CN104502599B - 25-hydroxy-vitamin D quantitative testing test paper bar and application thereof - Google Patents
25-hydroxy-vitamin D quantitative testing test paper bar and application thereof Download PDFInfo
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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
- G01N33/577—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
Abstract
The present invention relates to a kind of 25 hydroxy-vitamine D quantitative testing test paper bar and application thereof, belong to medical agent field.The test strips that the present invention provides includes sample pad, pad, NC film, adsorptive pads and PVC liner plate, and described sample pad is coated with the emulsion particle and/or 1 of 25 OH VD activation, 25 (OH)2The emulsion particle of VD activation, the anti-25 OH VD monoclonal antibodies of colloid gold label and anti-VDBP monoclonal antibody it is coated with on described pad, described NC film includes T line and C line successively, it is coated with 25 OH VD BSA and the people VDBP of part expression on described T line, described C line is coated with sheep anti-mouse antibody.Use test strips of the present invention, it is not necessary to sample preprocessing, it is possible to quickly, the content of vitamin D in Accurate Determining sample.
Description
Technical field
The present invention relates to a kind of 25-hydroxy-vitamin D quantitative testing test paper bar and application thereof, belong to field of medicine preparations.
Background technology
In recent years, along with deepening continuously of research, people have had more understanding to vitamin D (VD), and VD is institute
Having a member unique in bioactive substance, have multiple effect, it is vitamin, but is substantially hormone,
It is also possible that cytokine.Because VD is derived from the required nutrient of food, and human body requirement is the least, institute
To say that it is vitamin.It is mainly the 7-DHC in skin under ultraviolet irradiation and changes, then
Play a role to whole body with blood operation, so belonging to steroids.People gradually recognize that it has widely
Physiological action, in addition to regulation alcium and phosphor metabolization, also has antiproliferative, anti-differentiation, regulation apoptosis, mediated immunity anti-
The secretion of multiple endocrine gland hormone, the effect of regulation hemopoietic tissue hemopoietic, should be regulated.These functions are the similar of VD
Thing bone triol (1,25-(OH)2VD) by combining with the cell with VD receptor (VDR) and through a series of lifes
Manage biochemical process and work.The bone triol of these kidney external sources, acts on pericellular a certain position, so pressing
According to its effect it is also assumed that be a kind of cytokine.In any case, VD and the like significance clinically
Increasingly being subject to people's attention, the most scientific and reasonable use will be our important topic of facing now.
Vitamin D is that one has to pass through metabolic transformation and can be only achieved the bioactive fat-soluble steroid of optimal calcification alcohol and spread out
Biological.The vitamin D3 of skin synthesis or the vitamin D of intestinal absorption are by vitamin D binding protein (vitamin D
Binding protein, VDBP) transport, need to be at liver 25-hydroxylase CYP2R1 and kidney 1 α-hydroxylase CYP27B1
Catalysis under form final active metabolite 1,25 1 dihydroxy vitamin d3 through 2 enzymatics through changing reaction
(1,25-(OH)2VD), performance biological function is combined with vitamin D receptor (vitamin D receptor, VDR).
VDBP is synthesized by liver, and molecular weight is 51335, containing 458 amino acid residues, has highly parent with vitamin D
And power.Each VDBP molecule contains a VD binding site, and it combines amino acid sites is 35-49 aminoacid sequence
The domain that row are constituted.Therefore, in blood, there is two kinds of forms, i.e. affine with VD and metabolite thereof knot in VDBP
Conjunction type VDBP (accounting for the 5% of VDBP total amount) closed and the sequestered VDBP of VD binding site vacancy.
The activity form of the vitamin D of human body is 1,25 dihydroxyvitamin D3s (1,25-hydroxyvitamin D3), is
Because it is to have two hydroxyls at 1 and 25, wherein 25 hydroxylations complete at liver, and 1 hydroxylation is complete at kidney
Become, and 25 hydroxyl VD3 can lack the indication index of VD as body.The major function of VD is to promote small intestinal calcium phosphorus
Absorption, promote the kidney heavily absorption to calcium phosphorus, regulate internal alcium and phosphor metabolization.So, even if lacking calcium in VD meals
Amount abundant, also have calcium deficiency phenomenon.But, VD is difficult to lack, because can synthesize in human body, shines
The sun just, but has people's just exception of serious liver, kidney diaseases, because the dihydroxylation process of above-mentioned VD3 cannot enter
OK.
1,25(OH)2The VD half-life in vivo is about 4h, and its concentration is lower about 1000 times than 25-OH VD, and is subject to
Serum PTH, Ca2+And the strict regulation and control of phosphate concn.1,25(OH)2VD level can not reflect body vitamin D shape
State, but its rising is relevant with hyperthyroidism.Therefore, serum 1,25 (OH) is measured2VD content to acquired or
Heritability 25-OH VD and phosphate metabolism are disorderly (as chronic nephropathy, the shortage disorder of heritability phosphate, tumor cause
Osteomalacia, lack property rickets, chronic granuloma formed obstacle) auxiliary diagnosis have important value.
