CN102998468B - 25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof - Google Patents
25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof Download PDFInfo
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Abstract
The invention discloses a 25-hydroxy-vitamin D3 detection kit, as well as a preparation method and applications thereof. The detection kit consists of a reagent I and a reagent II which are independent from each other, wherein the reagent I comprises the following components: a 25-hydroxy-vitamin D3-BSA conjugate, a biobuffer, a chelating agent, a surface active agent, a coagulant, a preservative, a stabilizer and water; the reagent II comprises the following components: rubber latex particles coated by a 25-hydroxy-vitamin D3 antibody, a biobuffer, a chelating agent, a surface active agent, a suspending agent, a preservative, a sealing agent, a stabilizer and water. The detection kit disclosed by the invention is high in detection sensitivity, high in specificity, accurate in quantification and wide in linear range, and is especially suitable for the detection of samples with extremely low 25-hydroxy-vitamin D3 content. In addition, the detection kit disclosed by the invention does not need to dilute the samples in advance in the detection process, facilitates the clinical use, and is easy to operate, low in detection cost and suitable for various full-automatic biochemical analyzers.
Description
Technical field
The present invention relates to kit of 25-hydroxyvitamin D3 content and preparation method thereof in a kind of human body, the invention still further relates to the using method that this 25-hydroxyvitamin D3 measures kit 25-hydroxyvitamin D3 content in human body, belong to the detection field of 25-hydroxyvitamin D3 content.
Background technology
25-hydroxyvitamin D3 is one of principal mode storing vitamin D in human body, and it is one of material that human homergy is movable indispensable.In general, the people in the whole world 2/3rds is deficient in vitamin D, wherein, more easily occurs the phenomenon of vitamin D deficiency in children, old man and pregnant woman.The shortage of vitamin D in body, children are suffered from, and possibility that rickets and old man suffer from osteoporosis increases greatly.Large quantity research shows in recent years, and the shortage of vitamin D directly has influence on the generation of people's in-vivo tumour, particularly colon cancer, breast cancer and prostate cancer; At present, the shortage of vitamin D has been proved to be the important symbol thing differentiating that gland cancer occurs, and clinical diagnosis will become an important index.In addition, the shortage of vitamin D also with autoimmune disease, infectious diseases and angiocardiopathy etc. have close relationship.At present, clinically conventional sense project and health check-up project have been become to the quantitative detection of vitamin D.For this reason, a kind of method that is quick, easy, sensitive, that detect vitamin D level is accurately and reliably set up particularly important.
In recent years, the quantitative detection of 25-hydroxyvitamin D3 mainly contained physical and chemical inspection method and immunological method.Physical and chemical inspection method mainly contains LC-MS-MS (LC-MS); Immunological method mainly contains radioimmunology (RIA) and enzyme linked immunosorbent assay (ELISA).Liquid chromatograph mass spectrography (LC-MS) although detection method highly sensitive, there is many drawbacks such as expensive equipment, sample pre-treatments complexity, complex operation, detection time be long.Although radioimmunology exists the advantages such as simple to operate, high specificity, sensitivity is high, there is the defect of the aspect such as radiation pollution and poor stability equally.Although accurate, highly sensitive, there is the strict and defect such as time-consuming of operation requirements, be not suitable for the needs of emergency treatment and the timely Diagnosis and Treat of clinical patient in enzyme linked immunosorbent assay (ELISA).
Up to now, still lack a kind of simple to operate fast, quantitatively accurately, the kit for 25-hydroxyvitamin D3 content in human body of the high or high specificity of susceptibility.
Summary of the invention
An object of the present invention is to provide a kind of 25-hydroxyvitamin D3 detection kit;
Two of object of the present invention is to provide a kind of method preparing described 25-hydroxyvitamin D3 detection kit.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Detect a kit for 25-hydroxyvitamin D3 content, be made up of reagent I independent of each other and reagent II; Wherein, the component of reagent I comprises: 25-hydroxyvitamin D3-BSA conjugate, biological buffer, sequestrant, surfactant, set accelerator, antiseptic, stabilizing agent and water;
The component of reagent II comprises: wrap by the latex particle of 25-hydroxyvitamin D3 antibody, biological buffer, sequestrant, surfactant, suspending agent, antiseptic, sealer, stabilizing agent and water.
