CN102692514A - Blood 25-hydroxy vitamin D3 adenocarcinoma assay kit and preparation method thereof - Google Patents
Blood 25-hydroxy vitamin D3 adenocarcinoma assay kit and preparation method thereof Download PDFInfo
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Abstract
The invention provides a blood 25-hydroxy vitamin D3 adenocarcinoma assay kit and a preparation method thereof, and relates to an assay kit. The kit comprises calibrators of different concentrations, an ELISA (enzyme-linked immuno sorbent assay) plate of a 25-hydroxy vitamin D3 coated antibody, horse radish peroxidase marked 25-hydroxy vitamin D3, a quality control product, a substrate chromogenic solution, a reaction stop buffer and a washing fluid. By adopting a series of chemical modification, the 25-hydroxy vitamin D3 is modified into 25-hydroxy vitamin D3 hemisuccinate ester. Hapten is transformed into complete antigen by an improved carbodiimide method and protein vector BSA (bovine serum albumin) protein coupling. Furthermore, the blood25-hydroxy vitamin D3 adenocarcinoma assay kit is developed by combining the ELISA method. The kit can be used in cancer assay in clinical application.
Description
Technical field
The present invention relates to a kind of detection kit, especially relate to a kind of blood 25-hydroxyvitamin D3 gland cancer detection kit and preparation method thereof.
Background technology
Before more than 100 years, vitamin D just is widely used in the generation of bone property diseases such as monitoring rickets, rickets after coming to light as keeping the basis of bone health.At present, the researcher finds that again vitamin D is also relevant with other multiple chronic disease, especially cancer.
1, vitamin D source and metabolism
Vitamin D (Vitamin D is fat-soluble steroid derivatives VitD), and five kinds of forms are arranged, with the health relation closer be VitD2 and VitD3.Wherein, the former derives from the ergosterol in the yeast cells, and the latter then derives from 7-dehydrocholesterol under the animal skins.
Usually as long as we accept sufficient sunlight every day, just can obtain to satisfy the VitD3 demand of human body 90%, some foods such as milk, cod-liver oil etc. then can supply VitD2 and VitD3, to satisfy 10% demand of human body remainder.Yet VitD does not have active, in human body, combines albumen or lipoprotein to combine to be transported to liver with VitD, (is also referred to as CYP27A1, CYP3A4, CYP2R1, CYP2J3) catalysis formation 25-(OH) VitD through the VitD-25-hydroxylase.It is the main circulation form of vitamin D in the blood plasma, and Chang Zuowei assesses the detection index (optimum or reference is 75-150nmol/L, although the reference value that adopt in many laboratories is 50-250nmol/L) of individual VitD nutrition condition.But 25-(OH) VitD inanimate object is active, needs under 25-(OH) VitD-1 α-hydroxylase effect, to be metabolized to 1 of biologically active, and 25-(OH) 2VitD brings into play effect.Discover that 25-(OH) 2VitD-1 α-hydroxylase does not exist only in the kidney; Also be present in the various kinds of cell such as prostate, mammary gland, rectum; So VitD mainly plays a role through two approach in vivo: one is the endocrine approach; Through 1 in the blood, 25-(OH) 2VitD regulates the calcium homeostasis of general; Another is autocrine or paracrine approach, and through 1 of part, 25-(OH) 2VitD and vitamin D receptor play a role, and do not rely on the calcium homeostasis of general, relate to cell proliferation, differentiation and immunological regulation etc.In addition, 1, the existence of 25-(OH) 2VitD can be induced the expression of 25-(OH) VitD-24-hydroxylase (CYP24), thus catalysis 25-(OH) VitD and 1,25-(OH) 2VitD forms cholecalciferol water-soluble, non-activity and discharges outward.
2, the molecular mechanism of vitamin D in cancer prevention
Vitamin D receptor (vitamin D receptor, VDR), as 1, the acceptor of 25-(OH) 2VitD; Do not exist only in and regulate the calcium phosphorus balance in kidney, the parathyroid gland, at prostate, rectum; Mammary gland, pancreas is also all expressed in immune various cells and the malignant cell thereof.1,25-(OH) 2VitD impels VDR and RXR to form heterodimer with after VDR combines, and final formed compound acts on vitamin D response element on the target gene, and (vitamin D – responsive elements VDREs), regulates target gene expression.
At present; Researchist (Sreeram V.Ramagopalan, Andreas Heger, Antonio J.Berlanga; Et al.A ChIP-seq defined genome-wide map of vitamin D receptor binding:Associations with disease and evolution.Genome Res; 2010, find that 20:1352-1360) vitamin D can influence the activity of 229 genes directly or indirectly.It is relevant with a series of diseases that these genes have been proved to be, and wherein the most noticeable is exactly that the gene expression relevant with the cancer incidence and development is regulated.For example, suppress the albumen Rb that cell was changed to the S phase by the G phase, p107 and p130 etc.; The cell cycle regulation signal path that suppresses EGFR and mediation thereof; The reduction anti-apoptosis factor (Bcl-2 Bcl-XL) expresses, and the short antiapoptotic factors of increase (Bax, expression Bak) waits and promotes the malignant cell apoptosis; Regulate expression such as VEGF and E-cadherin, suppress angiogenesis, stop malignant cell to shift; And inhibition forms relevant inflammatory reaction etc. with cancer, thereby reduces the risk of falling ill of cancer.
3, vitamin D and gland cancer epidemiological study
In the 1980's, Cedric Garland passes through relatively different geographic carcinoma of the rectum fatal rates of the U.S. and amount of sunlight with Frank Garland two brothers, proposes ultraviolet irradiation and the VitD role in reducing risk of cancer first.Subsequently, more epidemiology researchs begin to focus on the relation between cancer incidence/mortality ratio and the VitD.At present, this vital role of VitD is accepted extensively in multiple gland cancer, wherein belongs to breast cancer, colon cancer, prostate cancer research especially at most.
