CN106990254A - 25 hydroxycholecalciferols determine kit and preparation method - Google Patents
25 hydroxycholecalciferols determine kit and preparation method Download PDFInfo
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- CN106990254A CN106990254A CN201611157508.8A CN201611157508A CN106990254A CN 106990254 A CN106990254 A CN 106990254A CN 201611157508 A CN201611157508 A CN 201611157508A CN 106990254 A CN106990254 A CN 106990254A
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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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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Abstract
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically a kind of 25 hydroxycholecalciferol determines kit and preparation method, provided with test card, it is characterised in that the test card is sequentially provided with from the bottom to top：Rare earth Eu is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad3+25 hydroxycholecalciferol monoclonal antibodies of fluorescent microsphere mark, a diameter of 200nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, it is stable under ground state, wavelength 615nm fluorescence is launched under 337nm excitation source effect；The monoclonal antibody is the monoclonal antibody mixed after purification, from the cell strain of monoclonal antibody for 26 25 different hydroxycholecalciferol epitopes, has the advantages that easy to operate, reaction is quick, sensitivity is high, high specificity.
Description
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, including protein-crosslinking technology, film layer analysis skill
Art, labelling immunoassay technology etc..Specifically one kind can be fast and accurately in the samples such as serum, blood plasma and whole blood
The 25-hydroxyvitamin D3 that 25-hydroxyvitamin D3 carries out quantitative analysis determines kit and preparation method.
Background technology：
The derivative of VD systems steroids is the essential element for promoting body Calcium and phosphorous absorption, with more than 10 kinds of shapes in nature
Formula is present, and two most important forms of vitamin are vitamin Ds3And vitamin D2, to the most nutritious meaning of animal.And VD exists
Only be the precursor of its active metabolite in vivo, be only converted into 25- hydroxyls D3 (25-OH-D3) and 1,25- dihydroxy D3 (1,
25- (OH) 2D3) after just have bioactivity, could effectively play its biochemical functions.25-OH-D3 is vitamin D in body
Interior chief active product, is to evaluate vitamin D nutrition level and diagnosis rickets reliably sensitive index.25-OH-D3's
Important physiological function is the eubolism for adjusting body Ca, P, maintains animal blood calcium, serum phosphorus levels, keeps the normal growth of bone
Development, prevents acalcicosis, such as osteomalacia, rickets.In human body, vitamine D3 and D2 are combined with vitamin D in blood plasma
Protein binding, and liver is transported to, both turn into 25(OH)VD, 95% detected in serum through 25 hydroxylatings
25(OH)VD above is 25(OH)VD 3, and vitamin D deficiency is Secondary Hyperparathyroidism
Common disease factor.Parathyroid hormone cellulose content is improved, the parathyroid hormone cellulose content of the elderly for the D that is deficient in vitamin particularly is improved
Malacosteon can be caused, bone conversion is added, reduce bone mass, and have the danger of fracture.25-hydroxyvitamin D3 content is low
It is also low relevant with bone density.Combined with other clinical datas, its result can judge Bone m etabolism as supplementary means.
Immuno analytical method is using the immune response between trace antigen and corresponding antibody with high specificity, to detect such as
It is living in the organisms such as hormone, medicine, protein, polypeptide, enzyme, tumor associated antigen, micro-element, virus, bacterium and metallic element
Property material.Immuno analytical method includes labelling immunoassay, non-marked immunoassay and instrument immunoassay.This kit is utilized
The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be the one kind for belonging to labelling immunoassay.
Fluoroimmunoassay (FIA) and radiommunoassay (RIA) since the advent of the world, experienced the development of decades, but
It is that people increasingly feel FIA because of naturally local too high, interference detection results；RIA uses isotope marks, has pole to human body
It is big to endanger and made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme is unstable in itself, pushes away
Wide application is restricted.The beginning of the eighties, people begin one's study and replace fluorescent material and isotope-labelled protein with rare earth element
Or antibody, TIME RESOLVED TECHNIQUE is incorporated into field of biological detection, new ultramicron time-resolved fluoroimmunoassay point is established
Analysis technology (Time resolved Fluoroimmunoassay, abbreviation TrFIA).The technology uses multidisciplinary advanced technology, collection
The characteristics of having tied other immunoassays, in fields such as immunology, molecular biology, cytology and medical science, obtains significant progress
And extensive use.