The present invention finds, in reagent I or reagent II, the consumption of each component all has the impact of highly significant for the accuracy detected, sensitivity and specificity; The present invention has chosen the optimum amount of each component in reagent I or reagent II by a large amount of optimization test finishing screens, adopts these preferred consumptions following effectively can improve the accuracy of testing result, sensitivity and specificity:
In every 1 liter of reagent I, the consumption of each component is preferably: 25-hydroxyvitamin D3-BSA conjugate 0.05-50mg, biological buffer 1-100g, sequestrant 0.1-10g, surfactant 0.1-5mL, antiseptic 0.1-10g, stabilizing agent 1-50g, set accelerator 1-50g, surplus is water;
Preferred further, in every 1 liter of reagent I, the consumption of each component is: 25-hydroxyvitamin D3-BSA conjugate 0.1-30mg, biological buffer 5-50g, sequestrant 0.5-5g, surfactant 0.5-4mL, antiseptic 0.5-5g, stabilizing agent 10-40g, set accelerator 10-40g, surplus is water;
Further preferred, in every 1 liter of reagent I, the consumption of each component is: 25-hydroxyvitamin D3-BSA conjugate 0.5-20mg, biological buffer 10-20g, sequestrant 0.7-2g, surfactant 0.7-2mL, antiseptic 0.6-1.5g, stabilizing agent 15-30g, set accelerator 12-30g, surplus is water;
Most preferred, in every 1 liter of reagent I, the consumption of each component is: 25-hydroxyvitamin D3-BSA conjugate 5mg, biological buffer 12g, sequestrant 1g, surfactant 0.5mL, antiseptic 1g, stabilizing agent 20g, set accelerator 15g, and surplus is water;
In every 1 liter of reagent II, the consumption of each component is preferably: wrap by the latex particle 0.1-10g of 25-hydroxyvitamin D3 antibody, biological buffer 1-100g, sequestrant 0.1-10g, surfactant 0.1-10mL, antiseptic 0.1-10g, suspending agent 5-50mL, sealer 5-50g, stabilizing agent 50-150g, surplus is water.
Preferred further, in every 1 liter of reagent II, the consumption of each component is: wrap by 25-hydroxyvitamin D3 antibody latex particle 0.5-5g, biological buffer 5-40g, sequestrant 0.5-5g, surfactant 1-7mL, antiseptic 0.5-5g, suspending agent 10-40mL, sealer 6-30g, stabilizing agent 80-140g, surplus is water.
Further preferred, in every 1 liter of reagent II, the consumption of each component is: wrap by 25-hydroxyvitamin D3 antibody latex particle 0.8-3g, biological buffer 8-15g, sequestrant 1-3g, surfactant 1-4mL, antiseptic 0.7-2g, suspending agent 15-30mL, sealer 8-15g, stabilizing agent 90-120g, surplus is water.
Most preferred, in every 1 liter of reagent II, the consumption of each component is: wrap by 25-hydroxyvitamin D3 antibody latex particle 1.8g, biological buffer 10.5g, sequestrant 2g, surfactant 3mL, antiseptic 1g, suspending agent 20mL, sealer 10g, stabilizing agent 100g, surplus is water.
In order to realize better Detection results, the pH value of reagent I range preferably from 7.0-8.0, the pH value of reagent II range preferably from 6.5-7.5.
" wrapping by the latex particle of 25-hydroxyvitamin D3 antibody " described in reagent II of the present invention be in phosphate buffer by 25-hydroxyvitamin D3 antibody by the surface of the methods combining such as physisorption or chemical crosslinking to carboxy-modified polystyrene latex particles, then through centrifugal, washing after gained; Wherein, described 25-hydroxyvitamin D3 antibody can be 25-hydroxyvitamin D3 monoclonal antibody or polyclonal antibody.