Expection and recovery epidemiological study show 25-(OH) VitD level when 50nmol/L is following, breast cancer, and colon cancer, and the ill risk of prostate cancer increases to 30%～50%.Grant investigates discovery, and 25% mortality ratio causes owing to living in high latitude area disappearance vitamin D in the European breast cancer.And 25-(OH) VitD level is compared at 25nmol/L with its content when 130nmol/L in the breast cancer serum, and 25-(OH) VitD level is when 85nmol/L compares with 15nmol/L in the colon cancer, and two kinds of gland cancer occurrence risk all reduce by 50%.A perspective study shows, in 1954 male sex's objects vitamin D intake and colon cancer risk be direct relation (promptly when the vitamin D intake be 6～94IU/ days, relative risk is 1.0; The vitamin D intake is 233～652IU/ days, and relative risk is 0.53, P＜0.05).Its colon cancer occurrence risk raise 25% [3] when the follow in 8 years found then that the basic horizontal of 25-in women participant's serum (OH) VitD is lower than 30nmol/L.The comprehensive many pieces of data analyses of William B.Grant [10] show that the dose-response relationship of VitD and breast cancer and colon cancer is J-shaped; The optimised quantity of 25-(OH) VitD is seemingly more than the 100nmol/L; And its level possibly brought into play bigger for the effect of cancer when rising to 250nmol/L.Other has the male sex of research contrast outside work and office work, finds that the former suffered from prostate cancer late 3～5 years relatively, because the outdoor male sex's VitD level will be higher than the office work male sex.Comprehensively show 25-(OH) VitD water article when 100nmol/L is above, breast cancer, colon cancer, carcinoma of the rectum occurrence risk can reduce.Therefore; According to individual solar radiation, age, sex; Body weight and VitD basic horizontal; Suggestion replenish every day 2000-4000IUVitD can keep 25-(OH) VitD water article more than 100nmol/L (JoEllen Welsh.Cellular and molecular effects of vitamin D on carcinogenesis.Archives of Biochemistry and Biophysics, 2011, doi:10.1016/j.abb.2011.10.019).
VitD is different for the incidence of various cancers and the effect of fatal rate.Recent one piece of summary (E.R.Bertone-Johnson; Vitamin D and breast cancer, Ann.Epidemiol.2009,19 (2009) 462-467) show; There is several factors can cause the difference of VitD effect; Comprising the age, women's menopause situation, tumour characteristic and VDR gene pleiomorphism.Discover that Fok I ff compares with the FF genotype on the VDR gene; The ill risk of breast cancer has increased by 14% (Raimondi S significantly; Johansson H, Maisonneuve P, Gandini S:Review and meta-analysis on vitamin D receptor polymorphisms and cancer risk.Carcinogenesis; 2009,30:1170 – 1180).Other has ANOVA showed significant BsmI Bb to compare with the bb genotype, and the ill risk of its prostate cancer has reduced by 17% significantly.Obviously, no matter the vitamin D level lacks or genetic mutation, and 1,25-(OH) 2VitD has received inhibition for the normal regulation of cell growth, has promoted the malignant cell growth, thereby has improved the occurrence risk of gland cancer.
4, the clinical practice of vitamin D in gland cancer
4.1 gland cancer biomarker
The World Health Organization (WHO) points out, if can early diagnosis also in time treat, and 95% tumour can be cured.This makes the discovery of biomarker become the main flow of cancer research.Along with the develop rapidly of emerging genomics, protein science technology, as DNA with organize micromatrix, SELDI-TOF, mass spectrum, ELISA, RT-PCR, FISH etc., many have the gland cancer molecular marker of application prospect to come to light.Roughly be divided into following several kinds according to its biological chemistry and immunological characteristic:
(1) gene class mark: microsatellite instability; Genic instabilities such as DNA supermethylation and SNP are Fundamentals of carcinogenesis; Cause the unconventionality expression of some oncogenes and product thereof; As individual difference, help as cancer risk assessment, detect and draft the molecular marked compound of therapeutic scheme.At present people have found that nearly 100 kinds relevant oncogene and product thereof take place, develop with tumour; Part has been applied to tumor markers; Relate to proto-oncogene (like Her-2) and tumor suppressor gene (like BRCA1; MTS1), they all present unconventionality expression in most cancers, are preferable broad-spectrum tumor molecular marked compounds.In addition, some metastasis related genes (like nm23) and apoptosis-related genes (like Bcl-2) are then relevant with invasive cancer, apoptosis, can be used as prognostic indicator.MLH1, these gene mismatch of MSH2 or MSH6 sudden change and the microsatellite instability proof that causes is relevant with carcinoma of the rectum prognosis, but owing to the low specificity and the microsatellite instability of carcinoma of the rectum mark, its use is not extensive.
(2) enzyme label: enzyme and isodynamic enzyme are to occur the earliest and one of the tumor markers that uses, show as in the tumour generating process, with cell differentiation or hyperplasia linked groups enzyme-specific or isodynamic enzyme class amount or (with) activity change.For example PSA PSA, human glandular kallikrein 2 (hk2), matrix metalloproteinase MMPs, lysosome cysteine proteinase CathepsinB and L, serine stretch protein enzyme uPA and its suppressant PAI-1,2 etc.
(3) Antigens label: mainly be divided into tumour antigen (CEA, PCNA, Ki-67 etc.) and sugar antigen (CA125, CA15-3 etc.).
(4) hormone and acceptor class label thereof: like calcitonin Calcitonin, ERs ER and PgR PR, thymosin extrasin thymosin β-15 etc.
(5) other protide: like osteopontin OPN,, be high level expression in the blood of prostate cancer and lung cancer patient, can be used as the gland cancer prognostic marker at metastatic breast cancer.Recently, FDA has ratified some new biomarkers based on urine, comprising nuclear matrix protein NMP22, as the diagnosis of bladder cancer mark.These tumor markerses are all being brought into play bigger practical value at aspects such as the assessment of gland cancer occurrence risk, diagnosis, judging prognosis, evaluation curative effect and people at highest risk's follow-up observations.