It is presently used for serum 1,25 (OH)2VD assay method has the methods such as HPLC, LC-MS/MS, RIA, CLIA,
Wherein due to reasons such as HPLC, LC-MS/MS sample process are difficult, testing cost is high, less in clinical practice.At present,
Due to highly sensitive, the feature such as specificity good, easy and simple to handle, testing cost is low of lateral chromatography technology, application is increasingly
Extensively.
Lateral chromatography technology, can be divided into two classes, a class to be based on enzymatic reaction develops the color by its principle, highly come with colour developing
Quantitatively；Another kind of then use colored marker such as emulsion particle, electroselenium, gold colloidal and liposome etc., during chromatography,
Label reacts the complex formed and is enriched with colour developing by the capture of the corresponding part being fixed on chromatographic material with determinand,
Presence or absence or how many next qualitative or quantitative according to the band that develops the color on chromatographic material.Immune layer based on enzymatic reaction develops the color
Analysis technology is quantitatively with enzymatic activity as foundation in test strips due to determinand, thus its measurement result by enzyme stability and
The impact of the environmental factorss such as sample substrate, pH value, incubative time and temperature.
Immune colloidal gold technique is consolidating of growing up after three big labelling techniques (fluorescein, radiosiotope and enzyme)
Phase label immunoassay technology.Occur in colloidal gold immunochromatographimethod (the colloidal gold in clinical diagnosis field the eighties
Immunochromatography assay, GICA) on the basis of quick diagnosis test strips is built upon gold-marking immunity diafiltration
A kind of immunoassay technology.It has simple and quick, result clearly, without complex operations skill and special installation, sensitive
Spending the advantages such as high, easy to carry, oneself becomes a new direction of clinical experiment diagnostic field development.Therefore, exploitation 25-
Hydroxy-vitamine D test paper bar has important value in the auxiliary of vitamin D deficiency diagnoses.
But, in serum, more than 80% 25-OH VD with VDBP is combined, and common immunologic detection method is to utilize sample
This pretreatment fluid (such as protein denaturants such as organic solvents) processes, and is measured by 25-OH VD after VDBP dissociates release；
Or use acid reaction system (pH4.0-6.0) to be measured；Or use the VDBP affine conjugate competition of high concentration
Release 25-OH VD, these methods there is Sample Dilution process or 25-OH VD release is incomplete or the parent of affine conjugate
Limit with the factor such as property.Therefore, research and development are a kind of without sample preprocessing, testing result 25-hydroxy-vitamin D accurately
Content assaying method becomes a current difficult problem.
Summary of the invention
Deficiency according to above-mentioned field and demand, the present invention provides a kind of detection with 25-hydroxy-vitamin D as object
Test strips, described test strips utilizes lateral chromatography principle, it is not necessary to Sample pretreatment, directly measures 25-OH VD in sample
Content.
The technical scheme that the present invention is claimed is as follows:
A kind of test strips for 25-hydroxy-vitamin D detection by quantitative, including sample pad, pad, NC film, water suction
Pad and PCV liner plate；Described sample pad, pad, NC film and adsorptive pads are set in turn in PCV lining by sample chromatography direction
On plate；
It is characterized in that: described sample pad uses has network structure and can to capture particle diameter be the particulate matter between 2.0-2.6 μm
Fiber mat, and be coated with 25-hydroxy-vitamin D activation emulsion particle and/or 1,25-dihydroxyvitamin D activation breast
Glue microgranule；
The anti-25-hydroxy-vitamin D monoclonal antibody of colloid gold label and the anti-human of colloid gold label it is coated with on described pad
Vitamin D binding protein monoclonal antibody；
Including on described NC film detecting line and nature controlling line, described detection line, near described pad, is coated with 25-hydroxy vitamin
D-bovine serum albumin conjugate and recombined human vitamin D binding protein, described nature controlling line, near adsorptive pads, is coated with goat-anti
Murine antibody.
The emulsion particle and/or 1 of 25-hydroxy-vitamin D activation in described sample pad, the latex of 25-dihydroxyvitamin D activation
The concentration that is coated of microgranule is 0.5-10 μ g/cm2。
On described pad, the concentration of the anti-25-hydroxy-vitamin D monoclonal antibody of colloid gold label is 0-10 μ l/cm2；Described glue
The concentration of the anti-human vitamin D binding protein monoclonal antibody of body gold labelling is 0-10 μ l/cm2。
On the detection line of described NC film, the concentration that is coated of 25-hydroxy-vitamin D-bovine serum albumin conjugate is 1.0-2.0 μ g/ bar,
The concentration that is coated of described recombined human vitamin D binding protein is 0-2.0 μ g/ bar；On described nature controlling line sheep anti-mouse antibody be coated dense
Degree is 0.5-2.0 μ g/ bar.
Described recombined human vitamin D binding protein is for having people's vitamin D binding protein 159-458 amino acids sequence
Polypeptide.
