CN110488004A - A kind of 25-hydroxy-vitamin D kit and its preparation method and application for detecting peripheral blood - Google Patents
A kind of 25-hydroxy-vitamin D kit and its preparation method and application for detecting peripheral blood Download PDFInfo
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- CN110488004A CN110488004A CN201910916842.4A CN201910916842A CN110488004A CN 110488004 A CN110488004 A CN 110488004A CN 201910916842 A CN201910916842 A CN 201910916842A CN 110488004 A CN110488004 A CN 110488004A
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
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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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The present invention relates to a kind of 25-hydroxy-vitamin D kits and its preparation method and application for detecting peripheral blood, the kit includes 25(OH)VD immunochromatographydetection detection card and releasing agent, the 25-hydroxy-vitamin D immunochromatographydetection detection card includes test strips, the test strips include detection line and nature controlling line, it is coated with 25-hydroxy-vitamin D comlete antigen in the detection line, goat-anti rabbit polyclonal antibody is coated on the nature controlling line.25-hydroxy-vitamin D kit high sensitivity provided by the present invention, stability is strong, the range of linearity is wide, has excellent accuracy and precision.
Description
Technical field
The invention belongs to in-vitro diagnosis field, be related to a kind of 25-hydroxy-vitamin D kit for detecting peripheral blood and
Preparation method and application.
Background technique
Vitamin D is steroid derivatives, belongs to liposoluble vitamin, has and adjusts alcium and phosphor metabolization, influences cell Proliferation point
The effects of change is to maintain a kind of essential steroid hormone of human health.The intracorporal vitamin D of people is mainly shone by sunlight
It is synthesized after penetrating, only small part is taken in from food, and the principal mode that vitamin D occurs in vivo is 25-hydroxy-vitamin D.
The main function of vitamin D is to adjust phosphorus, calcium metabolism, promotes the absorption and reabsorption phosphorus of phosphorus and calcium in enteron aisle
The mode of calcium, the phosphorus calcium level in Lai Tigao blood, the method for calcification, with calcitonin, thyroid hormone and other hormones
Collective effect makes sclerotin become hard.In addition to traditional bone effect, additionally it is possible to influence tumour, diabetes, the heart
The non-bone effect such as vascular diseases, autoimmune disease.
Currently, clinically the detection common method of 25-OH-D have chemiluminescence, colloidal gold immunochromatographimethod, enzyme-linked immunization,
Electrochemical luminescence, these detection methods are largely required to handle the whole blood sample of acquisition, be taken a long time, and are operated numerous
Trivial, used blood sample demand is big and is venous blood.However for patients such as infant, children, large-area burns
For, it adopts the blood sample more difficulty of sufficient demand and is not easy to extract vein repeatedly.In contrast, the acquisition of peripheral blood operates letter
Just, fast, blood sampling volume is less, does not need professional's operation, the wound caused by human body is smaller, and some inspections clinically
Surveying index and venous blood does not have difference, is the source of preferable blood sample.
At present clinically, the detection of 25-hydroxy-vitamin D is almost seldom detected using peripheral blood.
Summary of the invention
In view of this, the main purpose of the present invention is to provide the 25-hydroxy-vitamin D kits for detecting peripheral blood
And its preparation method and application.
To achieve the goals above, the present invention provides the following technical scheme that
It is a kind of for detecting the 25-hydroxy-vitamin D kit of peripheral blood, including 25(OH)VD immunochromatography inspection
Card and releasing agent are surveyed, the 25-hydroxy-vitamin D immunochromatographydetection detection card includes test strips, and the test strips include detection line
And nature controlling line, 25-hydroxy-vitamin D comlete antigen is coated in the detection line, and it is more that goat-anti rabbit is coated on the nature controlling line
Clonal antibody；The releasing agent of 1L includes each component of following content: dithiothreitol (DTT), the 0.1g~2g of 154mg~771mg
Lauryl sodium sulfate, the ethylenediamine tetra-acetic acid of 2.92g~14.61g, the sodium citrate of 11.76g~29.41g, 12.9g~
The ethyl alcohol of the trisodium citrate of 25.81g, 10g~50g, surplus is distilled water.