4.2VitD detect the application in the gland cancer diagnosis
The existing kind more than 100 of the tumor markers of having named at present, the mark of clinical practice reaches about 20, but because of individual event tumor markers susceptibility and specificity are lower, accurately diagnosing tumour and predict prognosis.Research both at home and abroad shows that multi-tumor marker associating detection of dynamic is to improve the diagnosing tumor feasible strategy.Therefore, we are intended to find more biomarker, hope to make diagnostic result more reliable.To sum up the general introduction of the epidemiology of VitD and gland cancer and with cancer relevant Study on Molecular Mechanism, the incidence and development of VitD and multiple gland cancer has the relation that can not cut.And VitD is as the necessary micromolecule organic compound of individual metabolism; Compare with big branchs subclass label such as gene and protide: 25-(OH) the VitD half life period of the non-activity of metabolism generation was 2 weeks in vivo; More stable; Reflection human body situation that comparatively speaking more can be real-time has indicative preferably; The researchist finds the VitD metabolin in the blood is stored in 24 ℃, 72h (the Bruce W Hollis.Measuring 25-hydroxyvitamin D in a clinical environment:challenges and needs.Am J Clin Nutr that still is kept perfectly; 2008; 88 (suppl): 507S – 10S), so its stability when having guaranteed that sample stores; The result error of having avoided gene or protein contamination, degraded etc. to cause in the test experience has predictability reliably; Simultaneously also simplified the sample process step, be easy to detect; And its level all presents significant change in multiple gland cancer, and certain susceptibility is arranged; Therefore, if more step further as the diagnoses and treatment development that one of predictor of cancer occurrence risk will promote cancer VitD.
VitD is also unstable in vivo, can be metabolized to 25-(OH) VitD and 1,25-(OH) 2VitD.25-in the serum (OH) the VitD half life period was 2 weeks, horizontal stable in human body, and 1; 25-(OH) the 2VitD half life period is 15h, is the activity form of VitD, regulated and control by multiple factor; In many target tissues, all can produce, though the VitD disappearance, 1; 25-(OH) 2VitD level also often is in normal or increases state; So whether decide VitD to lack with it is nonsensical, (Michael F.Holick.High Prevalence of Vitamin D Inadequacy and Implications for Health.Mayo Clin Proc, 2006; 81 (3): 353-373) so clinically with in the serum 1,25-(OH) VitD content is as the barometer of individual VitD situation.
At present, 25-(OH) VitD method mainly contains physical and chemical inspection method and immunological method in the human body.Physical and chemical inspection method: liquid chromatograph mass spectrography technology (LC-MS), high-efficient liquid phase chromatogram technology (HPLC).These two kinds of methods all can detect 25-(OH) VitD2 and 25-(OH) VitD3 content respectively, have high duplication, usually with HPLC as " golden standard " method.But these methods are loaded down with trivial details time-consuming; Require sample big relatively; Need the technician of professional training accurately to operate; And LC-MS can't distinguish 25-(OH) VitD3 and its nonactive isomers, and therefore this problem especially it is noted that when neonate's content detection and be not suitable for clinical labororatory.Immunological method: radiommunoassay (RIA), competitive protein binding assay (CPBA), radio receptor assay (RRA) and enzyme-linked immunoassay (EIA); Owing to have sensitivity, advantage such as quick, special, easy; On market, use and become a kind of inevitable trend; Like Luo Shi Elecsys25-hydroxycholecalciferol, detect and used anti-VitD3 polyclonal antibody, whether diagnose into the interior vitamin V itD3 of human body sufficient; Combine with other clinical data, judge the bone metabolism situation as supplementary means.
A situation arises yet present detection method all only is applied to estimate individual treating bone diseases clinically, still hope to study 25-(OH) VitD detection method in order to prediction gland cancer occurrence risk.Sensitive based on immunology, special fast, characteristics such as easy, but the immunological method of a kind of detection by quantitative 25-(OH) VitD3 is set up in this laboratory.25-(OH) VitD3 is a kind of fat-soluble micromolecular compound, and is simple in structure, belongs to haptens, do not possess immunogenicity, need be connected on the macro-molecular protein carrier, could prepare artificial comlete antigen.So this prepared in laboratory goes out 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA antigen; Prepare polyclonal antibody with it; Just carrying out MONOCLONAL ANTIBODIES SPECIFIC FOR at present; Expectation is following to be assembled into 25-(OH) the VitD3 detection kit that can supply clinical practice, in order to the occurrence risk property of estimation gland cancer, makes it become the first road threshold that detects the gland cancer patient.
At present, increasing research shows that 25-hydroxyvitamin D3 can be used as an index of gland cancer, and still, at present, the feasible commercial prod that is applied to the gland cancer detection that 25-hydroxy-vitamin D is detected of a cover still relatively lacks.The process of most of technology carrier albumen and the coupling of 25-hydroxyl hemisuccinic acid ester has been used water, compares organic phase, and the efficient of coupling is not high, complicated operating process, and the immune effect of antigen is relatively poor.Context of detection at vitamin D also has commercial product; For example Luo Shi 25-hydroxyvitamin D3 electrochemiluminescence immunoassays " ECLIA "; Britain idas25-hydroxy-vitamine D detection kit (ELISA); Kit is used for the assessment of vitamin D deficiency situation, and does not carry out the explanation about the diagnostic research of gland cancer aspect as yet.
Summary of the invention
The object of the present invention is to provide a kind of blood 25-hydroxyvitamin D3 gland cancer detection kit and preparation method thereof.
Said blood 25-hydroxyvitamin D3 gland cancer detection kit comprises:
The calibration object of variable concentrations (calibration object is designated as CAL): the human serum that contains 25-hydroxyvitamin D3.Said 25-hydroxyvitamin D3 concentration can adopt 0nmol/L, 6nmol/L, 18nmol/L, 30nmol/L, 50nmol/L, 180nmol/L, 330nmol/L.
Be coated with the ELISA Plate (ELISA Plate of antibody is designated as AbPLAT) of the antibody of 25-hydroxyvitamin D3: the antibody of anti--25 hydroxycholecalciferols of hole endoperidium, said ELISA Plate can adopt 12 * 8, is packaged in the Fresco Bag of band drying agent.
Indicate the 25-hydroxyvitamin D3 (25-hydroxyvitamin D3 that indicates horseradish peroxidase is designated as VD3+HRP) of horseradish peroxidase.
Quality-control product (quality-control product is designated as CTRL): contain the human serum dried frozen aquatic products of 25-hydroxyvitamin D3 and sodium azide, the percentage composition of said sodium azide is 0.07%～0.09%.