TrFIA make use of trivalent rare earth ion and chelate with unique fluorescent characteristic be tracer replace fluorescent material,
Enzyme, isotope, chemiluminescent substance, labelled antibody, antigen, hormone, polypeptide, protein, nucleic acid probe and biological cell treat anti-
Answer system (such as antigen-antibody reaction, nucleic acid probe hybridization, biotin-labeled pentylamine reaction and the killing of target cell pairing effect cell
Effect etc.) occur after, with TrFIA detectors determine reaction product in fluorescence intensity.According to product fluorescence intensity and relatively glimmering
The ratio of luminous intensity, judges the concentration of analyte in reaction system, so as to reach quantitative analysis.In common fluoremetry,
Due to containing a variety of fluorescent components in test sample, background fluorescence is (caused by the colloidal solid and solvent molecule in sample
The non-specific fluorescence that scattered light and Proteins in Serum and other compounds are sent) intensity is big, disturb strong, as fluorescence point
The bottleneck that analysis method is promoted on a large scale.Why TrFIA can turn into after the new sensitive detection method of EIA, RIA latter,
Depend primarily on the wavelength resolution used in the unique fluorescence feature of lanthanide series, detection and time-delay technique and dissociation-
Enhancing technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, has in 17, be usually used in TrFIA mainly have europium (Eu),
Samarium (Sm), terbium (Tb), dysprosium (Dy).Lanthanide series has unique fluorescence radiation feature, compared with common fluorescent, lanthanide ion chela
Compound fluorescence decay time is long, is the 10 of conventional fluorescent3-106Times.If the fluorescence decay time of lanthanide ion chelate is in 60-
900 μ s, conventional Eu3+Fluorescence decay time is the fluorescence decay time of fluorogen in 714 μ s, common fluorescent immunoassay
The fluorescence decay time for having some protein in 1-100 μ s, sample is only 1-10 μ s, therefore utilizes TIME RESOLVED TECHNIQUE, delay
Measured after certain time, just can obtain Eu3+Specific fluorescence signal.Simultaneously because decay time is long, Eu3+Label is in measurement
It can be repeatedly excited in time, ground state is transitted to by excitation state quickly after exciting every time, just has fluorescence to send, then again can quilt
Remotivate, so it is per second have 1000 times excite so that the relative specific activity of TrFIA fluorescent markers is very high.Lanthanide series
The maximum of fluorescence spectrum is characterized in that the Stokes displacements between exciting light and transmitting light are larger, Eu3+Excitation wavelength is 337nm, hair
The a length of 615nm of ejected wave, Stokes displacements are up to 278nm；While Eu3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is non-
It is often sharp, instrument adjustment can be made to be determined in extremely narrow wave-length coverage, the interference of background fluorescence is thus almost completely eliminated,
Then passage time delay and wavelength resolution, distinguish out (therefore referred to as time resolution) by strong specificity fluorescent and background fluorescence, make to do
Disturb reach it is almost nil.
In view of the application of above labeling method and detection technique, this kit has good detection specific, higher
Sensitivity, the simplicity of operation and stable fluorescent marker ensure that the accuracy of detection.
The content of the invention：
The present invention is for shortcoming and defect present in prior art, it is proposed that a kind of utilization fluorescence immune chromatography it is sensitive
Property, with reference to fluorescence immune chromatography analyzer realize sensitivity it is high, fast and simple, can be with the 25- hydroxy vitamins of accurate quantitative analysis
D3 determines kit and preparation method.