Can by the method for physisorption or chemical crosslinking by the surface of 25-hydroxyvitamin D3 antibody in conjunction with polystyrene latex particles.The antibody of physisorption is after long-time placement, and the easy desorption of antibody protein, causes detection sensitivity to reduce, and repeatability is poor.The present invention preferably adopts the surface by the method for chemical crosslinking, 25-hydroxyvitamin D3 antibody being attached to carboxy-modified polystyrene latex particles, and the antibody of combination can be made so more stable.
Preferably, a kind of bag of preparing, by the method for 25-hydroxyvitamin D3 monoclonal antibody or polyclonal antibody latex particle, comprises the following steps:
1. polystyrene latex particles phosphate buffer is disperseed, then adds 6-aminocaprolc acid solution and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), after stirring, centrifugally abandon supernatant; Carboxy-modified polystyrene latex solution is obtained again with phosphate buffer dilution dispersion precipitation latex;
2. by 25-hydroxyvitamin D3 monoclonal antibody or polyclonal antibody dried frozen aquatic products deionized water dissolving, antibody-solutions is made;
3. antibody-solutions is got, after phosphate buffer dilution, add the carboxy-modified polystyrene latex solution that step is 1. prepared, add EDC again, at 0-4 DEG C of stirring reaction, add glycine buffer and stir, cessation reaction, centrifugally abandon supernatant, use phosphate buffer washing precipitation, obtain bag by 25-hydroxyvitamin D3 antibody latex particle.
With the effect of soda acid during reagent I or the biological buffer described in reagent II mainly play, so all biological buffer of the present invention can be applicable to the various buffering agents of acid-base function in can playing, it can be such as trishydroxymethylaminomethane (Tris), Pehanorm base ethyl sulfonic acid (TES), 2-(N-morpholine) ethyl sulfonic acid (MES), N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid (HEPES), Pehanorm base propane sulfonic acid (TAPS), piperazine-1, 4-dihydroxy propane sulfonic acid (POPSO), 3-(N-morpholine) the various conventional biological buffer such as propane sulfonic acid (MOPS), in order to reach better Detection results, biological buffer described in reagent I is preferably trishydroxymethylaminomethane, biological buffer described in reagent II is preferably 3-(N-morpholine) propane sulfonic acid.
The effect that reagent I or the sequestrant described in reagent II play is the metallic ion that complexing detects in sample, to reduce the interference that metallic ion causes; Therefore, it is possible to the various sequestrants of complexation of metal ions all can as the sequestrant in kit of the present invention, such as: disodium ethylene diamine tetraacetate, aminotriacetic acid, diethylene-triamine pentaacetic acid and salt thereof etc. all can be used as the sequestrant in kit of the present invention; In order to reach better Detection results, the preferred disodium ethylene diamine tetraacetate of the present invention (EDTA) is as the sequestrant in reagent I and reagent II.
Reagent I or the antiseptic described in reagent II, mainly in order to the product preventing bacteria breed from causing goes bad, prevent the antiseptic of bacteria breed effect all can be applicable to the present invention so any one can play; In order to reach better Detection results, preferably, reagent I of the present invention and the antiseptic described in reagent II are sodium azide.
Reagent I or the surfactant described in reagent II mainly play the dissolving promoting each component in reagent system and detect each material in sample, reach and reduce sample turbidity to the effect of the impact of measurement result; Therefore, the existing surfactant with solubilization all can be used as the surfactant described in reagent I or reagent II, such as, polysorbas20, polysorbate40, triton x-100, Nonidet P40 etc. all can be used as the sequestrant in kit of the present invention.In order to reach better effect, the surfactant described in reagent I is preferably triton x-100.