Substrate colour developing liquid: comprise substrate A (substrate A is designated as SUBSA) and substrate B (substrate B is designated as SUBSB), said substrate A is tetramethyl benzidine and sodium acetate mixed solution, and said substrate B is the WS of hydrogen peroxide.
Reaction terminating liquid (reaction terminating liquid is designated as STOP): the hydrochloric acid that adopts 0.35～0.5M.
Washing fluid (washing fluid is designated as WASHBUF): adopt the phosphate buffer that contains Tween-20, the percentage composition of said Tween-20 is 0.1%.
The preparation method of said blood 25-hydroxyvitamin D3 gland cancer detection kit may further comprise the steps:
1) calibration object (calibration object is designated as CAL) of preparation variable concentrations: contain the human serum of 25-hydroxyvitamin D3,25-hydroxyvitamin D3 concentration is respectively, 0nmol/L, 6nmol/L, 18nmol/L, 30nmol/L, 50nmol/L, 180nmol/L, 330nmol/L.
2) preparation is coated with the ELISA Plate of the antibody of 25-hydroxyvitamin D3, and concrete grammar is following:
(1) using the carbonate of 0.05M pH9.0 to encapsulate damping fluid, will to resist antibody dilution to the content of-25 hydroxycholecalciferols be the mixed liquor of 1～10 μ g/ml.
(2) in the reacting hole of each 96 hole ELISA Plate, add the antibody mixed liquor that 0.1ml resists-25 hydroxycholecalciferols, 4 ℃ are spent the night.
(3) discard solution in the hole next day, with water-soluble the washing 3 times of phosphate-buffered of containing Tween-20, and each 3min.
3) preparation indicates the 25-hydroxyvitamin D3 of horseradish peroxidase, and concrete grammar is following:
(1) takes by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, add the 0.2ml 0.1M sodium metaperiodate WS again, get mixed solution;
(2) mixed solution is packed in the bag filter; Sodium-acetate buffer dialysis to 1mM pH4.4; 4 ℃ are spent the night, and take out aldehyde radical HRPO solution, add 20 μ l 0.2M pH9.5 carbonate aqueous buffer solutions again; Regulate pH value to 9.0～9.5, add 10mg 25-hydroxyvitamin D3 hemisuccinic acid ester then in 1ml 0.01M carbonate aqueous buffer solution;
(3) add 0.1ml 4mg/ml sodium borohydride aqueous solution, mixing is put in the bag filter of packing into behind 4 ℃ of 2h again; To 0.15MpH7.4 phosphate-buffered WS dialysis, 4 ℃ spend the night after, add the equal-volume saturated ammonium sulfate; Put 4 ℃ of 1h, the centrifugal 0.5h of 3000rpm abandons supernatant again; Sediment is washed with the semi-saturation ammonium sulfate solution, and last sediment is dissolved in the phosphate-buffered WS of 2.5ml 0.15M pH7.4;
(4) solution that step (3) is obtained is packed in the bag filter, with phosphate buffer (0.15M, pH7.4) dialysis; After removing ammonium ion, 10, the centrifugal 30min of 000rpm removes deposition; Supernatant is the 25-hydroxyvitamin D3 that indicates horseradish peroxidase, after the packing, and stored frozen;
4) preparation quality-control product (quality-control product is designated as CTRL): 25-hydroxyvitamin D3 is dissolved into human serum, after the freeze drying, weighs.For ease of standing storage, add sodium azide according to 0.07%～0.09% percentage composition;
5) preparation substrate A: take by weighing TMB 17.2mg (solid), add DMSO 1ml dissolving, add sodium-acetate buffer (0.1M, pH5.5) 66ml then;
6) preparation substrate B: get the hydrogen peroxide urea 0.64ml of monohydrate potassium 2.1g, ADSP 2.82g and 0.75%, be settled to 100ml with distilled water, pH4.5～5.0;
7) preparation reaction terminating liquid (reaction terminating liquid is designated as STOP): the hydrochloric acid WS of getting 0.5M.
8) preparation washing fluid (washing fluid is designated as WASHBUF): get the phosphate buffer that contains Tween-20, the percentage composition of said Tween-20 is 0.1%.
Beneficial effect of the present invention is following:
The present invention adopts a series of chemical modification, and 25-hydroxyvitamin D3 is modified to 25-hydroxyvitamin D3 hemisuccinic acid fat the most at last.Through improvement carbodlimide method and protein carrier BSA albumen coupling, finally become comlete antigen by haptens.And the method for desmoenzyme linked immunosorbent assay is developed blood 25-hydroxyvitamin D3 gland cancer detection kit.Practical application of the present invention is to be used for the detection of cancer clinically, and the hematology of cancer detects and remain technical barrier so far, and commercialization blood testing Products Development also is extremely important.
Description of drawings
Fig. 1 is that the structure of blood 25-hydroxyvitamin D3 gland cancer detection kit embodiment is formed synoptic diagram.
Fig. 2 is the MALDI-TOF mass spectrogram of the BSA of not coupling 25-hydroxy-vitamin D.
Fig. 3 is the MALDI-TOF mass spectrogram of 25-hydroxy-vitamin D coupling BSA in organic phase.
Fig. 4 is the MALDI-TOF mass spectrogram of 25-hydroxy-vitamin D at aqueous phase coupling BSA.
In Fig. 2～4, horizontal ordinate is specific charge m/z, and ordinate is intensity I ntensity.
Embodiment
Following examples will combine accompanying drawing that the present invention is further described.
Referring to Fig. 1, said blood 25-hydroxyvitamin D3 gland cancer detection kit embodiment comprises:
1, the calibration object of variable concentrations (calibration object is designated as CAL): contain the human serum of 25-hydroxyvitamin D3,25-hydroxyvitamin D3 concentration is respectively 0nmol/L, 6nmol/L, 18nmol/L; 30nmol/L, 50nmol/L, 180nmol/L, 330nmol/L; Be designated as CAL-1 respectively, CAL-2, CAL-3, CAL-4; CAL-5, CAL-6, CAL-7.
2, be coated with the ELISA Plate (ELISA Plate of antibody is designated as AbPLAT) of the antibody of 25-hydroxyvitamin D3: the antibody of anti--25 hydroxycholecalciferols of hole endoperidium, 12 * 8 of ELISA Plates are packaged in the Fresco Bag of band drying agent.