The emulsion particle and/or 1 of 25-hydroxy-vitamin D activation in described sample pad, the latex of 25-dihydroxyvitamin D activation
The concentration that is coated of microgranule is 2.0 μ g/cm2；The anti-25-hydroxy-vitamin D monoclonal antibody of colloid gold label on described pad
Concentration is 10 μ l/cm2, the concentration of the anti-human vitamin D binding protein monoclonal antibody of described colloid gold label is 10 μ l/cm2；
On the detection line of described NC film, the concentration that is coated of 25-hydroxy-vitamin D-bovine serum albumin conjugate is 1.0 μ g/ bars, described
The concentration that is coated of recombined human vitamin D binding protein is 1.0 μ g/ bars；On described nature controlling line, the concentration that is coated of sheep anti-mouse antibody is
0.8 μ g/ bar.
A kind of method for 25-hydroxy-vitamin D detection by quantitative, it is characterised in that: use arbitrary described test strips inspection
The total content of 25-hydroxy-vitamin D in test sample product, its step is as follows:
(1) testing sample is dripped in sample pad, room temperature reaction 15min；
(2) after nature controlling line develops the color, test strips is put into reading bar instrument, read the concentration of 25-OH VD in sample.
A kind of detection card for 25-hydroxy-vitamin D detection by quantitative, comprises arbitrary described test strips and cartridge, its
It is characterised by: described cartridge includes lid and cassette bottom；Described test strips is arranged at the cassette bottom of described cartridge；Described cartridge
Having well and observation window on lid, described well is just to the sample pad in described test strips, and described observation window is the most right
Detection line in described test strips and nature controlling line.
The present invention provides one for 25-hydroxy-vitamin D (25-OH based on solid phase isolation technics with immunity lateral chromatography technology
VD) test strips of detection by quantitative, the structure such as including sample pad, pad, NC film, adsorptive pads and PCV liner plate.Described
Test strips uses special lateral chromatography system, it is not necessary to Sample pretreatment, directly measures the total content of 25-OH VD in sample.
The sample pad of test strips of the present invention for having cancellated fiber mat, can capture particle diameter be between 2.0-2.6 μm
Grain thing, it is coated with the emulsion particle (LP-25-OH VD) and/or 1 of 25-hydroxy-vitamin D activation, and 25-dihydroxy is tieed up
Emulsion particle (LP-1, the 25-(OH) of raw element D activation2VD).Described LP-25-OH VD be 25-OH VD-BSA with
Activity emulsion particle (LP) is obtained by 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) method covalent coupling.
Described sample pad can capture VDBP free in sample, makes sequestered VDBP separate with conjunction type VDBP.It is former
Reason is to have the emulsion particle that the fixing affine conjugate of VDBP (such as 25-OH VD) of cancellated sample pad activates
Utilize higher (Ka=5 × 10 of chromatographic theory competitor release effect 25-OH VD, VDBP and 25-OH VD affinity8M-1) and
There is (18～65 μ g/L) in VDBP high concentration, and competition binding thing can not discharge 25-OH VD completely, thus captures trip
Release VDBP, and conjunction type VDBP and free 25-OH VD continue (adsorptive pads direction) swimming forward.
The anti-25-hydroxyl dimension being coated with gold colloidal (Colloidal Gold, CG) labelling on the pad of test strips of the present invention is raw
Element D monoclonal antibody (CG-anti-25-OH VD) and the anti-human vitamin D binding protein monoclonal antibody of colloid gold label
(CG-anti-VDBP).The 25-OH VD that described CG-anti-25-OH VD can dissociate in sample is combined,
CG-anti-VDBP can be combined with the people VDBP in sample.
Include the most successively on the NC film of test strips of the present invention detecting line (T line) and nature controlling line (C line), described T line
On be coated with 25-OH VD-BSA and recombined human VDBP (rProtein), described 25-OH VD-BSA can be with sample middle reaches
From 25-OH VD competition binding CG-anti-25-OH VD, formed 25-OH VD-BSA-CG-anti-25-OH VD be combined
Thing, the amount of this complex is relevant to 25-OH VD content free in sample；Described recombined human VDBP is the people that part is expressed
VDBP, lacks VD binding domain amino acid sequence, therefore can not be combined with 25-hydroxy-vitamin D, it is possible to tie with sample
Mould assembly VDBP competition binding CG-anti-VDBP, formed rProtein-CG-anti-VDBP complex, the amount of this complex with
In sample, the content of conjunction type 25-OH VD is correlated with.Being coated with sheep anti-mouse antibody on described C line, this antibody can be with
CG-anti-25-OH VD and CG-anti-VDBP combines, and in order to the effectiveness of result of determination, develops the color for effectively, colourless is
Invalid.
The adsorptive pads of test strips of the present invention and PCV liner plate, respectively test strips provide chromatography swimming power, support and protect.