Preferably triton x-100 and polysorbas20 are used in combination as surfactant in reagent II, wherein, in every 1 liter of reagent II, the consumption of triton x-100 is 0.05 ~ 2.5mL, is preferably 0.15 ~ 2mL, again be preferably 0.5-1mL, most preferably be 0.5mL; In every 1 liter of reagent II, the consumption of polysorbas20 is 0.5 ~ 5mL, is preferably 1 ~ 4mL, is again preferably 2-3mL, most preferably is 2.5mL.
Reagent I or the stabilizing agent Main Function described in reagent II are the stability keeping each component in kit, extend the shelf life of product.Conventional stabilizing agent is not protein-contg pure chemicals, and as pure chemicals such as sodium chloride, potassium chloride, sucrose, the reagent I in the present invention and the stabilizing agent in reagent II are preferably sucrose.
Set accelerator described in reagent I is the quick aggegation in order to promote interfering material in blood sample, reduce the interference to testing result, what set accelerator conventional in detection kit was conventional is Macrogol 6000 or PEG 8000, and in detection kit of the present invention, these two kinds of compositions all can be selected.
The Main Function of the suspending agent described in reagent II is being uniformly distributed of each material in maintenance system, the measurement result error that minimizing reagent is uneven and cause, conventional suspending agent has ethylene glycol, glycerine, lactose and maltose etc., and the suspending agent in reagent II of the present invention is preferably ethylene glycol.
The Main Function of the sealer described in reagent II is the generation in order to avoid nonspecific reaction, and reduce the false positive interference in testing process, the sealer in reagent II of the present invention is selected from bovine serum albumin(BSA) (BSA).
Often kind of component used in detection kit of the present invention all obtains from biological reagent company or pharmaceuticals's purchase by commercial sources.
Another object of the present invention is to provide a kind of method preparing described detection 25-hydroxyvitamin D3 kit, comprising:
(1) reagent I is prepared: by each components dissolved in distilled water or distilled water, mix, constant volume; (2) reagent II is prepared: by each components dissolved in distilled water or distilled water, mix, constant volume; (3) by reagent I and the independent packing of reagent II, sealing, to obtain final product.
Another object of the present invention is to provide the detection method that described 25-hydroxyvitamin D3 detection kit detects 25-hydroxyvitamin D3 content in sample, and the method comprises the following steps:
To 2 μ L sample to be detected, (calibration tube makees sample with calibration object, blank is sample with distilled water) in add 125 μ L reagent I fully mix, in 37 DEG C of constant temperature 5 minutes, in mixed system, add 25 μ L reagent II again, mixing, 37 DEG C of constant temperature are after 1 minute, blank tube returns to zero, wavelength 570nm, measures each pipe absorbance A and measures each pipe absorbance A 2 after 1,5 minutes.Calculate Δ A=A1-A2.According to multiple spot calibration object concentration and corresponding absorbance changing value Δ A, adopt multiple spot gamma correction pattern determination working curve, sample absorbance change concentration value corresponding on working curve is mensuration concentration.
Detection kit of the present invention adopts double reagent pattern to efficiently avoid the interference of nonspecific reaction, ensure that the reliable and stable of measurement result.This detection kit adopts latex immunoturbidimetry as Cleaning Principle, have simple to operate, quick, quantitatively accurately, the advantage such as high, the high specificity of susceptibility, the needs that Emergency call etc. detects can be met, have purposes more and more widely clinically.
Accompanying drawing explanation
Fig. 1 is 5-hydroxycholecalciferol standard working curve.