3, indicate the 25-hydroxyvitamin D3 (25-hydroxyvitamin D3 that indicates horseradish peroxidase is designated as VD3+HRP) of horseradish peroxidase.
4, quality-control product (quality-control product is designated as CTRL, is abbreviated as C): contain the human serum dried frozen aquatic products of 25-hydroxyvitamin D3 and sodium azide, the percentage composition of said sodium azide is 0.07%～0.09%.
5, substrate colour developing liquid: comprise substrate A (substrate A is designated as SUBSA) and substrate B (substrate B is designated as SUBSB), said substrate A is tetramethyl benzidine and sodium acetate mixed solution, and said substrate B is the WS of hydrogen peroxide.
6, reaction terminating liquid (reaction terminating liquid is designated as STOP): the hydrochloric acid of 0.35～0.5M.
7, washing fluid (washing fluid is designated as WASHBUF): contain the phosphate buffer of Tween-20, the percentage composition of said Tween-20 is 0.1%.
The calibration object of said variable concentrations, the ELISA Plate that is coated with the antibody of 25-hydroxyvitamin D3, the 25-hydroxyvitamin D3 that indicates horseradish peroxidase, quality-control product, substrate colour developing liquid, reaction terminating liquid and washing fluid are located in the box body H.
Below provide the preparation method of blood 25-hydroxyvitamin D3 gland cancer detection kit:
1) calibration object (calibration object is designated as CAL) of preparation variable concentrations: contain the human serum of 25-hydroxyvitamin D3,25-hydroxyvitamin D3 concentration is respectively, 0nmol/L, 6nmol/L, 18nmol/L, 30nmol/L, 50nmol/L, 180nmol/L, 330nmol/L.
2) preparation is coated with the ELISA Plate of the antibody of 25-hydroxyvitamin D3, and concrete grammar is following:
(1) using the carbonate of 0.05M pH9.0 to encapsulate damping fluid, will to resist antibody dilution to the content of-25 hydroxycholecalciferols be the mixed liquor of 1～10 μ g/ml.
(2) in the reacting hole of each 96 hole ELISA Plate, add the antibody mixed liquor that 0.1ml resists-25 hydroxycholecalciferols, 4 ℃ are spent the night.
(3) discard solution in the hole next day, with water-soluble the washing 3 times of phosphate-buffered of containing Tween-20, and each 3min.
3) preparation indicates the 25-hydroxyvitamin D3 of horseradish peroxidase, and concrete grammar is following:
(1) takes by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, add the 0.2ml 0.1M sodium metaperiodate WS again, get mixed solution;
(2) mixed solution is packed in the bag filter; Sodium-acetate buffer dialysis to 1mM pH4.4; 4 ℃ are spent the night, and take out aldehyde radical HRPO solution, add 20 μ l 0.2M pH9.5 carbonate aqueous buffer solutions again; Regulate pH value to 9.0～9.5, add 10mg 25-hydroxyvitamin D3 hemisuccinic acid ester then in 1ml 0.01M carbonate aqueous buffer solution;
(3) add 0.1ml 4mg/ml sodium borohydride aqueous solution, mixing is put in the bag filter of packing into behind 4 ℃ of 2h again; To 0.15MpH7.4 phosphate-buffered WS dialysis, 4 ℃ spend the night after, add the equal-volume saturated ammonium sulfate; Put 4 ℃ of 1h, the centrifugal 0.5h of 3000rpm abandons supernatant again; Sediment is washed with the semi-saturation ammonium sulfate solution, and last sediment is dissolved in the phosphate-buffered WS of 2.5ml 0.15M pH7.4;
(4) solution that step (3) is obtained is packed in the bag filter, with phosphate buffer (0.15M, pH7.4) dialysis; After removing ammonium ion, 10, the centrifugal 30min of 000rpm removes deposition; Supernatant is the 25-hydroxyvitamin D3 that indicates horseradish peroxidase, after the packing, and stored frozen;
4) preparation quality-control product (quality-control product is designated as CTRL): 25-hydroxyvitamin D3 is dissolved into human serum, after the freeze drying, weighs.For ease of standing storage, add sodium azide according to 0.07%～0.09% percentage composition;
5) preparation substrate A: take by weighing TMB 17.2mg (solid), add DMSO 1ml dissolving, add sodium-acetate buffer (0.1M, ph5.5) 66ml then;
6) preparation substrate B: get the hydrogen peroxide urea 0.64ml of monohydrate potassium 2.1g, ADSP 2.82g and 0.75%, be settled to 100ml with distilled water, pH4.5～5.0;
7) preparation reaction terminating liquid (reaction terminating liquid is designated as STOP): the hydrochloric acid WS of getting 0.5M.
8) preparation washing fluid (washing fluid is designated as WASHBUF): get the phosphate buffer that contains Tween-20, the percentage composition of said Tween-20 is 0.1%.
Embodiment 1
25-hydroxyvitamin D3-3-hemisuccinic acid ester-BSA's is synthetic
In this synthetic method, 3 of chemical activation 25-hydroxyvitamin D3, and with BSA albumen coupling as carrier.This is synthetic through 25-hydroxyvitamin D3-hemisuccinic acid ester and 25-hydroxyl-cholecalciferol-intermediate steps such as 3-half succinimide ester.The 25-hydroxyvitamin D3 structural formula is following:
1.125-the preparation of hydroxycholecalciferol-3-hemisuccinic acid ester
Get 10mg (25umol) 25-hydroxyvitamin D3 and be dissolved in the 1ml anhydrous pyridine, the succinic anhydride of itself and 125mg (1.25mmol) was at room temperature stirred 4 days in the darkroom.After the ethyl acetate dilution of reaction mixture with 10ml, use the water of 2x10ml respectively, 0.1M salt acid elution is used water washing subsequently once more.
Use the anhydrous sodium sulfate drying organic phase, filter and solvent removed in vacuo.Use the high vacuum dry remaining solid.Obtain 10.5mg (productive rate: colorless solid 84%).