The present invention can be reached by following measures：
A kind of 25-hydroxyvitamin D3 determines kit, provided with test card, it is characterised in that the test card is from the bottom to top
It is sequentially provided with：Rare-earth fluorescent is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad
Microballoon mark anti-25-hydroxyvitamin D3 monoclonal antibody-microballoon coupled complex, the rare-earth fluorescent microballoon it is a diameter of
100-250nm, rare-earth fluorescent microballoon is stable under ground state containing the one or more in rare earth lanthanide, 300-400nm's
Launch the fluorescence that wave-length coverage is 550-650nm under excitation source effect；The monoclonal antibody is the list mixed after purification
Clonal antibody, from the cell strain of monoclonal antibody for 2-6 different 25-hydroxyvitamin D3 epitopes.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 150-200nm；The rare-earth fluorescent microballoon
Preferably comprise one or more of rare earth lanthanides；The antibody that rare-earth fluorescent microballoon is marked on pad is preferably derived from for 2
The monoclonal cell cell line of individual different epitopes.
Pad of the present invention is made using following steps：Glass fibre membrane is soaked in 150mM Tris-HCL processing
In liquid (X-100 containing 1.0%Triton, 2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 small
When, it is standby, glass fibre membrane is placed on the three-dimensional specking platforms of Bio-DotXYZ3050, connect with Bio-Jet Quanti300 are non-
The anti-25-hydroxyvitamin D3 monoclonal antibody coupled complex that rare-earth fluorescent microballoon is marked is sprayed onto by the micro- quantitation nozzle of touch
Glass fibre membrane, 37 DEG C drying 1 hour after be made.
The anti-25-hydroxyvitamin D3 monoclonal antibody of rare-earth fluorescent microballoon mark in the present invention on pad is adopted
It is made with following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：Mouse is immunized with 25-hydroxyvitamin D3 sterling, using standard
Method for preparing monoclonal antibody prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line obtained is entered
Row pairing screening, the monoclonal antibody cell line for kit is selected according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify anti-25- hydroxyls dimension life
Plain D3 monoclonal antibodies, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, pH 9.5 carbonate delays
Fliud flushing, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbon
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In the above-mentioned carbonate buffer solution for washing and being resuspended to 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The preparation of the anti-25-hydroxyvitamin D3 monoclonal antibody of rare-earth fluorescent microballoon mark：Choose and come from 2
The monoclonal antibody of the monoclonal cell cell line of different epitopes, according to mass ratio 1:1 by 2mg 25- hydroxy vitamins
D3 monoclonal antibodies are with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, and then the rare-earth fluorescent microballoon with above-mentioned aldehyde radical is mixed
Close, 4 DEG C of reactions are stayed overnight；Then, sodium borohydride is added to final concentration 5mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid
(50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are stayed overnight；Then 50mM Tris-HCL, pH7.4 are used
Buffer solution using centrifugal process wash 3 times, be resuspended in 100 μ l 50mM Tris-HCL buffer solutions (contain 1.2%NaCL,
0.5%BSA, 0.1%Tween 20), 4 DEG C be kept in dark place it is standby.
The nitrocellulose filter of the present invention for being coated with detection line and nature controlling line is made by following steps：
Step 1：Using the cell different from anti-25-hydroxyvitamin D3 cell strain of monoclonal antibody used on pad
Strain, prepared using the ascites production technology of standard and purify anti-25-hydroxyvitamin D3 monoclonal antibody, be stored in -20 DEG C it is standby
With；
Step 2：Coating dilution is used respectively by the anti-25-hydroxyvitamin D3 monoclonal antibody in above-mentioned mouse source and sheep anti-Mouse
IgG antibody adjusts concentration to 1-3mg/ml, and film liquid amount is 0.5-2 μ l/cm, parallel using them as detection line with nature controlling line
It is sprayed on nitrocellulose filter and is coated with, detection line and nature controlling line is subsequently placed in baking oven at intervals of 3-7mm, 37 DEG C of bakings
It is dry 2 hours.
Sample pad of the present invention is made by following steps：Glass fibre membrane is soaked in containing 1.0%Triton X-
In 100,2.5%BSA, 0.15M Tris buffer solutions, pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, baking oven is subsequently placed in
In, 37 DEG C dry 2 hours.
Present invention also offers the 25-hydroxyvitamin D3 preparation method that a kind of kit as described above is realized, its feature
It is to comprise the following steps：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Card Reader：IC-card is placed on dry type fluorescence immunity analyzer labeling position, relevant information is read, it is described glimmering
Light immunochromatographiassays assays instrument is a kind of Systems for optical inspection, and the measurement range to 25-hydroxyvitamin D3 is 3.00-120ng/
mL；Step 3：Sample-adding：Serum/plasma：100 μ L serum/plasmas samples are taken vertically to be added dropwise at test card sample-adding；Whole blood：Take 150
μ L whole blood samples are vertically added dropwise at test card sample-adding；It is careful not to suck bubble during sampling.