In a concrete scheme of the invention, wherein the test strips further include PVC board, it is fixed in the PVC board
Sequentially connected sample pad, labeling pad, coating pad and water absorption pad, the packet, which is covered with, is successively arranged detection line and nature controlling line,
The sample pad and labeling pad are connected as one.
In a concrete scheme of the invention, wherein the coating pads and is connected with marker close to one end of detection line
Pad, one end close to nature controlling line are connected with water absorption pad.
In a concrete scheme of the invention, wherein being coated with goat-anti 25-hydroxy-vitamin D list in the labeling pad
The latex fluorescent microsphere of clonal antibody label and the latex fluorescent microsphere of rabbit igg label, the goat-anti 25(OH)VD Dan Ke
The molar ratio of the fluorescent latex microballoon of the fluorescent latex microballoon and rabbit igg label of grand antibody label is 1:0.2~4.
In a concrete scheme of the invention, wherein 25-hydroxy-vitamin D detection card further includes setting examination for blocking
Paper slip gets stuck.
In a concrete scheme of the invention, wherein described get stuck includes:
Kerve is connected to the PVC board；
Upper cover is connected to the kerve, the well for being loaded in the sample pad is provided on the upper lid；
Observation window is set on lid and for detection line and the acquisition of the data of nature controlling line.
It is a kind of for detecting the preparation method of the 25-hydroxy-vitamin D kit of peripheral blood, comprising the following steps:
1) preparation of coating pad: 25-hydroxy-vitamin D comlete antigen and goat-anti rabbit polyclonal antibody are coated with respectively to nitre
On acid cellulose film, drying for standby；
2) preparation of labeling pad: by the latex fluorescent microsphere of goat-anti 25(OH)VD labeling of monoclonal antibody and rabbit
After the latex fluorescent microsphere mixing of IgG label, it is sprayed on glass fibre element film, drying for standby；
3) test strips are assembled: the bonding coating pad in PVC board, and overlapped in the one end for the nature controlling line being covered with close to the packet
Water absorption pad, overlap joint labeling pad and its sample pad of connection in the one end for the detection line being covered with close to the packet；Then it is cut
At the test strips of required width, the reagent strip is put into gets stuck later.
It is a kind of for detecting the application method of the 25-hydroxy-vitamin D kit of peripheral blood, comprising the following steps:
1) blood sample taken and releasing agent are diluted according to the volume ratio of 1~10:10；
2) blood sample in 30~100 μ L steps 1) after dilution is taken to be added drop-wise to the 25-hydroxy-vitamin D detection card
In well (sample pads of corresponding test strips), it is stored at room temperature 5~20min, then enters 25-hydroxy-vitamin D detection card insertion
It is detected to fluorescence immunity analyzer, can get testing result immediately.
In a concrete scheme of the invention, wherein the blood sample is selected from peripheral blood.
In a concrete scheme of the invention, wherein the peripheral blood is selected from finger tip blood or Ear lobe blood.
Beneficial effects of the present invention:
1,25-hydroxy-vitamin D kit provided by the present invention can be used for venous blood (serum, blood plasma, whole blood), end
The detection of tip blood and diagnosis for vitamin D deficiency related disease, are especially able to carry out the detection of peripheral blood, and sample is used
Measure it is small, detection can be completed within 5~20min, linear detection range be 5ng/mL~120ng/mL, greatly improve inspection
Efficiency is surveyed, can effectively solve the problem that infant, children, the rare problem of Patients with Big Area Burn blood sampling.
2,25-hydroxy-vitamin D kit provided by the present invention, high sensitivity, stability are strong, the range of linearity is wide,
With excellent accuracy and precision.
3, after blood sample is using the release dilution agent in the present invention, without falling into a long wait, pattern detection can be carried out,
Easy to operate, the sample process time is short, reduces time cost, is suitable for clinical quick diagnosis detection.
4,25-hydroxy-vitamin D test strips provided by the present invention, sample pad and labeling pad are connected as one, system
It is standby simple, reduce red tape, reduces the production cost of kit.