1.225-the preparation of hydroxycholecalciferol-3-hemisuccinic acid ester-N-hydroxy-succinamide
Get 10.0mg (20umol) 25-hydroxyvitamin D3-3-hemisuccinic acid ester and be dissolved in the anhydrous methylene chloride of 7ml, and be mixed together with the N-hydroxy-succinamide of 2.8mg and the N of 3.7mg (3-dimethylaminopropyl)-N '-ethyl-carbodiimide (EDC).Stirred overnight under argon gas is with the water washing organic phase twice of 10ml, with about 1g anhydrous sodium sulfate drying and filtration.Use solvent removed in vacuo, remaining reaction product is dry 3h in high vacuum, obtains the N-hydroxy-succinamide ester that need not about 10mg further purified and can be used for puting together.
1.325-the synthesizing of hydroxycholecalciferol-3-hemisuccinic acid ester-BSA at aqueous phase
66ul is equipped with in 25-hydroxyvitamin D3-hemisuccinic acid ester of 1.0mg, the EDC of 0.5mg and N-hydroxy-succinamide (NHS) adding of 0.5mg, and 0.01Mm/L in the round-bottomed flask of phosphate buffer, stirs 1h under room temperature.The ester that adds the DMSO dissolving NHS connection of 200ul.(be dissolved in phosphate buffer (0.01mol/L, pH7.2) in) solution 1mL, stirring reaction spends the night under room temperature to add 0.25mg/mL BSA then.Phosphate buffer (the 0.01mol/L that reactant liquor is used; PH7.2) 4 ℃ of dialysis 48h, liquid is changed 6 times in the centre, and every separated 8h changes liquid 1 time; Collect dialysate; Cooperate sephadex HiTrapTM Desalting to cross the post desalting and purifying 2 times with GE AKTA protein purification appearance, promptly make artificial comlete antigen 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA, subsequent use in-20 ℃ of preservations.
1.425-the synthesizing in organic phase of hydroxycholecalciferol-3-hemisuccinic acid ester-BSA
25-hydroxyvitamin D3-hemisuccinic acid ester of 1.0mg, the EDC of 0.5mg and the NHS adding of 0.5mg are equipped with in the round-bottomed flask of 66ul absolute ethyl alcohol, under room temperature, stir 1h, rotary evaporation solvent (10-15min).The ester that adds the DMSO dissolving NHS connection of 200ul.Add 0.25mg/mL BSA (being dissolved among the PBS of pH 7.2 of 0.01mol/L) solution 1mL then, stirring reaction spends the night under room temperature.Reactant liquor is with 4 ℃ of dialysis of PBS 48h of 0.01mol/L pH 7.2; Liquid is changed 6 times in the centre; Every separated 8h changes liquid 1 time, collects dialysate, cooperates sephadex HiTrapTMDesalting to cross the post desalting and purifying 2 times with GE akata purifying appearance; Promptly make artificial comlete antigen 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA, subsequent use in-20 ℃ of preservations.
1.5 the mensuration of synthetic 25-hydroxyvitamin D3-3-hemisuccinic acid ester-BSA coupling rate
Use the MALDI-TOF-MS mass spectrometer to carrying out the mass spectrum checking at the synthetic 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA of aqueous phase, 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA and pure BSA synthetic in organic phase.In the organic phase; The Mr of BSA is 66255.413 (the mass spectrum result sees Fig. 2); The Mr of 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA is 71125.177 (the mass spectrum result sees Fig. 3); Mr after the dehydration of 25-hydroxyvitamin D3-hemisuccinic acid ester is 500.34-17=483.34, and the result shows, coupling has 10 [(71125.177-66255.413)/483.34=10] 25-hydroxyvitamin D3s-hemisuccinic acid ester micromolecule on each carrier protein BSA molecule.Aqueous phase; The Mr of BSA is 66255.41; The Mr of 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA is 66903.266 (the mass spectrum result sees Fig. 4), and coupling has 2 [(66903.266-66255.413)/483.34=2] 25-hydroxyvitamin D3s-hemisuccinic acid ester micromolecule on each carrier protein BSA molecule.
Embodiment 2
2.1 immunity produces the antibody of anti-25-hydroxy-vitamin D
With 4 of the female BALB/C mices in 6 ages in week of 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA immunity; First immunisation is with aseptic PBS solution dissolving 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA and mix fully emulsified with the Freund's complete adjuvant (FCA) of equivalent; Immunizing dose is 50 μ g (0.3mL)/only, the subcutaneous branch 4 in the abdominal cavity, back～6 injections; First immunisation is mixed fully emulsified immune mouse with aseptic PBS solution dissolving 25-hydroxyvitamin D3-hemisuccinic acid ester-BSA and with the Freund (FIA) of equivalent after 3 weeks equally, and immunizing dose is that 30 μ g (0.3mL)/only, the injection site is the same.Altogether immunity is 5 times, each 2 weeks at interval, last 1 time through the intravenous injection booster immunization, docking blood sampling in the 3rd day also prepares and contains antibody serum.To contain antibody serum and pack in the bag filter, put 4 ℃ of refrigerators, the Tris-Cl WS (0.01mol/L, pH8.6) dialysed overnight.
2.2DEAE-cellulose extraction method antibody purification
The DE52 cellulose is through acid, alkali treatment, in the Tris-Cl WS (0.01mol/L, pH8.6) balance.Chromatographic column is fixed on the titration stand, post heelpiece one circular nylon yarn, outlet connects a thin plastic pipe and closes water outlet.To be soaked in Tris-Cl (0.01mol/L, the DE52 in pH8.6) pours in the post along glass bar, when treating that DE52 natural subsidence 3～5cm is high; Absorb 0.01mol/L, pH8.6Tris-Cl, or unclamp the water delivering orifice screw clamp; Control flow velocity 1～2ml/min adds DE52 simultaneously continuously to desired height.After treating the complete sedimentation of DE52, cylinder is put a circular filter paper sheet.Unclamp the water delivering orifice screw clamp, (the control flow velocity is 15/min, when treating that effluent reaches consistent with the pH value of eluent, stops balance for 0.01mol/L, pH8.6) balance with the Tris-Cl WS.Remove cylinder liquid with the suction pipe suction, add sample with capillary syring along post jamb, sample volume is equivalent to 1%～2% of bed volume, and protein concentration is 50～70mg.Breakdown water delivering orifice screw clamp slowly gets in the post bed sample, and with a small amount of eluant stripper column wall; After treating that liquid gets into the post bed, (the control flow velocity is 14～16/min for 0.01mol/L, pH8.6) wash-out with the Tris-Cl WS.Fraction collector is collected, and ultraviolet monitoring is measured every pipe 280nmOD value respectively with ultraviolet spectrophotometer, describes the eluent peak.Each pipe of crest segment on the 280nm OD of eluent and following crest segment is merged respectively, and the bag filter of packing into is concentrated into volume required with polyglycol.Antibody purified is saved in 20 ℃.