Step 4：Detection, can be using automatic test or test both of which is detected immediately；Automatic test：By test card
On the carrier for inserting dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis detection to test card automatically；
Immediately test：Test card room temperature is placed after 15min, on the carrier for inserting dry type fluorescence immunity analyzer, clicks on test immediately.
The present invention provides 25- hydroxyls prepared by a kind of fluorescence immune chromatography technology of utilization rare earth carboxyl latex microballoon mark
Base vitamine D3 determines kit, while being adapted to serum, blood plasma and whole blood sample, and is adapted to clinically single part detection, relatively
In the qualitative colloid gold reagent of 25-hydroxyvitamin D3, the 25- hydroxycholecalciferol contents in detection sample can be quantified, with more
Clear and definite Clinical significance of MG, with easy to operate, reaction is quick, sensitivity height, high specificity, suitable Site Detection and economy
Practical the advantages of.
Brief description of the drawings：
Accompanying drawing 1 is the structural representation of test card in the present invention.
Accompanying drawing 2 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 3 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 4 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
Embodiment：
The present invention is further illustrated with reference to the accompanying drawings and examples：
As shown in Figure 1, determined present invention firstly provides a kind of 25-hydroxyvitamin D3 in kit, box provided with examination
Paper card, the test card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads
5, rare earth Eu is wherein adsorbed with pad3+Anti- 25- hydroxycholecalciferols monoclonal antibody-microballoon of fluorescent microsphere mark is even
Join compound, a diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, in ground state
Lower stabilization, launches wavelength 615nm fluorescence under 337nm excitation source effect；The monoclonal antibody is to mix after purification
The monoclonal antibody of conjunction, from the Monoclonal Antibody Cell for 2-6 different 25-hydroxyvitamin D3 epitopes
Strain.
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 150nm；The rare-earth fluorescent microballoon preferably comprises dilute
Native lanthanide series europium (Eu3+)；The antibody that rare-earth fluorescent microballoon is marked on pad is preferably derived from for 2 not synantigen tables
The monoclonal cell cell line of position.
Embodiment 1：
Each part that 25-hydroxyvitamin D3 determines test card in kit can be made by following measures：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M Tris buffer solutions,
In pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in baking oven, 37 DEG C dry 2 hours.
2nd, the preparation of the pad 3 of absorption fluorescent microsphere labelled antibody：
Glass fibre membrane is soaked in (X-100 containing 1.0%Triton, 2.5% in 150mM Tris-HCL treatment fluids
BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby, and glass fibre membrane is placed on into Bio-
On DotXYZ3050 three-dimensional specking platforms, the quantitation nozzle that declined with Bio-Jet Quanti300 noncontacts is by rare earth Eu3+Fluorescence is micro-
The anti-25-hydroxyvitamin D3 monoclonal antibody coupled complex of ball mark is sprayed onto glass fibre membrane, and 37 DEG C of drying are made after 1 hour
.
The aldehyde radical of rare-earth fluorescent nanoparticle：5mg rare-earth fluorescent nanoparticles are taken, with 20mM, pH9.5 carbonate
Buffer solution, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned of 100 μ l
In carbonate buffer solution, the glucan of 500 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugation
Method wash and be resuspended in 100 μ l above-mentioned carbonate buffer solution, be placed in 4 DEG C it is standby；
Rare earth Eu3+The preparation of the anti-25-hydroxyvitamin D3 monoclonal antibody of fluorescent microsphere mark：Choose from 2 not
The monoclonal antibody of the monoclonal cell cell line of synantigen epitope, according to mass ratio 1:1 by the anti-25-hydroxyvitamin D3s of 2mg
Monoclonal antibody, in 4 DEG C of dialysed overnights, is then mixed with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical,
4 DEG C of reactions are stayed overnight；Then, sodium borohydride is added to final concentration 5mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid
(50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are stayed overnight；Then 50mM Tris-HCL, pH7.4 are used
Buffer solution using centrifugal process wash 3 times, be resuspended in 100 μ l 50mM Tris-HCL buffer solutions (contain 1.2%NaCL,
0.5%BSA, 0.1%Tween 20), 4 DEG C be kept in dark place it is standby.
