried
With；
S6, by 1mg/mL sheep anti-mouse iggs polyclonal antibody and 1mg/mL mouse source d-dimer monoclonal antibody use coating buffer respectively
Coating, is individually fixed on nitrocellulose filter using ribbon 1.0mm by the use of metal spraying stroke film instrument and is used as nature controlling line and detection line；
S7, paste with overlapping successively on end liner：Sample pad, bonding pad, nitrocellulose filter, water absorption pad, and cut into specific
The immunofluorescence chromatograph test strip of width；
The test strips cut in S7, be loaded by S8, crosses case pressing machine.
10. preparation method according to claim 7, it is characterised in that the activation buffer is MES, pH of 75mM
5.5；The coupling buffer is PB, pH 7.5 of 20mM；The confining liquid is 20%BSA；The coating buffer includes：20mM
PBS, 1.2% isopropanol, 0.4% Dextran 200 00,1.5%BSA, 0.5%Tween-80,0.03%Proclin300, pH
7.0。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711176368.3A CN108020661A (en) | 2017-11-22 | 2017-11-22 | A kind of d-dimer immunofluorescence quantification kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711176368.3A CN108020661A (en) | 2017-11-22 | 2017-11-22 | A kind of d-dimer immunofluorescence quantification kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108020661A true CN108020661A (en) | 2018-05-11 |
Family
ID=62080017
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711176368.3A Pending CN108020661A (en) | 2017-11-22 | 2017-11-22 | A kind of d-dimer immunofluorescence quantification kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108020661A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111638363A (en) * | 2020-04-30 | 2020-09-08 | 吉林省格瑞斯特生物技术有限公司 | 3-deoxyfructose rapid quantitative fluorescence detection device and preparation method thereof |
| CN112213498A (en) * | 2020-09-28 | 2021-01-12 | 河南迈达斯实业有限公司 | D-dimer determination kit and application thereof |
| CN113866426A (en) * | 2021-09-26 | 2021-12-31 | 上海艾瑞德生物科技有限公司 | Kit for detecting D-dimer and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090142856A1 (en) * | 2007-12-03 | 2009-06-04 | Robert Hudak | Use of unique fluorochromes in rapid test systems |
| US20120028245A1 (en) * | 2008-12-30 | 2012-02-02 | Jin Po Lee | Quantitative analyte assay device and method |
| CN106370869A (en) * | 2016-08-31 | 2017-02-01 | 中山市创艺生化工程有限公司 | Storage liquid for antibody-fluorescent microsphere coupling and application of storage liquid |
| CN106370840A (en) * | 2016-08-31 | 2017-02-01 | 中山市创艺生化工程有限公司 | Procalcitonin immunofluorescence quantitation test strip and preparation method thereof |
| CN106405078A (en) * | 2016-08-31 | 2017-02-15 | 中山市创艺生化工程有限公司 | D-dimer immunofluorescent quantitative test strip and preparation method thereof |
| CN106442966A (en) * | 2016-08-31 | 2017-02-22 | 中山市创艺生化工程有限公司 | C-reactive protein immunofluorescence quantitative test strip and preparation method thereof |
-
2017
- 2017-11-22 CN CN201711176368.3A patent/CN108020661A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090142856A1 (en) * | 2007-12-03 | 2009-06-04 | Robert Hudak | Use of unique fluorochromes in rapid test systems |
| US20120028245A1 (en) * | 2008-12-30 | 2012-02-02 | Jin Po Lee | Quantitative analyte assay device and method |
| US20150140552A1 (en) * | 2008-12-30 | 2015-05-21 | Jin Po Lee | Quantitative analyte assay device and method |
| CN106370869A (en) * | 2016-08-31 | 2017-02-01 | 中山市创艺生化工程有限公司 | Storage liquid for antibody-fluorescent microsphere coupling and application of storage liquid |
| CN106370840A (en) * | 2016-08-31 | 2017-02-01 | 中山市创艺生化工程有限公司 | Procalcitonin immunofluorescence quantitation test strip and preparation method thereof |
| CN106405078A (en) * | 2016-08-31 | 2017-02-15 | 中山市创艺生化工程有限公司 | D-dimer immunofluorescent quantitative test strip and preparation method thereof |
| CN106442966A (en) * | 2016-08-31 | 2017-02-22 | 中山市创艺生化工程有限公司 | C-reactive protein immunofluorescence quantitative test strip and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111638363A (en) * | 2020-04-30 | 2020-09-08 | 吉林省格瑞斯特生物技术有限公司 | 3-deoxyfructose rapid quantitative fluorescence detection device and preparation method thereof |
| CN112213498A (en) * | 2020-09-28 | 2021-01-12 | 河南迈达斯实业有限公司 | D-dimer determination kit and application thereof |
| CN113866426A (en) * | 2021-09-26 | 2021-12-31 | 上海艾瑞德生物科技有限公司 | Kit for detecting D-dimer and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106370869B (en) | A kind of storing liquid and its application of antibody-fluorescent microballoon conjugate | |
| CN106405078B (en) | A kind of d-dimer immunofluorescence quantitative test paper item and preparation method thereof | |
| CN102692504A (en) | D-dimer quantitative fluorescence immunoassay test strip and preparation method thereof | |
| JP5738328B2 (en) | Method for producing agglutination reagent, method for measuring analyte, test kit and analysis device | |
| CN102680692A (en) | Immunochromatographic quantitative test reagent based on near-infrared fluorescent marker | |
| CN106442966B (en) | A kind of c reactive protein immunofluorescence quantitative test paper item and preparation method thereof | |
| CN106370840B (en) | A kind of Procalcitonin immunofluorescence quantitative test paper item and preparation method thereof | |
| Pei et al. | Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor | |
| CN103492879B (en) | Method for affixing antibodies to self-assembled monolayer | |
| CN107132359B (en) | Pepsinogen Cgene and Pepsinogen II detection method and its kit | |
| CN108020661A (en) | A kind of d-dimer immunofluorescence quantification kit and preparation method thereof | |
| CN105388292A (en) | Reagent kit and method for joint detection of PCT, CRP and IL-6 | |
| CN105785041A (en) | Test strip for quantitatively detecting calprotectin, preparation method thereof and determining method for calprotectin concentration | |
| CN101566631A (en) | Magnetic immunochromatographic test strip for combined detection of HIV-1+2 antibody and P24 antigen and preparation method thereof | |
| CN107589266A (en) | A kind of VEGF latex enhancing immune is than turbid kit and its application | |
| CN106959372A (en) | Serum amyloid A protein and the two-in-one measure kit of C reactive proteins and preparation method | |
| CN107957495A (en) | A kind of CK-MB detection kits and its application method | |
| CN102707056B (en) | Immunofluorescence test strip component for quickly and quantitatively detecting myocardial creatine kinase isozyme, detection card component comprising same and preparation method | |
| CN209102726U (en) | Placenta growth factor fluorescence immune chromatography test paper bar, test board and kit | |
| CN206038685U (en) | Immunity side direction chromatography detecting system | |
| CN107703313A (en) | One kind detection myoglobins immunofluorescence quantitative test paper bar | |
| CN104407134A (en) | Method and kit for detecting A type influenza virus H1 subtype | |
| CN104950111A (en) | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit | |
| CN109541235A (en) | Heparin-binding protein and Procalcitonin two joint detection reagent box and preparation method thereof | |
| CN108918865A (en) | Fluorescence immune chromatography test paper bar and reagent card |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180511 |