Embodiment 3
The detecting operation step of blood 25-hydroxyvitamin D3 gland cancer detection kit (enzyme-linked immunosorbent assay method)
3.1 prepare the glass tube marked, the corresponding pipe of each calibration object CAL, quality-control product CTRL and sample.
3.2 calibration object CAL, quality-control product CTRL and sample are respectively got the test tube that 10ul joins respective markers respectively.
3.3 each test tube adds the horseradish peroxidase solution VD3+HRP of the 25-hydroxyvitamin D3 of 0.4ml.Place on the vortex oscillation device and mix 15～30s.
3.4 with add calibration object, quality-control product or the sample that 200ul has diluted in the corresponding ELISA Plate AbPLAT micropore.Stick preservative film, under 18-25c, hatch 2h.
3.5 wash plate 3 times with dcq buffer liquid WASHBUF, manual and automatic washer all can.Attention: before getting into next procedure, on thieving paper, firmly pat inverted plate to remove unnecessary washing fluid.
3.6 every hole adds the substrate A (SUBSA) of 200ul and the equal-volume mixed solution of substrate B (SUBSB).Stick preservative film and under 37 ℃, hatch 30min.
3.7 every hole adds 150ul stop buffer hydrochloric acid.
Read absorptance 3.8 add inherent 450nm (with reference to the 650nm) wavelength of the 30min of stop buffer.
Embodiment 4
The checking of kit effect and accuracy
With 25-OH, the VD3 detection kit contrasts (Luo Shi diagnosis Elecsys and cobas e analyser) with the radioimmunoassay of the detection 25-hydroxy-vitamin D that obtains to generally acknowledge and other hydroxylation products.Select 80 samples of the big 25-hydroxy-vitamin D scope [9.3-151.2nmol/L] of representative to detect through every kind of method.Table 1 is seen in the testing result statistical study.Through comparing in the sensing range of big 25-hydroxyvitamin D3, the statistical indicator of each item as a result of the radioimmunoassay of the present invention and Luo Shi hydroxylation product is approaching.
Table 1 new invention kit detects the comparison of effect and Luo Shi testing result
Embodiment 5
Be directed to the formulation of the term of reference of gland cancer detection
5.125-the influence factor of hydroxycholecalciferol contents level
The optimum range that detects with regard to 25-hydroxyvitamin D3 is not at present reached common understanding yet.Its level receives influence of various factors: diet, and one day your different time section, Exposure to Sunlight, in season, sunblock lotion or protective clothing at the age, are used, the colour of skin in the geographic position.Therefore, be not the ideal path of setting up term of reference to healthy population sampling detection.
5.2 the formulation of the term of reference that gland cancer detects
Aspect osteoporosis, American National osteoporosis foundation suggestion guarantees that the level of the 25-hydroxyvitamin D3 of bone health should be greater than 75nmol/L; Then 25-hydroxy-vitamin D was not enough or lack when equally, the foundation of American National ephrosis was thought less than 75nmol/L.Clinically, many experts are defined as cholecalciferol when the 25-hydroxyvitamin D3 level is lower than 50nmol/L in the serum and lack, and when 52.5-72.5nmol/L, measure vitamin d insufficiency, think then that when 75nmol/L is above vitamin D is normal.Scope (the Imtiaz of the vitamin D that is directed to breast cancer detection that 25-hydroxyvitamin D3 term of reference (table 2) list of references is recommended in the gland cancer that the present invention formulates; S.; Et al.; Vitamin D deficiency in newly diagnosed breast cancer patients.Indian J Endocrinol Metab, 2012.16 (3): 409-413 :).
25-hydroxyvitamin D3 term of reference in table 2 gland cancer of the present invention
| Disease | Term of reference (nmol/L) |
| Breast cancer | ≤50 |
| Colon cancer | ≤50 |
| Sdenocarcinoma of stomach | ≤50 |
Embodiment 6
Use the 25-hydroxyvitamin D3 detection kit to detect the content of 25-hydroxyvitamin D3 in the gland cancer patient body
6.1 the use kit detects 80 breast cancer disease human blood samples according to operation and 80 non-gland cancer human blood samples carry out the detection of ELISA.(18.9～86.7nmol/L), non-breast cancer patient has represented bigger 25-hydroxyvitamin D3 scope (50.6～213.6nmol/L) to the bigger 25-hydroxyvitamin D3 scope of selected breast cancer sample representative.Table 3 is seen in the testing result statistical study.
Table 380 a breast cancer disease human blood sample and 80 non-gland cancer human blood sample 25-hydroxyvitamin D3 detection level statistical analysis tables
6.2 the use kit detects 80 colon cancer patients according to operation and 80 non-gland cancer human blood samples carry out the detection of ELISA.(13.9～236.7nmol/L), the normal person has represented bigger 25-hydroxyvitamin D3 scope (50.6～213.6nmol/L) to the big 25-hydroxyvitamin D3 scope of selected colon cancer sample representative.Table 4 is seen in the testing result statistical study.Table 480 a colon cancer patient blood sample and 80 non-gland cancer human blood sample 25-hydroxyvitamin D3 detection level statistical analysis tables
Carry out the detection of ELISA 6.3 use kit to detect 20 gastric glands, carninomatosis people and 80 non-gland cancer human blood samples according to operation.(23.27～60.9nmol/L), the normal person has represented bigger 25-hydroxyvitamin D3 scope (50.6～213.6nmol/L) to the big 25-hydroxyvitamin D3 scope of selected sdenocarcinoma of stomach sample representative.Table 5 is seen in the testing result statistical study.