3rd, it is coated with the preparation of the nitrocellulose filter 4 of detection line and nature controlling line：
Using the cell line different from anti-25-hydroxyvitamin D3 cell strain of monoclonal antibody used on pad, use
The ascites production technology of standard prepares and purifies anti-25-hydroxyvitamin D3 monoclonal antibody, be stored in -20 DEG C it is standby；
The anti-25-hydroxyvitamin D3 monoclonal antibody in above-mentioned mouse source and goat anti-mouse igg are resisted with coating dilution respectively
Body adjusts concentration to 1.5mg/ml, and film liquid amount is 1.5 μ l/cm, is sprayed on using them as detection line is parallel with nature controlling line
It is coated with nitrocellulose filter, detection line and nature controlling line are subsequently placed in baking oven at intervals of 4mm, 37 DEG C dry 2 hours.
The assembling of test card：Paste treated sample pad 2 successively in PVC board 1, be adsorbed with rare-earth fluorescence labeling
The pad 3 of antibody, the nitrocellulose filter 4 and adsorptive pads 5 for being coated with detection line and nature controlling line, obtain test paper big after assembling
Plate, it is wide to cut into 4mm as requested, loads in plastic clip and form test card test paper.
The equipment and raw material selected in above steps preferably following raw material：
25-hydroxyvitamin D3 specific pairs antibody；25-hydroxyvitamin D3 quality-control product：Britain's Landau laboratory diagnosis
Co., Ltd；Rare-earth fluorescent microballoon：Shanghai Zhen Zhun bio tech ltd；Nitrocellulose (NC) film：Millipore is public
Take charge of product；Bovine serum albumin(BSA) (BSA), polyethylene glycol PEG20000, caseinhydrolysate：Sigma products, other common agents are equal
For AR.
Embodiment 2：Accuracy test
From above-mentioned test card and fluorescence immune chromatography analyzer (model：NEO-007),
The setting of fluorescence immunity analyzer parameter：Set after test card technological parameter, take on fluorescence immunity analyzer
The above-mentioned test card assembled, respectively with 4,10,20,40,60,80,100ng/mL 25-hydroxyvitamin D3 calibration object, use
Test card is measured, and obtains the fluorescence intensity level of each calibration object, result is input in the parameter of analyzer, completes analyzer
Parameter setting.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 300 parts of latex enhancing immune turbidimetry definite value samples
Sheet, wherein 100 parts of serum sample, 100 parts of plasma sample, 100 parts of whole blood sample, 25-hydroxyvitamin D3 content distribution are interval
For between 3.00-120ng/mL.
Detection method：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Card Reader：Check whether the lot number of IC-card and kit is consistent.IC-card is placed on dry type fluorescence immunoassay point
Analyzer labeling position, reads relevant information；
Step 3：Sample-adding：Serum/plasma：100 μ L serum/plasmas samples are taken vertically to be added dropwise at test card sample-adding, whole blood：
150 μ L whole blood samples are taken vertically to be added dropwise at test card sample-adding；It is careful not to suck bubble during sampling.
Step 4：Detection, can be detected, automatic test using automatic test or instant test both of which：By test card
On the carrier for inserting dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis detection to test card automatically；
Immediately test：Test card room temperature is placed after 15min, on the carrier for inserting dry type fluorescence immunity analyzer, clicks on test immediately.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, all clinical samples are detected by detection method, and analyze inspection
Survey result.
Result of the test：
As shown in accompanying drawing 2 to accompanying drawing 4, using the detected value of experimental system as Y-axis, the test value using contradistinction system is painted as X-axis
Scatter diagram processed, and carry out correlation analysis.Clinical sample detection is to 300 parts of clinical definite value pattern detections, sample mean deviation
Less than 10%, maximum deviation is less than 20%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection reagent prepared
Box is functional, is suitable for clinical detection, meets the differentiation needs of different clients difference detection occasion.