Detailed description of the invention
Fig. 1 is test strips schematic diagram in 25-hydroxy-vitamin D immunochromatographydetection detection card provided by the present invention；
Fig. 2A is a kind of internal structure of upper cover in 25-hydroxy-vitamin D immunochromatographydetection detection card provided by the present invention
Schematic diagram；
Fig. 2 B is a kind of internal structure of kerve in 25-hydroxy-vitamin D immunochromatographydetection detection card provided by the present invention
Schematic diagram；
Fig. 3 is 25-hydroxy-vitamin D kit detection process schematic diagram of the present invention；
Fig. 4 is the standard curve schematic diagram of 25(OH)VD in embodiment 1；
Fig. 5 detects finger tip peripheral blood for 25-OH-D kit of the present invention in implementation 3 and Siemens 25-OH-D kit is examined
Survey the dependency graph of the testing result of venous blood (serum).
Fig. 6 detects finger tip peripheral blood for 25-OH-D kit of the present invention in implementation 6 and Siemens 25-OH-D kit is examined
Survey the dependency graph of the testing result of venous blood (serum)；
Wherein, 1-PVC plate, 2- coating pad, 3- labeling pad, 4- water absorption pad, 5- detection line, 6- nature controlling line, 7- marker
Junction, 8- sample, 9- sample pad, 11- upper cover, 12- kerve, 13- well, 14- detection window, 15- test strips placement region,
16- fixed column, 17- location hole, the first limited section 18-, the second limited section 19-, 20- third limited section, 21- blood sample, 22-
Releasing agent, the blood sample after 23- dilution, 24-25- hydroxy-vitamine D detection card, 25- fluorescence immune chromatography instrument.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below with reference to embodiment, still
It should be appreciated that these descriptions are only further explanation the features and advantages of the present invention, rather than to the claims in the present invention
Limitation.
Following material or reagent are unless stated otherwise commercially available.
The preparation of embodiment 1:25- hydroxy-vitamine D (25-OH-D) kit
The preparation of 1.1 25-hydroxy-vitamin Ds (25-OH-D) immunochromatographydetection detection card
1) goat-anti 25-hydroxy-vitamin D (25-OH-D) labeling of monoclonal antibody latex fluorescent microsphere
Take 1mL latex fluorescent microsphere (purchased from Merck Millipore company) that 1mg carbodiimides (EDC) is added, 1mg
3h is stirred at room temperature in n-hydroxysuccinimide (NHS), and 0.1mg goat-anti 25-hydroxy-vitamin D monoclonal antibody, room is then added
Temperature is lower to stir 1h, adds 10mg BSA confining liquid, continues to stir 1h.At 2~8 DEG C, according to 11000r/min revolving speed from
Heart 30min removes supernatant.Finally, obtained after taking the phosphate buffer solution (pH=7.4) of 1mL 0.2M to be added to centrifugation
In solid sediment, the Proclin300 of 1 μ L is added, 1min is vibrated on the ultrasonic oscillator of 40Hz and is uniformly mixed, so
It is saved at 4 DEG C afterwards stand-by.
2) rabbit igg polyclonal antibody marks latex fluorescent microsphere
Take 1mL latex fluorescent microsphere (purchased from Merck Millipore company) that 1mg carbodiimides (EDC) is added, 1mg
3h is stirred at room temperature in n-hydroxysuccinimide (NHS), and 0.1mg sheep rabbit igg polyclonal antibody is then added, stirs 1h at room temperature,
10mg BSA confining liquid is added, continues to stir 1h.At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, is removed
Supernatant.Finally, the phosphate buffer solution (pH=7.4) of 1mL 0.2M is taken to be added to the solid sediment obtained after centrifugation
In, the Proclin300 of 1 μ L is added, 1min is vibrated on the ultrasonic oscillator of 40Hz and is uniformly mixed, is then protected at 4 DEG C
It deposits stand-by.