Table 580 a colon cancer patient blood sample and 80 non-gland cancer human blood sample 25-hydroxyvitamin D3 detection level statistical analysis tables
6.4 the use kit detects 20 lung cancer patients according to operation and 80 non-gland cancer human blood samples carry out the detection of ELISA.(23.7～76.8nmol/L), the normal person has represented bigger 25-hydroxyvitamin D3 scope (50.6～213.6nmol/L) to the big 25-hydroxyvitamin D3 scope of selected lung cancer sample representative.Table 6 is seen in the testing result statistical study.
Table 680 a colon cancer patient blood sample and 80 non-gland cancer human blood sample 25-hydroxyvitamin D3 detection level statistical analysis tables
6.5 the use kit detects 320 gland cancer patient blood samples according to operation and 80 non-gland cancer human blood samples carry out the detection of ELISA.(18.9～86.7nmol/L), non-gland cancer people has represented bigger 25-hydroxyvitamin D3 scope (213.60～50.60nmol/L) to the big 25-hydroxyvitamin D3 scope of selected gland cancer patient's sample representative.
Table 780 a colon cancer patient blood sample and 80 non-gland cancer human blood sample 25-hydroxyvitamin D3 detection level statistical analysis tables
Testing result analysis in table 7.
Claims (4)
1. blood 25-hydroxyvitamin D3 gland cancer detection kit is characterized in that comprising:
The calibration object of variable concentrations: the human serum that contains 25-hydroxyvitamin D3;
Be coated with the ELISA Plate of the antibody of 25-hydroxyvitamin D3: the antibody of anti--25 hydroxycholecalciferols of hole endoperidium;
Indicate the 25-hydroxyvitamin D3 of horseradish peroxidase;
Quality-control product: contain the human serum dried frozen aquatic products of 25-hydroxyvitamin D3 and sodium azide, the percentage composition of said sodium azide is 0.07%～0.09%;
Substrate colour developing liquid: comprise substrate A and substrate B, said substrate A is tetramethyl benzidine and sodium acetate mixed solution, and said substrate B is the WS of hydrogen peroxide;
Reaction terminating liquid: the hydrochloric acid that adopts 0.35～0.5M;
Washing fluid: adopt the phosphate buffer that contains Tween-20, the percentage composition of said Tween-20 is 0.1%.
2. blood 25-hydroxyvitamin D3 gland cancer detection kit as claimed in claim 1 is characterized in that said 25-hydroxyvitamin D3 concentration adopts 0nmol/L, 6nmol/L, 18nmol/L, 30nmol/L, 50nmol/L, 180nmol/L, 330nmol/L.
3. blood 25-hydroxyvitamin D3 gland cancer detection kit as claimed in claim 1 is characterized in that said ELISA Plate adopts 12 * 8, is packaged in the Fresco Bag of band drying agent.
4. the preparation method of blood 25-hydroxyvitamin D3 gland cancer detection kit as claimed in claim 1 is characterized in that may further comprise the steps:
1) calibration object of preparation variable concentrations: contain the human serum of 25-hydroxyvitamin D3,25-hydroxyvitamin D3 concentration is respectively, 0nmol/L, 6nmol/L, 18nmol/L, 30nmol/L, 50nmol/L, 180nmol/L, 330nmol/L;
2) preparation is coated with the ELISA Plate of the antibody of 25-hydroxyvitamin D3, and concrete grammar is following:
(1) using the carbonate of 0.05M pH9.0 to encapsulate damping fluid, will to resist antibody dilution to the content of-25 hydroxycholecalciferols be the mixed liquor of 1～10 μ g/ml;
(2) in the reacting hole of each 96 hole ELISA Plate, add the antibody mixed liquor that 0.1ml resists-25 hydroxycholecalciferols, 4 ℃ are spent the night;
(3) discard solution in the hole next day, with water-soluble the washing 3 times of phosphate-buffered of containing Tween-20, and each 3min;
3) preparation indicates the 25-hydroxyvitamin D3 of horseradish peroxidase, and concrete grammar is following:
(1) takes by weighing the 5mg horseradish peroxidase and be dissolved in the 1ml distilled water, add the 0.2ml 0.1M sodium metaperiodate WS again, get mixed solution;
(2) mixed solution is packed in the bag filter; Sodium-acetate buffer dialysis to 1mM pH4.4; 4 ℃ are spent the night, and take out aldehyde radical HRPO solution, add 20 μ l 0.2M pH9.5 carbonate aqueous buffer solutions again; Regulate pH value to 9.0～9.5, add 10mg 25-hydroxyvitamin D3 hemisuccinic acid ester then in 1ml 0.01M carbonate aqueous buffer solution;
(3) add 0.1ml 4mg/ml sodium borohydride aqueous solution, mixing is put in the bag filter of packing into behind 4 ℃ of 2h again; To 0.15MpH7.4 phosphate-buffered WS dialysis, 4 ℃ spend the night after, add the equal-volume saturated ammonium sulfate; Put 4 ℃ of 1h, the centrifugal 0.5h of 3000rpm abandons supernatant again; Sediment is washed with the semi-saturation ammonium sulfate solution, and last sediment is dissolved in the phosphate-buffered WS of 2.5ml 0.15M pH7.4;
(4) solution that step (3) is obtained is packed in the bag filter, with the phosphate buffer dialysis, remove ammonium ion after; 10, the centrifugal 30min of 000rpm removes deposition, and supernatant is the 25-hydroxyvitamin D3 that indicates horseradish peroxidase; After the packing, stored frozen;
4) preparation quality-control product: 25-hydroxyvitamin D3 is dissolved into human serum, after the freeze drying, weighs.For ease of standing storage, add sodium azide according to 0.07%～0.09% percentage composition;
5) preparation substrate A: take by weighing TMB 17.2mg, add DMSO 1ml dissolving, add sodium-acetate buffer 66ml then;
6) preparation substrate B: get the hydrogen peroxide urea 0.64ml of monohydrate potassium 2.1g, ADSP 2.82g and 0.75%, be settled to 100ml with distilled water, pH4.5～5.0;
7) preparation reaction terminating liquid (reaction terminating liquid is designated as STOP): the hydrochloric acid WS of getting 0.5M;
8) preparation washing fluid: get the phosphate buffer that contains Tween-20, the percentage composition of said Tween-20 is 0.1%.
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