Fig. 2 is the correlation test result of kit of the present invention and enzyme-linked immunosorbent assay measured value.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of Preparative Example 125-hydroxycholecalciferol-BSA conjugate
1. take the 25-hydroxyvitamin D3-hemisuccinic acid ester (being purchased from Shanghai Han Zun biotech firm) of 2.0mg, the 1-ethyl-3-(3-dimethylaminopropyl of 0.8mg) N-hydroxy-succinamide (NHS) of carbodiimides (EDC) and 2.0mg adds in the round-bottomed flask that the anhydrous methenyl choloride of 1mL is housed, after at room temperature, stirred under nitrogen atmosphere reaction 2h;
2. after reaction terminating, in round-bottomed flask, add 1mL3mg/mL BSA solution, spend the night in room temperature, stirred under nitrogen atmosphere reaction;
3. dialyse 48 hours for PBS4 DEG C of above-mentioned reactant liquor 0.01mol/L pH7.2, liquid is changed 6 times in centre, changes liquid 1 time every 8 hours, collects dialysate, uses sephadex HiTrap
tMdesalting crosses post desalting and purifying 2 times, vacuum freeze drying, i.e. obtained 25-hydroxyvitamin D3-BSA conjugate 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA.
The preparation of Preparative Example 225-hydroxycholecalciferol polyclonal antibody
1. 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA 3 monthly ages of immunity are used, the male Japan large ear rabbit of body weight 1.5kg, first immunisation is dissolved 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA by aseptic PBS solution and is mixed fully emulsified with the Freund's complete adjuvant (FCA) of equivalent, immunizing agent consumption is 1mg(0.5mL)/only, divide 4-6 injection location in dorsal sc;
2. first immunisation is after 2 weeks, dissolve 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA by aseptic PBS solution equally and mix fully emulsified immune rabbit with the Freund's incomplete adjuvant (FIA) of equivalent, immunizing dose is 500 μ g(0.5mL)/only, injection site is the same.
3. all rabbits immune 6 times altogether, every minor tick 2 weeks, adopts indirect ELSIA and indirect competition ELSIA to measure rabbit anti-serum and tires and specificity after the 4th immunity, 6th immunity is by lumbar injection booster immunization, after last immune 7 days, Culling heart blood, obtains a large amount of rabbit anti-serums.Rabbit polyvalent antibody can be obtained 25-hydroxyvitamin D3 polyclonal antibody through affinity chromatography column separating purification.
Preparative Example 3 wraps by the preparation of 25-hydroxyvitamin D3 polyclonal antibody latex particle
1. 1.0g polystyrene latex particles (purchased from PolyMicrospheres company) dry thing is taken, add 100mL0.12M phosphate buffer (pH7.5) dispersion, add 6-aminocaprolc acid solution and the 40mg1-ethyl-3-(3-dimethylaminopropyl of the 0.01M of 20mL again) carbodiimides (EDC), at room temperature stir 3 hours, centrifugally abandon supernatant, with 0.12M phosphate buffer washing precipitation 2 times, make the Carboxylated Polystyrene latex being connected to aminocaproic acid arm, carboxy-modified polystyrene latex solution is obtained again with 100mL0.12M phosphate buffer dilution dispersion precipitation latex,
2. by 250 μ g25-hydroxycholecalciferol polyclonal antibody dried frozen aquatic products 0.5mL deionized water dissolvings, antibody-solutions is obtained;
3. after antibody-solutions 25mL0.12M phosphate buffer 2. made for step being diluted, add the latex solution that 25mL step is 1. made, add 10mg EDC again, 5 DEG C of stirring reactions 8 hours, add 1mL0.15M glycine buffer and stir 40 minutes, cessation reaction, centrifugally abandon supernatant, with 0.12M phosphate buffer washing precipitation 2 times, obtain bag by the latex particle of 25-hydroxyvitamin D3 polyclonal antibody.
The preparation of embodiment 125-hydroxycholecalciferol detection kit:
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
Bag is by the latex particle 2.0g of 25-hydroxyvitamin D3 polyclonal antibody
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The preparation of embodiment 225-hydroxycholecalciferol detection kit
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The preparation of embodiment 325-hydroxycholecalciferol detection kit:
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The preparation of embodiment 425-hydroxycholecalciferol detection kit
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The preparation of embodiment 525-hydroxycholecalciferol detection kit:
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The preparation of embodiment 625-hydroxycholecalciferol detection kit
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The preparation of embodiment 725-hydroxycholecalciferol detection kit
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The preparation of embodiment 825-hydroxycholecalciferol detection kit
1, the preparation of reagent I:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
2, the preparation of reagent II:
By described with measuring each component:
By above-mentioned each components dissolved in distilled water or distilled water, be settled to 1 liter, sealing is preserved, for subsequent use.