In ELISA test strip sample of the present invention, the principle of vitamin D content is: free 25-OH VD and NC film T line
On be coated thing 25-OH VD-BSA competition binding CG-anti-25-OH VD, combine sequestered 25-OH VD's
CG-anti-25-OH VD continues swimming forward, and the thing sheep anti-mouse antibody that is coated on NC film C line is combined；Conjunction type
It is coated thing recombined human VDBP competition binding CG-anti-VDBP on VDBP Yu T line, combines conjunction type VDBP
CG-anti-VDBP continues swimming forward, is coated thing sheep anti-mouse antibody with C line and is combined.This method is in competition law, i.e. sample
25-OH VD content is the highest, and CG-anti-25-OH VD and CG-anti-VDBP being combined on T line is the fewest, colour developing
The most shallow, absorbance is the lowest, otherwise the highest, T line the depth developed the color i.e. can determine that the content of VD.And C line combines
CG-anti-25-OH VD and CG-anti-VDBP is only used as availability deciding, and colour developing is then effective, otherwise the most invalid.By
In 1,25-(OH)2The structure of VD to 25-OH VD is the most similar, the result that 25-OH VD test strips the most of the present invention measures
It is 1,25-(OH)2The total content of VD and 25-OH VD, but due to 1,25-(OH)2VD concentration in blood is the lowest
In 25-OH VD, therefore the impact on 25-OH VD measurement result can be ignored.
In order to improve the accuracy of ELISA test strip system of the present invention, inventor is coated thing from sample pad, sample pad is coated the dense of thing
Degree, the aspect such as the concentration of colloidal gold labeled monoclonal antibody and package amount, T linear protein kind and package amount is optimized on pad.
In some embodiments of the invention, described sample pad is coated thing preferred LP-25-OH VD and/or LP-1,25-(OH)2VD,
Being coated concentration is 2.0 μ g/cm2；On described pad, the concentration of CG-anti-25-OH VD is 10 μ l/cm2, described
The concentration of CG-anti-VDBP is 10 μ l/cm2；On described NC film T line, the concentration that is coated of 25-OH VD-BSA is 1.0 μ g/
Bar, the concentration that is coated of described recombined human VDBP is 1.0 μ g/ bars, and on described nature controlling line, the concentration that is coated of sheep anti-mouse antibody is 0.8 μ g/
Bar.
The present invention also provides for a kind of method for 25-hydroxy-vitamin D detection by quantitative, uses the test strips that the present invention provides
Detecting, only need to be added in sample pad by a small amount of sample drop, ambient temperatare puts 15min, after nature controlling line develops the color, and will
Test strips puts into reading bar instrument, automatically substitutes into the calibration song that instrument is preset after reading the absorbance of bar instrument reading detection line and nature controlling line
Line (four parameter fittings) calculates, and directly gives the concentration of 25-hydroxy-vitamin D.The method is without carrying out sample
Pre-treatment, easy and simple to handle, the most quick and precisely, it is simple to the mensuration of clinical VD content.
In sum, the test strips for 25-hydroxy-vitamin D detection by quantitative that the present invention provides can be fast and accurately
The content of vitamin D in detection sample.Compared with existing detection method, test strips of the present invention is used to carry out VD content
Detection has the advantage that (1) is easy and simple to handle: owing in blood, more than 80% 25-OH VD with VDBP is combined, conventional
Method needs to carry out sample preprocessing, makes 25-OH VD detect after exposing, and test strips of the present invention uses two kinds of antibody
Capture sequestered 25-OH VD and conjunction type 25-OH VD respectively, it is not necessary to sample preprocessing, simplifies experimental procedure, saves
Time；It is (2) the most accurate: if common test strips does not carry out sample preprocessing, to be only able to detect free 25-OH VD,
And test strips of the present invention can detect sequestered and conjunction type 25-OH VD simultaneously, accuracy is high.
Accompanying drawing explanation
Fig. 1. the structural representation of test strips of the present invention, wherein 1-PVC liner plate, 2-sample pad, 3-pad, 4-T line, 5-NC
Film, 6-C line, 7-adsorptive pads；
Fig. 2 .rProtein Yu anti-VDBP, 25-OH VD binding ability；
Fig. 3 .NC film T/C line is coated schematic diagram；
Fig. 4. ELISA test strip result of the present invention and the fitting a straight line of IDS test kit testing result.
Detailed description of the invention
Below by way of specific embodiment, the present invention is explained, it should be noted that following embodiment is only used as this
Bright being explained further and illustrating, and limits the present invention never in any form.
Test agents useful for same:
Activity emulsion particle (Latex Particle): the active group of its particle surface can with protein covalent bond,
It is purchased from Merck company, article No. 39510001.
The conjugate that 25-OH VD-BSA: i.e. 25-OH VD with BSA is obtained by covalent bond coupling, is purchased from Shang Haihui
This is biological.
1,25-(OH) 2VD-BSA: the conjugate that i.e. 1,25-(OH) 2VD with BSA is obtained by covalent bond coupling, purchases
In this biology of Shanghai favour.
25mM MES (2-(N-morpholinyl) ethyl sulfonic acid (sigma, M3671) buffer: weigh 4.88g MES in 900mL
In ultra-pure water, regulate pH5.0 after being completely dissolved, be settled to 1L.
EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) solution: sigma, 39391.
PH7.510mM PBS: weigh 0.27g potassium dihydrogen phosphate (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g phosphorus
Acid disodium hydrogen (Na2HPO4) (traditional Chinese medicines, 100203008), 8g sodium chloride (NaCl) (traditional Chinese medicines, 10019308), 0.2g
Potassium chloride (KCl) (traditional Chinese medicines, 10016308), in 900mL ultra-pure water, regulates pH to 7.5, then after being completely dissolved
It is settled to 1L with volumetric flask.
10mM Tris-HCl: weigh 1.576g (traditional Chinese medicines, xw020034) in 900mL ultra-pure water, after being completely dissolved
It is adjusted to required pH value, is then settled to 1L.
1% citrate buffer solution: weigh 1.0g trisodium citrate (traditional Chinese medicines, 10019428) in 100mL ultra-pure water,
To being completely dissolved.
5%BSA: weigh 5g BSA (amresco, 0332) in 100ml PBS solution, to being completely dissolved.
TBS:50mM Tris HCl, 150mM NaCl, 1.5mM EDTA, 10mM DTT, 0.5%Triton
X-100,0.2mg/ml lysozyme, pH 7.4.
2M H2SO4: take 100mL concentrated sulphuric acid in 800ml ultra-pure water.
Anti-VDBP: anti-human VDBP monoclonal antibody, is purchased from company Santa cruz, article No. sc-365441.
Anti-25-OH VD: the most anti-25-hydroxy-vitamin D antibody, a kind of can the exempting from of combination affine with 25-OH VD
Epidemic disease globulin, is purchased from Meridian, article No. K24124M.
Read bar instrument: C10066-10, shore pine.
Fiber mat: as sample pad, Fusion5, GE.
The not specified experiment reagent of the present invention is this area conventional reagent, can be prepared by this area conventional method or
Commercially available, specification is the pure level of laboratory.
Embodiment 1, the preparation of 25-hydroxy-vitamin D quantitative testing test paper bar
(1) expression of VDBP recombiant protein and checking
The rProtein recombiant protein i.e. people VDBP partial sequence 159-458 aminoacid expression side by gst fusion protein
At E.coli system expression, (expression vector is PET28a to formula, and the E.coli cell of employing is BL21 (DE3), is purchased from
Beijing Quanshijin Biotechnology Co., Ltd).Antibacterial is at the LB containing 100mg/ml ampicillin (23YT-Amp)
Culture medium is cultivated, with the scaling cultivation of 1:10 after 37 DEG C of incubated overnight.When bacterial concentration (A600) reaches 0.8-0.9
Time, add 0.5mM IPTG and carry out abduction delivering.After 25 DEG C are cultivated 12-15h, centrifugal collection thalline also buffers with TBS
Liquid is resuspended.Ultrasonication antibacterial liquid, 40w, 10s, 10s, 50 times；12000rpm, 4 DEG C of centrifugal 15min, in collection
Clear liquid.Using GE GSTrap FF purification column (GE, 71-5016-96AK) to carry out protein purification, protein concentration is used
A280/A260 is measured.
Microwell plate (being purchased from Shenzhen bright China of gold), 1.0ug/ml (pH7.5 it is coated respectively with anti-VDBP and 25-OH VD-BSA
10mM PBS), 100ul/ hole.After placing 2 hours under the conditions of 37 DEG C, discard liquid in hole, add 5%BSA, 150ul/
Hole.After placing 1 hour under the conditions of 37 DEG C, discard liquid in hole, stand-by.
The rProtein of variable concentrations is added in above-mentioned microwell plate, 100ul/ hole, hatch 1 hour for 37 DEG C；Then hole is discarded
Interior liquid, washes plate 5 times with PBST (pH7.510mM PBS contains 0.05%Tween-20), adds the anti-of HRP labelling
GST antibody (is purchased from Yi Qiao Divine Land, Beijing, 11213-MM05；With PBST with the dilution proportion of 1:10000), 100ul/
Hole, hatches 0.5 hour for 37 DEG C；Then discard liquid in hole, wash plate 5 times with PBST, add substrate and (be purchased from Beijing rope
Lay is precious, PR1200) 100ul/ hole, 37 DEG C hatch 10 minutes after add 2M H2SO4 stop buffer, 50ul/ hole, measure it
Absorbance (A450nm).
Result is as in figure 2 it is shown, recombiant protein rProtein can be combined with anti-VDBP, and is not combined with 25-OH VD.
(2) preparation of the emulsion particle of 25-OH VD activation
1, the activation of emulsion particle (LP)
Take 1mg emulsion particle, with 1mL 25mM MES (pH5.0) buffer solution 3 times (mixing 10min) every time；
(1) preparation 10mg/mL EDC (M=191.7g/mol, C=52mM) solution (EDC is dissolved in cold 25mM MES,
pH5.0)。
(2) Latex Particle is resuspended with 440 μ L 25mM MES (pH5.0).