3) preparation of coating pad
25-hydroxy-vitamin D comlete antigen and goat-anti rabbit polyclonal antibody are used to the phosphate-buffered of 0.2mol/L respectively
Solution (pH=7.4) is diluted to 1mg/mL, is then carried out drawing a film on nitrocellulose filter with a stroke film gold spraying instrument.Contain 25- hydroxyl
The line of base vitamin D comlete antigen contains the line of goat-anti rabbit polyclonal antibody as nature controlling line 6, then 37 as detection line 5
DEG C, coating pad 2 is made in dry 4h in the environment of humidity < 30%.
4) preparation of labeling pad
1. glass fibre element film is impregnated 6h in the phosphate buffer (pH=7.4) of 0.2M, then done at 35 DEG C
Dry 8h, it is spare.
2. by the latex fluorescent microsphere and rabbit igg of the goat-anti 25(OH)VD labeling of monoclonal antibody that molar ratio is 1:1
The latex fluorescent microsphere of label after mixing, according to the velocity spray of 10 μ L/cm on glass fibre element film, is then placed within
Labeling pad 3 is made in dry 2h at 45 DEG C.Goat-anti 25(OH)VD monoclonal antibody mark is coated in the labeling pad 3
The position of the latex fluorescent microsphere of latex fluorescent microsphere and the rabbit igg label of note is known as marker junction 7.
The purpose of setting flag object junction 7 is: the blood sample 8 after release dilution agent is added drop-wise in sample pad 9, In
Under the action of water absorption pad 4, blood sample is chromatographed forward, the 25(OH)VD and mark in 7 blood sample of marker junction
Remember the latex fluorescent microsphere reaction of the coated goat-anti 25(OH)VD labeling of monoclonal antibody in object junction, the object after reaction
Matter, do not participate in reaction goat-anti 25(OH)VD labeling of monoclonal antibody latex fluorescent microsphere, rabbit igg label latex
Fluorescent microsphere is micro- as blood sample continues to chromatograph, the unreacted goat-anti 25(OH)VD monoclonal antibody at detection line
The fluorescent latex microballoon of label reacts with detection line coating 25-hydroxy-vitamin D comlete antigen and rests on detection line, in Quality Control
The latex fluorescent microsphere that rabbit igg marks at line reacts with the coated goat-anti rabbit polyclonal antibody of nature controlling line and rests on nature controlling line,
Other substances continue to chromatograph, finally by the signal (being denoted as T) of the fluorescent microsphere of fluorescence immune chromatography instrument difference acquisition testing line
With the signal (being denoted as C) of the fluorescent microsphere of nature controlling line, T/C value is calculated, reads blood from the standard curve of 25-hydroxy-vitamin D
The concentration of 25-hydroxy-vitamin D in liquid sample.
5) assembling of immunochromatographydetection detection card
Then the bonding coating pad 2 first in PVC board 1 overlaps water suction in one end of the nature controlling line 6 on coating pad 2
Pad 4, close to packet be covered with detection line 5 one end overlap joint labeling pad 3 and its connection sample pad 9, with cutting machine by its
The test strips of 4mm ± 0.1mm are cut into, are later inserted into the test strips in getting stuck, layer is immunized to be prepared into 25-hydroxy-vitamin D
Analysis detection card；It is described to get stuck selected from the prior art, for example, described get stuck (as shown in Fig. 2A, Fig. 2 B) may include: kerve 12,
It is connected to the PVC board 1；Upper cover 11 is connected to the kerve 12, is provided in the upper cover 11 for the sample
The well 13 being loaded on pad 9；Observation window 14 is set in upper cover 11, is acquired for detection line 5 and the data of nature controlling line 6.
As shown in Figure 2 B, the kerve 12 includes: symmetrical multiple location holes 17, Duo Gesuo positioned at its inner surface
It states between location hole 17 equipped with multiple for limiting the first limited section 18 of test strips transverse shifting and for limiting test strips
Second limited section 19 of longitudinal movement；Symmetrically arranged first limited section 18 is enclosed with second limited section 19 is set as paper slip
Placement region 15 (dashed region), for placing test strips；
As shown in Figure 2 A, the upper cover 11 includes: the multiple positioning columns 16 matched with multiple location holes 17, in this way
With the use of upper cover 11 and kerve 12 to be fixed together；The upper cover 11 further includes for limiting moving up and down for test strips
Third limited section 20.