The range of linearity detection experiment of test example 1 detection kit of the present invention
1, for examination kit: the kit prepared by embodiment 1-8
Prepare 5 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L, the 25-hydroxyvitamin D3 standard solution of 100 μ g/L, measure kit with 25-hydroxyvitamin D3 prepared by embodiment of the present invention 1-6, draw its standard working curve, the results are shown in Figure 1.
Kit of the present invention detects 25-hydroxyvitamin D3 content in sample, comprise the following steps: to 2 μ L sample to be detected, (calibration tube makees sample with calibration object, blank is sample with distilled water) in add 125 μ L reagent I fully mix, in 37 DEG C of constant temperature 5 minutes, then in mixed system, add 25 μ L reagent II, mixing, 37 DEG C of constant temperature are after 1 minute, and blank tube returns to zero, wavelength 570nm, measure each pipe absorbance A and measure each pipe absorbance A 2 after 1,5 minutes.Calculate Δ A=A1-A2.With absorbance difference Δ A for ordinate, standard concentration is ordinate, makes " concentration-absorbance difference " standard working curve, sees Fig. 1.
Test example 2 kit accuracy of the present invention and degree of accuracy detection experiment
1, for examination kit: the kit prepared by embodiment 1-6;
2, test method and result
Accuracy and precision is order of accuarcy and the credibility of reaction detection kits result.This test is using the 25-hydroxy-vitamin D calibration object of IDS company of Britain as tested object, and evaluate the accuracy and precision of detection kit of the present invention, test findings is in table 1.
Table 1 kit accuracy of the present invention and degree of accuracy detection experiment result
From the test findings of table 1, the result that detection kit of the present invention measures IDS company of Britain calibration object gained is very accurate, and the degree of accuracy measured is fine.
The correlation test of test example 3 kit of the present invention and enzyme-linked immunosorbent assay (Elisa) measured value
1, for examination kit: the kit prepared by embodiment 1
2, test method and result
Compared by the measured value measuring the content of 25-hydroxyvitamin D3 in EDTA anticoagulate plasma with the kit of the embodiment of the present invention 1 and enzyme-linked immune analytic method, test findings for shown in Fig. 2, line retrace analysis of going forward side by side.As can be seen from Figure 2 kit of the present invention and enzyme-linked immunosorbent assay have good correlativity in EDTA anticoagulate plasma 25-hydroxyvitamin D3 mensuration.
Claims (9)
1. detect a kit for 25-hydroxyvitamin D3 content, it is characterized in that: be made up of reagent I independent of each other and reagent II; Wherein, the component of reagent I comprises: 25-hydroxyvitamin D3-BSA conjugate, biological buffer, sequestrant, surfactant, set accelerator, antiseptic, stabilizing agent and water; The component of reagent II comprises: wrap by the latex particle of 25-hydroxyvitamin D3 antibody, biological buffer, sequestrant, surfactant, suspending agent, antiseptic, sealer, stabilizing agent and water; Described biological buffer is trishydroxymethylaminomethane or 3-(N-morpholine) propane sulfonic acid, described sequestrant is disodium ethylene diamine tetraacetate, described surfactant is triton x-100 or polysorbas20, described suspending agent is ethylene glycol, described antiseptic is sodium azide, described stabilizing agent is sucrose, and described set accelerator is Macrogol 6000, and described sealer is bovine serum albumin.