(3) add 10 μ L EDC solution, fully mix, slowly shake, 30min, room temperature.
(4) centrifuge tube reaction completed centrifugal (3000rmp, 5min), removes supernatant, and with 1mL 25mM MES,
PH 5.0 washs 3 times.
2, Latex Particle is coated
(1) with 300 μ L 25mM MES, pH 5.0 resuspended Latex Particle；
(2) add 10ul 10mg/ml 25-Hydroxy Vitamin D3-BSA (Shanghai favour this biological) (25mM MES,
PH5.0) solution；
(3) use 25mM MES, pH 5.0 to make reaction system constant volume be 500 μ L, fully mix；
(4) slowly shake, room temperature 30-120min or 4 DEG C 2 hours；
(5) centrifuge tube reaction completed is centrifugal (3000rmp, 5min), removes supernatant.
3, Latex Particle washing and preservation
(1) wash 4 times with 500 μ L 25mM MES (pH 5.0)；
(2) 100 μ L 0.1-0.5%BSA (amresco, 0332), 30-60min, room temperature are added；
(3) wash 4 times with 100 μ L 50mM Tris-HCl (pH 6.8, containing 1%Tween-20) (sigma, 44112),
Obtain the emulsion particle (LP-25-OH VD) of 25-OH VD activation；
(4) add 1mL 5%BSA to preserve.
(3) preparation of gold mark conjugate
(1) take 100mL 0.01%HAuCl4 (traditional Chinese medicines, 10010711) solution, be heated to boiling.
(2) 2.0mL 1% citric acid three sodium solution, heated and stirred 15min are added.
(3) turning off heating turn-knob, suitable speed stirs to room temperature, obtains colloidal gold solution (Colloidal Gold, CG).
(4) gold colloidal measuring 20.0mL is placed in the small beaker of 100.0mL, is slowly added to 0.4mg when stirring
Albumen to be marked (such as anti-VDBP, anti-25-OH VD), stirs 30min.
(5) add 5%BSA to final concentration of 1%, continue stirring 30min.
(6) it is centrifuged 30min with 12000r/min, abandoning supernatant, resuspended with PBST.
(7) it is centrifuged 30min with 12000r/min, abandoning supernatant, resuspended with PBST.
(8) being centrifuged 30min, abandoning supernatant with 12000r/min, precipitation 1%BSA is resuspended, obtains 1.0mL and concentrates
Thing (i.e. gold mark conjugate), puts 4 DEG C of refrigerators standby.
(4) pretreatment of sample pad, pad
(1) sample pad (Fusion 5, GE) and pad (Shanghai Jinbiao Bio-Tech Co., Ltd.) are soaked with 1%BSA
30min, 37 DEG C of drying.
(2) emulsion particle (LP-25-OH VD, the 2.0ug/cm that 25-OH VD is activated2) coat in sample pad, 37 DEG C
Dry.
(3) depletion mark conjugate is added drop-wise to (10uL/cm on pad2), 37 DEG C of drying.
(5) T/C line (detection line/nature controlling line) is coated
C line (nature controlling line): be coated NC film (135s, Millipore) C line (see Fig. 3 a, figure with 1mg/mL sheep anti-mouse antibody
3b), every 1.0 μ L, 37 DEG C are dried.
T line (detection line): by 1.0mg/mL 25-hydroxy-vitamin D-bovine serum albumin conjugate and the recombined human of 1.0mg/mL
Vitamin D binding protein is coated NC film (135s, Millipore) (see Fig. 3 c, Fig. 3 d), often according to the ratio mixing of 1:5
Bar is coated 1.0 μ g mixture, and 37 DEG C are dried.
(6) test strips is assembled
By sample pad, pad, NC film, adsorptive pads (CH37M, Shanghai Jinbiao Bio-Tech Co., Ltd.), PVC lining
Plate (Shanghai Jinbiao Bio-Tech Co., Ltd.), cartridge (Jin Canhua), from down to up, carry out assembling (see figure from inside to outside
1)。
Embodiment 2, sample pad are coated the selection of thing
It is coated thing to improve the performance of ELISA test strip system of the present invention by preferred sample pad.According to measurement result, use 2
Example clinical sample (25-OHD3 content high level H, low value L) adds recovery sample according to table 1 preparation, calculates
The response rate, select optimum is coated thing.
Table 1 adds recovery sample preparation methods
Screening technique is as follows:
(1) LP-25-OH VD, LP-1,25-(OH) are selected respectively2VD or 25-OH VD-BSA is coated sample pad
(2.0ug/cm2), method for coating is with embodiment 1；
(2) chromatograph test strip preparation method is with embodiment 1；
(3) 100 μ L samples, room temperature reaction 15min are added；
(4) according to reading bar instrument calculating 25-OH VD, then response rate R is calculated by formula (1).
Wherein: R is the response rate；
V is for adding sample volume；
V0 is the volume of substrate sample；
C is the detectable concentration of mixing sample；
C0 is the detectable concentration of substrate sample；
Cs is the concentration adding sample.