The top of the coating pad 2 is equipped with the observation window 14 for data acquisition, to expose all detection line 5 and matter
Line 6 is controlled, for collecting its testing result；And the observation window 14 is opened in the upper cover 11 and test strips placement region 15
The corresponding position in middle part.The upper cover 11 offers well at position corresponding with the sample pad 9, to be used for sample
Sample 8 is added dropwise on product pad 9.The detection line is apart from well 15-25mm.
1.2. the preparation of releasing agent
By the dithiothreitol (DTT) (DTT) of 308mg, the lauryl sodium sulfate (SDS) of 1g, 5.84g ethylenediamine tetra-acetic acid
(EDTA), the sodium citrate of 17.65g, the trisodium citrate of 15.48g and 20g ethyl alcohol are added in beaker, while waterside is added to stir
To whole dissolutions, it then is released agent quantitatively to 1000mL with water, it is spare.
The detection of the 25-hydroxy-vitamin D kit of 2 embodiment 1 of embodiment
2.1 detection process (see Fig. 3)
1) blood sample 21 taken such as finger tip blood is diluted with releasing agent 22 according to the volume ratio of 3:10；
2) blood sample 23 in 60 μ L steps 1) after dilution is taken to be added drop-wise to the 25-hydroxy-vitamin D detection card 24
In well (sample pads 9 of corresponding test strips), it is stored at room temperature 15min and carries out immunochromatography reaction, then 25- hydroxyl is tieed up and is given birth to
Plain D detection card 24 is inserted into fluorescence immunity analyzer 25 and is detected, and can get testing result immediately.
2.2. the foundation of standard curve
The 25-hydroxy-vitamin D standard items (0,5,15,30,60,120ng/mL) for preparing various concentration, it is real with the present invention
The 25-hydroxy-vitamin D kit for applying the preparation of example 1 is successively detected according to the detection process in 2.1, and blank detection limit is not high
In 5ng/mL, detection range is 5~120ng/mL, and testing result is as shown in table 1, and it is quasi- to carry out curve using tetra- parameter of Logistic
It closes, degree of fitting R2=0.9961, standard curve as shown in figure 4, T/C value and calibration object in the range that concentration is 5~120ng/mL
Inside have good correlation.
1 detection data of table
| Normal concentration ng/mL | 0 | 5 | 15 | 30 | 60 | 120 |
| T/C value | 4.2345 | 2.8456 | 2.0914 | 1.3456 | 1.1378 | 0.7854 |
3 control experiment of embodiment
It is examined using the 25-hydroxy-vitamin D of the 25-hydroxy-vitamin D kit and Siemens prepared in Examples 1 and 2
Experiment is compared in test agent box.It is examined by 20 patient's finger tip peripheral bloods of acquisition and venous blood (serum, blood plasma, whole blood)
Survey, with determine the 25-hydroxy-vitamin D kit prepared in the present patent application whether suitable for finger tip peripheral blood 25- hydroxyl
The detection of vitamin D, wherein finger tip blood repeats detection 2 times.Testing result is as shown in table 2 below.
2 detection data of table
25-OH-D kit detection finger tip peripheral blood detects quiet with Siemens's 25-OH-D kit in the embodiment of the present invention 1
The testing result of arteries and veins blood (serum) carries out correlation analysis as shown in figure 5, regression equation is y=1.0113x+0.3081, R2=
0.9982, n=20, it is quiet to show that 25-OH-D kit detection finger tip peripheral blood of the present invention is detected with Siemens's 25-OH-D kit
The significant correlation of testing result of arteries and veins blood (serum) has same equivalence.It is related to detection 2 results progress that finger tip blood examination surveys 1
Property analysis, regression equation y=1.0010x-0.2952, R2=0.9996, n=20 show the 25- hydroxyl of the embodiment of the present invention 1
Base vitamin D kit has good stability.And kit of the present invention detection finger tip peripheral blood, venous blood (serum, blood plasma,
Whole blood) testing result carry out correlation analysis, regression equation is respectively as follows:
Y=1.006x+1.4477, R2=0.9926；Y=0.9983x+1.6160, R2=0.9904；Y=1.0119+
0.7650, R2=0.9912.Show 25-OH-D kit detection finger tip peripheral blood and detection venous blood (serum, blood of the present invention
Slurry, whole blood) the significant correlation of testing result, have same equivalence.