2. according to kit according to claim 1, it is characterized in that: in every 1 liter of reagent I, the consumption of each component is: 25-hydroxyvitamin D3-BSA conjugate 0.05-50mg, biological buffer 1-100g, sequestrant 0.1-10g, surfactant 0.1-5mL, antiseptic 0.1-10g, stabilizing agent 1-50g, set accelerator 1-50g, surplus is water;
In every 1 liter of reagent II, the consumption of each component is: wrap by the latex particle 0.1-10g of 25-hydroxyvitamin D3 antibody, biological buffer 1-100g, sequestrant 0.1-10g, surfactant 0.1-10mL, antiseptic 0.1-10g, suspending agent 5-50mL, sealer 5-50g, stabilizing agent 50-150g, surplus is water.
3. according to kit according to claim 2, it is characterized in that: in every 1 liter of reagent I, the consumption of each component is: 25-hydroxyvitamin D3-BSA conjugate 0.1-30mg, biological buffer 5-50g, sequestrant 0.5-5g, surfactant 0.5-4mL, antiseptic 0.5-5g, stabilizing agent 10-50g, set accelerator 10-40g, surplus is water;
In every 1 liter of reagent II, the consumption of each component is: wrap by the latex particle 0.5-5g of 25-hydroxyvitamin D3 antibody, biological buffer 5-40g, sequestrant 0.5-5g, surfactant 0.5-5mL, antiseptic 0.5-5g, suspending agent 10-40mL, sealer 6-30g, stabilizing agent 80-140g, surplus is water.
4. according to kit according to claim 3, it is characterized in that: in every 1 liter of reagent I, the consumption of each component is: 25-hydroxyvitamin D3-BSA conjugate 0.5-20mg, biological buffer 10-20g, sequestrant 0.7-2g, surfactant 0.7-2mL, antiseptic 0.6-1.5g, stabilizing agent 15-30g, set accelerator 12-30g, surplus is water;
In every 1 liter of reagent II, the consumption of each component is: wrap by 25-hydroxyvitamin D3 antibody latex particle 0.8-3g, biological buffer 8-15g, sequestrant 1-3g, surfactant 1-4mL, antiseptic 0.7-2g, suspending agent 15-30mL, sealer 8-15g, stabilizing agent 90-120g, surplus is water.
5. according to the kit of claim 1-4 described in any one, it is characterized in that: described bag is prepared by the following method by the latex particle of 25-hydroxyvitamin D3 antibody: surface 25-hydroxyvitamin D3 antibody being attached to polystyrene latex particles, again through centrifugal, washing, to obtain final product.
6. according to kit according to claim 5, it is characterized in that: described bag is prepared by the following method by the latex particle of 25-hydroxyvitamin D3 antibody:
1. polystyrene latex particles phosphate buffer is disperseed, then add 6-aminocaprolc acid solution and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides, stir, centrifugally abandon supernatant; Carboxy-modified polystyrene latex solution is obtained with phosphate buffer dilution dispersion precipitation latex;
2. by the water-soluble solution of 25-hydroxyvitamin D3 monoclonal antibody or polyclonal antibody, antibody-solutions is made;
3. antibody-solutions is got, dilute with phosphate buffer, add the carboxy-modified polystyrene latex solution that step is 1. prepared, add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides again, at 0-4 DEG C of stirring reaction, add glycine buffer and stir, cessation reaction, centrifugally abandon supernatant, washing precipitation, to obtain final product.
7., according to the kit of claim 1-4 described in any one, it is characterized in that: the scope of the pH value of reagent I is 7.0-8.0; The scope of the pH value of reagent II is 6.5-7.5.
8. prepare a method for claim 1-4 kit described in any one, comprising:
(1) reagent I is prepared: by each components dissolved in distilled water, mix;
(2) reagent II is prepared: by each components dissolved in distilled water, mix;
(3) by reagent I and the independent packing of reagent II, sealing, to obtain final product.
9. prepare a method for claim 1-4 kit described in any one, comprising:
(1) reagent I is prepared: by each components dissolved in distilled water, mix;
(2) reagent II is prepared: by each components dissolved in distilled water, mix;
(3) by reagent I and the independent packing of reagent II, sealing, to obtain final product.
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