The present invention provides a kind of 25-hydroxyvitamin D3 fast quantification of utilization rare-earth fluorescent immunochromatography technique preparation and exempted from
Epidemic disease chromatographs detection kit, while being adapted to serum/plasma and whole blood sample, and is adapted to clinically single part detection, relative to 25-
The qualitative colloid gold reagent of hydroxycholecalciferol, can quantify the 25- hydroxycholecalciferol contents in detection sample, with clearer and more definite
Clinical significance of MG, with easy to operate, reaction quick, sensitivity height, high specificity, suitable Site Detection and economical and practical
The advantages of.
Claims (6)
1. a kind of 25-hydroxyvitamin D3 determines kit, provided with test card, it is characterised in that the test card from the bottom to top according to
It is secondary to be provided with：It is micro- that rare-earth fluorescent is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad
Ball mark anti-25-hydroxyvitamin D3 monoclonal antibody-microballoon coupled complex, the rare-earth fluorescent microballoon it is a diameter of
100-250nm, rare-earth fluorescent microballoon is stable under ground state containing the one or more in rare earth lanthanide, 300-400nm's
Launch the fluorescence that wave-length coverage is 550-650nm under excitation source effect；The monoclonal antibody is the list mixed after purification
Clonal antibody, from the cell strain of monoclonal antibody for 2-6 different 25-hydroxyvitamin D3 epitopes.
2. a kind of 25-hydroxyvitamin D3 according to claim 1 determines kit, it is characterised in that the pad
The diameter range of rare-earth fluorescent microballoon is 150-200nm；The rare-earth fluorescent microballoon is doped with rare earth lanthanide Eu3+, with reference to
The antibody sources that rare-earth fluorescent microballoon is marked on pad are in the monoclonal cell cell line for 2 different epitopes.
3. a kind of 25-hydroxyvitamin D3 according to claim 1 determines kit, it is characterised in that the pad is adopted
It is made with following steps：Glass fibre membrane is soaked in 150mM Tris-HCL treatment fluids (X-100 containing 1.0%Triton,
2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby, and glass fibre membrane is placed on
On Bio-DotXYZ3050 three-dimensional specking platforms, the quantitation nozzle that declined with Bio-Jet Quanti300 noncontacts is by rare earth Eu3+It is glimmering
The anti-25-hydroxyvitamin D3 monoclonal antibody coupled complex of light microballoon mark is sprayed onto glass fibre membrane, and 37 DEG C dry 1 hour
After be made.
4. a kind of 25-hydroxyvitamin D3 according to claim 1 determines kit, it is characterised in that the institute on pad
The 25-hydroxyvitamin D3 monoclonal antibody for stating rare-earth fluorescent microballoon mark is made using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：Mouse is immunized with 25-hydroxyvitamin D3 sterling, using the Dan Ke of standard
Grand preparation method for antibody prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line obtained is matched somebody with somebody
To screening, the monoclonal antibody cell line for kit is preferably gone out according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify 25-hydroxyvitamin D3 list
Be stored in after clonal antibody, packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, pH9.5 carbonate buffer solution,
Washed 3 times using centrifugal process, centrifugal speed is 12000rpm, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbonate
In buffer solution, the glucan of 500 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, is washed using same centrifugal process
Be resuspended in 100 μ l above-mentioned carbonate buffer solution, be placed in 4 DEG C it is standby；
Step 4：The preparation of the 25-hydroxyvitamin D3 monoclonal antibody of rare-earth fluorescent microballoon mark：Choose and resist from 2 differences
The monoclonal antibody of the monoclonal cell cell line of former epitope, according to mass ratio 1:1 by 2mg25- hydroxycholecalciferol monoclonals
Antibody, in 4 DEG C of dialysed overnights, is then mixed with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, and 4 DEG C anti-
It should stay overnight；Then, sodium borohydride is added to final concentration 5mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid (50mM
Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are stayed overnight；Then 50mM Tris-HCL, pH7.4 buffering is used
Liquid is washed 3 times using centrifugal process, be resuspended in 100 μ l 50mM Tris-HCL buffer solutions (contain 1.2%NaCL, 0.5%BSA,
0.1%Tween 20), 4 DEG C be kept in dark place it is standby.