Therefore the embodiment of the present invention 1 25-hydroxy-vitamin D kit can satisfy peripheral blood, venous blood (serum,
Blood plasma, whole blood) adjuvant clinical diagnosis demand.
The preparation of 4 25-hydroxy-vitamin D kit of embodiment
4.1 the preparation of 25-hydroxy-vitamin D immunochromatographydetection detection card
Process one in " preparations of 1.1 25-hydroxy-vitamin D immunochromatographydetection detection cards " of the process and embodiment 1
It causes.
The preparation of 4.2 releasing agents
By the dithiothreitol (DTT) (DTT) of 463mg, the lauryl sodium sulfate (SDS) of 1.8g, 8.77g ethylenediamine tetrem
The ethyl alcohol of sour (EDTA), the sodium citrate of 17.65g, the trisodium citrate of 15.48g and 40g is added in beaker, while adding waterside
It stirs to whole dissolutions, is then released agent quantitatively to 1000mL with water, it is spare.
The detection of the 25-hydroxy-vitamin D kit of 5 embodiment 4 of embodiment
The process is consistent with the process in " the 1.3. detection " of embodiment 1.
6 control experiment of embodiment
Using the 25-hydroxy-vitamin D kit and the total 25-hydroxy-vitamin D of Siemens prepared in the embodiment of the present invention 4
Experiment is compared in assay kit.It is detected by 20 patient's peripheral bloods of acquisition and venous blood (serum), to determine this
The 25-hydroxy-vitamin D immunochromatography prepared in inventive embodiments 4 whether suitable for finger tip peripheral blood 25-hydroxy-vitamin D
Detection, wherein finger tip blood repeat detection 2 times.Testing result is as shown in table 3 below.
3 detection data of table
25-OH-D kit detection finger tip peripheral blood detects quiet with Siemens's 25-OH-D kit in the embodiment of the present invention 4
The testing result of arteries and veins blood (serum) carries out correlation analysis as shown in fig. 6, regression equation is y=1.0079x-0.7548, R2=
0.9917, n=20, it is quiet to show that 25-OH-D kit detection finger tip peripheral blood of the present invention is detected with Siemens's 25-OH-D kit
The significant correlation of testing result of arteries and veins blood (serum) has same equivalence.
1 and detection 2 results progress correlation analysis, regression equation y=1.0002x-0.1368, R are surveyed in finger tip blood examination2=
0.9993, n=20, show having good stability for 25-hydroxy-vitamin D kit of the invention.Therefore 25- hydroxyl of the invention
Vitamin D kit can satisfy the demand of peripheral blood adjuvant clinical diagnosis.
It is attached: required solution allocation
(1) 0.2M phosphate buffer solution
NaH2PO4.H2O 5.24g；
Na2HPO4.2H2O 28.83g；
Purified water is settled to 1000mL.
Embodiment described above be only for absolutely prove the present invention and for embodiment, protection scope of the present invention is unlimited
In this.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in protection of the invention
Within the scope of.Protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of for detecting the 25-hydroxy-vitamin D kit of peripheral blood, which is characterized in that exempt from including 25(OH)VD
Epidemic disease chromatography detection card and releasing agent, the 25-hydroxy-vitamin D immunochromatographydetection detection card includes test strips, the test strips packet
Detection line and nature controlling line are included, 25-hydroxy-vitamin D comlete antigen is coated in the detection line, is coated on the nature controlling line
Goat-anti rabbit polyclonal antibody；The releasing agent of 1L includes each component of following content: the dithiothreitol (DTT) of 154mg~771mg,
The lauryl sodium sulfate of 0.1g~2g, the ethylenediamine tetra-acetic acid of 2.92g~14.61g, 11.76g~29.41g citric acid
Sodium, the trisodium citrate of 12.9g~25.81g, the ethyl alcohol of 10g~50g, surplus are distilled water.