Result is as shown in table 2, uses LP-25-OH VD, LP-1,25-(OH)2Both VD or one of both are coated
Sample pad effect is preferable.
The different sample pad of table 2 is coated the response rate of thing
Embodiment 3, the concentration that is coated of LP-25-OH VD select
Owing to the concentration that is coated of LP-25-OH VD affects the capture rate of free VDBP in human serum, therefore need it is wrapped
Optimized further by concentration.Optimization method is with embodiment 2.
Screening technique is as follows:
(1) select variable concentrations LP-25-OH VD to be coated sample pad, be coated step with embodiment 1；
(2) chromatograph test strip preparation method is with embodiment 1；
(3) 100 μ L samples, room temperature reaction 15min are added；
(4) according to reading bar instrument calculating 25-OH VD, then response rate R is calculated by formula (1).
Experimental result is as shown in table 3, and LP-25-OH VD concentration represents with 25-OH VD-BSA concentration, optimum
LP-25-OH VD concentration is 2.0ug/cm2。
The response rate of the different LP-25-OH VD concentration of table 3
On embodiment 4, pad, the concentration of GC-anti-25-OH VD, GC-anti-VDBP selects
Owing to the concentration of GC-anti-25-OH VD, GC-anti-VDBP affects free 25-OH VD and combination in human serum
The measurement result of 25-OH VD, therefore needs to optimize its concentration further.Optimization method is with embodiment 3.
Result is as shown in table 4, and the concentration that is coated optimum for GC-anti-25-OH VD is 10ul/cm2, GC-anti-VDBP is excellent
Selecting concentration range is 0-10ul/cm2, the optimum concentration that is coated is 10ul/cm2。
The response rate under the conditions of table 4 pad variable concentrations GC-anti-25-OH VD, GC-anti-VDBP
On embodiment 5, NC film T line, the concentration of 25-OH VD-BSA, rProtein selects
Human serum dissociates 25-OH VD owing to the concentration of 25-OH VD-BSA, rProtein affects and combines 25-OH VD
Measurement result, therefore need its concentration is optimized.Optimization method is with embodiment 3.
Result is as shown in table 5, and wherein 25-OH VD-BSA preferred concentration range is 1.0-2.0ug/ bar, and optimum is coated concentration
For 1.0ug/ bar；RProtein preferred concentration range is 0-2.0ug/ bar, and the optimum concentration that is coated is 1.0ug/ bar.
The response rate under the conditions of table 5T line variable concentrations 25-OH VD-BSA, rProtein
Embodiment 6, clinical sample measure
The test strips prepared by above-mentioned optimal conditions, measures and measures test kit (Immunodiagnostic with IDS 25OH VD
Systems Limited, state's food medicine prison tool (entering) word 2011 the 2403360th) clinical sample 40 example of assignment, with IDS
It is X-axis that 25OH VD measures kit results, with test strips measurement result of the present invention as Y-axis, carries out fitting a straight line.
Assay method is as follows:
(1) 100 μ L testing samples are dripped in sample pad, room temperature reaction 15min；
(2) after nature controlling line develops the color, test strips is put into reading bar instrument (C10066-10, shore pine), reads 25-OH in sample
The concentration of VD.
As shown in Figure 4, the linear equation that experiment obtains is Y=0.98X+0.4 to result, and linearly dependent coefficient r is 0.91.By
This shows, measurement result is fine with IDS measurement result dependency, uses test strips of the present invention to measure 25-OH VD in sample
Content accuracy high.
Claims (8)
1. for a test strips for 25-hydroxy-vitamin D detection by quantitative, including sample pad, pad, NC film, adsorptive pads
And PVC liner plate；Described sample pad, pad, NC film and adsorptive pads are set in turn in PVC liner plate by sample chromatography direction
On；
It is characterized in that: described sample pad uses has network structure and can to capture particle diameter be the particulate matter between 2.0-2.6 μm
Fiber mat, and be coated with 25-hydroxy-vitamin D activation emulsion particle and/or 1,25-dihydroxyvitamin D activation breast
Glue microgranule；
The anti-25-hydroxy-vitamin D monoclonal antibody of colloid gold label and the anti-human of colloid gold label it is coated with on described pad
Vitamin D binding protein monoclonal antibody；
Including on described NC film detecting line and nature controlling line, described detection line, near described pad, is coated with 25-hydroxy vitamin
D-bovine serum albumin conjugate and recombined human vitamin D binding protein, described nature controlling line, near adsorptive pads, is coated with goat-anti
Murine antibody.
Test strips the most according to claim 1, the emulsion particle and/or 1 of 25-hydroxy-vitamin D activation in described sample pad,
The concentration that is coated of the emulsion particle of 25-dihydroxyvitamin D activation is 0.5-10 μ g/cm2。
Test strips the most according to claim 1, the anti-25-hydroxy-vitamin D monoclonal anti of colloid gold label on described pad
The concentration of body is 10 μ l/cm2；The concentration of the anti-human vitamin D binding protein monoclonal antibody of described colloid gold label is
0-10μl/cm2。
Test strips the most according to claim 1, on the detection line of described NC film, 25-hydroxy-vitamin D-bovine serum albumin is even
The concentration that is coated of connection thing is 1.0-2.0 μ g/ bar, and the concentration that is coated of described recombined human vitamin D binding protein is 0-2.0 μ g/ bar；
On described nature controlling line, the concentration that is coated of sheep anti-mouse antibody is 0.5-2.0 μ g/ bar.