5. a kind of 25-hydroxyvitamin D3 according to claim 1 determines kit, it is characterised in that described to be coated with inspection
The nitrocellulose filter of survey line and nature controlling line is made by following steps：
Step 1：Using the cell line different from 25-hydroxyvitamin D3 cell strain of monoclonal antibody used on pad, use
The ascites production technology of standard prepares and purifies 25-hydroxyvitamin D3 monoclonal antibody, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by the anti-25-hydroxyvitamin D3 monoclonal antibody in above-mentioned mouse source and goat anti-mouse igg
Antibody adjusts concentration to 1mg/ml, and film liquid amount is 1 μ l/cm, and nitre is sprayed on using them as detection line is parallel with nature controlling line
It is coated with acid cellulose film, detection line and nature controlling line are subsequently placed in baking oven at intervals of 4mm, 37 DEG C dry 2 hours.
6. a kind of 25-hydroxyvitamin D3 according to claim 1 determines kit, it is characterised in that the sample pad is led to
Following steps are crossed to be made：Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M Tris delay
In fliud flushing, pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in baking oven, 37 DEG C dry 2 hours.
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| CN201611157508.8A CN106990254A (en) | 2016-12-15 | 2016-12-15 | 25 hydroxycholecalciferols determine kit and preparation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107478851A (en) * | 2017-08-23 | 2017-12-15 | 成都和合医学检验所有限公司 | The method of 25 hydroxycholecalciferol contents in Quantitative detection blood of human body |
| CN108181465A (en) * | 2018-02-09 | 2018-06-19 | 广东优尼德生物科技有限公司 | Detect the fluorescent chromatographic kit and its quantitative detecting method of 25-hydroxy-vitamin D |
| CN109975559A (en) * | 2019-04-29 | 2019-07-05 | 厦门稀土材料研究所 | A kind of kit and method of time-resolved fluorescence quantitative detection 25(OH)VD |
| CN112378723A (en) * | 2020-11-19 | 2021-02-19 | 深圳市易瑞生物技术股份有限公司 | Sample pad for separating and concentrating target and application thereof |
| CN114184796A (en) * | 2021-12-03 | 2022-03-15 | 广州达泰生物工程技术有限公司 | Kit and method for quantitatively detecting full-segment parathyroid hormone |
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| CN104714033A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | Procalcitonin detection kit and detection method |
| CN104714025A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | NT-proBNP detection kit and detection method |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
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| CN104714033A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | Procalcitonin detection kit and detection method |
| CN104714025A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | NT-proBNP detection kit and detection method |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107478851A (en) * | 2017-08-23 | 2017-12-15 | 成都和合医学检验所有限公司 | The method of 25 hydroxycholecalciferol contents in Quantitative detection blood of human body |
| CN108181465A (en) * | 2018-02-09 | 2018-06-19 | 广东优尼德生物科技有限公司 | Detect the fluorescent chromatographic kit and its quantitative detecting method of 25-hydroxy-vitamin D |
| CN108181465B (en) * | 2018-02-09 | 2024-01-23 | 广东优尼德生物科技有限公司 | Fluorescent chromatography kit for detecting 25-hydroxyvitamin D and quantitative detection method thereof |
| CN109975559A (en) * | 2019-04-29 | 2019-07-05 | 厦门稀土材料研究所 | A kind of kit and method of time-resolved fluorescence quantitative detection 25(OH)VD |
| CN109975559B (en) * | 2019-04-29 | 2023-07-11 | 厦门稀土材料研究所 | Kit and method for time-resolved fluorescence quantitative detection of 25-hydroxy vitamin D |
| CN112378723A (en) * | 2020-11-19 | 2021-02-19 | 深圳市易瑞生物技术股份有限公司 | Sample pad for separating and concentrating target and application thereof |
| CN114184796A (en) * | 2021-12-03 | 2022-03-15 | 广州达泰生物工程技术有限公司 | Kit and method for quantitatively detecting full-segment parathyroid hormone |
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Application publication date: 20170728 |