2. 25-hydroxy-vitamin D kit as described in claim 1, which is characterized in that the test strips further include PVC board,
Sequentially connected sample pad, labeling pad, coating pad and water absorption pad are fixed in the PVC board, the packet, which is covered with, successively to be set
There are detection line and nature controlling line, the sample pad and labeling pad are connected as one.
3. 25-hydroxy-vitamin D kit as claimed in claim 2, which is characterized in that the coating is padded close to detection line
One end is connected with labeling pad, and one end close to nature controlling line is connected with water absorption pad.
4. 25-hydroxy-vitamin D kit as claimed in claim 3, which is characterized in that be coated with sheep in the labeling pad
The latex fluorescent microsphere of latex fluorescent microsphere and the rabbit igg label of anti-25-hydroxy-vitamin D labeling of monoclonal antibody, the sheep
The molar ratio of the fluorescent latex microballoon of fluorescent latex microballoon and the rabbit igg label of anti-25(OH)VD labeling of monoclonal antibody
For 1:0.2~4.
5. 25-hydroxy-vitamin D kit according to any one of claims 1-4, which is characterized in that the 25- hydroxyl dimension
Raw element D detection card further includes that getting stuck for test strips is set for card.
6. 25-hydroxy-vitamin D kit as claimed in claim 5, which is characterized in that described get stuck include:
Kerve is connected to the PVC board；
Upper cover is connected to the kerve, the well for being loaded in the sample pad is provided on the upper lid；
Observation window is set on lid and for detection line and the acquisition of the data of nature controlling line.
7. a kind of for detecting the preparation method of the 25-hydroxy-vitamin D kit of peripheral blood, which is characterized in that including following
Step:
1) preparation of coating pad: 25-hydroxy-vitamin D comlete antigen and goat-anti rabbit polyclonal antibody are coated with respectively fine to nitric acid
It ties up on plain film, drying for standby；
2) preparation of labeling pad: by the latex fluorescent microsphere and rabbit igg mark of goat-anti 25(OH)VD labeling of monoclonal antibody
After the latex fluorescent microsphere mixing of note, it is sprayed on glass fibre element film, drying for standby；
3) test strips are assembled: the bonding coating pad in PVC board, and in the overlap joint water suction of the one end for the nature controlling line being covered with close to the packet
Pad, overlap joint labeling pad and its sample pad of connection in the one end for the detection line being covered with close to the packet；Then it is cut into institute
The test strips of width are needed, later install the reagent strip into getting stuck.
8. a kind of for detecting the application method of the 25-hydroxy-vitamin D kit of peripheral blood, which is characterized in that including following
Step:
1) blood sample taken and releasing agent are diluted according to the volume ratio of 1~10:10；
2) blood sample in 30~100 μ L steps 1) after dilution is taken to be added drop-wise to the sample-adding of the 25-hydroxy-vitamin D detection card
Kong Zhong is stored at room temperature 5~20min and carries out immunochromatography reaction, and 25-hydroxy-vitamin D detection card is then inserted into fluorescence and is exempted from
Epidemic disease analyzer is detected, and can get testing result immediately.
9. application method as claimed in claim 8, which is characterized in that the blood sample is selected from peripheral blood.
10. application method as claimed in claim 9, which is characterized in that the peripheral blood is selected from finger tip blood or Ear lobe blood.
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| CN114460314A (en) * | 2022-04-13 | 2022-05-10 | 普迈德（北京）科技有限公司 | Detection kit and detection method for multivitamin D analogues |
| CN115856326A (en) * | 2023-03-01 | 2023-03-28 | 山东康华生物医疗科技股份有限公司 | 25 hydroxy vitamin D detect reagent box |
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