Test strips the most according to claim 1, described recombined human vitamin D binding protein combines for having people's vitamin D
The polypeptide of albumen 159-458 amino acids sequence.
Test strips the most according to claim 1, the emulsion particle and/or 1 of 25-hydroxy-vitamin D activation in described sample pad,
The concentration that is coated of the emulsion particle of 25-dihydroxyvitamin D activation is 2.0 μ g/cm2；On described pad, colloid gold label is anti-
The concentration of 25-hydroxy-vitamin D monoclonal antibody is 10 μ l/cm2, the anti-human vitamin D binding protein of described colloid gold label
The concentration of monoclonal antibody is 10 μ l/cm2；25-hydroxy-vitamin D-bovine serum albumin coupling on the detection line of described NC film
The concentration that is coated of thing is 1.0 μ g/ bars, and the concentration that is coated of described recombined human vitamin D binding protein is 1.0 μ g/ bars；Described matter
On control line, the concentration that is coated of sheep anti-mouse antibody is 0.8 μ g/ bar.
7. the method for 25-hydroxy-vitamin D detection by quantitative, it is characterised in that: use the arbitrary institute of claim 1-6
The total content of 25-hydroxy-vitamin D in the ELISA test strip sample stated, its step is as follows:
(1) testing sample is dripped in sample pad, room temperature reaction 15min；
(2) after nature controlling line develops the color, test strips is put into reading bar instrument, read the concentration of 25-hydroxy-vitamin D in sample.
8., for a detection card for 25-hydroxy-vitamin D detection by quantitative, comprise the arbitrary described test strips of claim 1-6
And cartridge, it is characterised in that: described cartridge includes lid and cassette bottom；Described test strips is arranged at the cassette bottom of described cartridge；
Having well and observation window on the lid of described cartridge, described well is just to the sample pad in described test strips, described
Observation window is just to the detection line in described test strips and nature controlling line.
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| CN105352958B (en) * | 2015-11-28 | 2019-02-26 | 美康生物科技股份有限公司 | Total 25-hydroxy-vitamin D detection kit |
| CN105891509B (en) * | 2016-05-03 | 2018-05-11 | 同昕生物技术（北京）有限公司 | For quantitatively detecting the lateral chromatography system of alpha-fetoprotein variant |
| CN107227351B (en) * | 2017-06-12 | 2020-02-07 | 深圳市慢性病防治中心 | Molecular beacon probe, primer pair and detection method for SNPs sites of GC genes |
| CN107656076A (en) * | 2017-08-11 | 2018-02-02 | 普菲特益斯生物科技（北京）有限公司 | A kind of time-resolved fluoroimmunoassay chromatography detection reagent of highly sensitive detection 25(OH)VD and preparation method thereof |
| CN108982882A (en) * | 2018-06-22 | 2018-12-11 | 卢氏实验室公司 | A kind of digital information vitamin D rapid detection system and method |
| CN109188001A (en) * | 2018-08-13 | 2019-01-11 | 深圳天辰医疗科技有限公司 | A kind of detection reagent of 25-hydroxy-vitamin D and preparation method thereof and kit |
| 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 |
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| EP1931711B1 (en) * | 2005-09-29 | 2009-04-08 | Roche Diagnostics GmbH | Antibodies against 25-hydroxyvitamin d |
| US20110097733A1 (en) * | 2009-10-27 | 2011-04-28 | Michel Anciaux | Process for the production of a hybridoma and antibody obtained therefrom, able to recognize more than one vitamin d metabolite |
| EP2372365A1 (en) * | 2010-04-01 | 2011-10-05 | Future Diagnostics B.V. | Direct immunoassay for vitamin D |
| WO2012129650A1 (en) * | 2011-03-25 | 2012-10-04 | Nanospeed Diagnostics Inc. | Lateral flow immunoassay for detecting vitamins |
| PL2759550T3 (en) * | 2013-01-28 | 2019-01-31 | Diasorin S.P.A. | Method and kit for detecting 1,25-dihydroxyvitamin D and related antibodies |
| CN103163306B (en) * | 2013-02-05 | 2015-06-17 | 北京九强生物技术股份有限公司 | 25 hydroxyl vitamin D detection kit and preparation method thereof |
| CN103983792B (en) * | 2014-05-29 | 2015-09-30 | 同昕生物技术（北京）有限公司 | A kind of immunoglobulin (Ig) lateral chromatography detection system |
| CN204008660U (en) * | 2014-06-30 | 2014-12-10 | 韩雅君 | A kind of kit of fast detecting vitamin D |
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