WO2020093989A1 - 一种以纳米硒作为吸附剂载体的dna免疫吸附剂及其制备方法和应用 - Google Patents

一种以纳米硒作为吸附剂载体的dna免疫吸附剂及其制备方法和应用 Download PDF

Info

Publication number
WO2020093989A1
WO2020093989A1 PCT/CN2019/115559 CN2019115559W WO2020093989A1 WO 2020093989 A1 WO2020093989 A1 WO 2020093989A1 CN 2019115559 W CN2019115559 W CN 2019115559W WO 2020093989 A1 WO2020093989 A1 WO 2020093989A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
selenium
solution
immunosorbent
nano
Prior art date
Application number
PCT/CN2019/115559
Other languages
English (en)
French (fr)
Inventor
陈填烽
林智明
Original Assignee
暨南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201811305882.7A external-priority patent/CN109317083B/zh
Priority claimed from CN201811305886.5A external-priority patent/CN109521193B/zh
Application filed by 暨南大学 filed Critical 暨南大学
Publication of WO2020093989A1 publication Critical patent/WO2020093989A1/zh

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the invention belongs to the technical field of biomedical materials. More specifically, it relates to a nano-drug loading system with glutathione responsiveness, its preparation method and application.
  • Systemic lupus erythematosus (systemic lupus erythematosus, SLE) is a chronic autoimmune disease with multiple system damage. The exact cause is unknown. It often causes irreversible damage to multiple organ systems, which can involve the skin, serous membrane, joints, kidneys and The central nervous system, etc., the pathological manifestation is the deposition of autoantibodies and immune complexes, which affects the patient's life span and quality of life.
  • SLE systemic lupus erythematosus
  • SLE systemic lupus erythematosus
  • Anti-dsDNA antibodies are the main antibodies involved in the pathogenesis of SLE, and their specificity can reach 90%. SLE specific antibody.
  • Immunoadsorption therapy is a new type of blood purification technology developed in recent years. It uses adsorbent materials to remove pathogenic antibodies (such as anti-dsDNA antibodies) in plasma to achieve the purpose of treating epidemics. Immunoadsorption is the removal of pathogenic factors through antigen-antibody immunoreaction or physicochemical action, mainly plasma adsorption, thereby avoiding the disadvantages of centrifugal and primary membrane plasma separation and discarding large amounts of plasma. In recent years, immunoadsorption has been increasingly used in the treatment of immune diseases.
  • DNA immunoadsorption therapy can specifically adsorb and remove anti-dsDNA antibodies, thereby effectively treating patients with SLE.
  • European Patent EP0272792A1 has prepared a DNA immunosorbent based on activated carbon or carbonized resin. They use activated carbon or carbonized resin as a carrier and calf thymus DNA (hereinafter referred to as DNA) as a ligand. DNA.
  • Anti-dsDNA antibodies can assist in the diagnosis of systemic lupus erythematosus (SLE) and the classification of lupus kidney.
  • Antinuclear antibodies ANA
  • ANA Antinuclear antibodies
  • screening of antinuclear antibodies is a sensitive method for detecting connective tissue diseases.
  • this screening method is a very good detection method for SLE (negative results can exclude active SLE), this method is not a specific detection method.
  • anti-dsDNA antibodies can almost rule out the possibility of other diseases, so the antibody is considered to be a sign of the disease.
  • a positive anti-dsDNA antibody is sufficient to prove SLE; however, a negative test for these antibodies does not completely exclude SLE in all cases.
  • H & E staining is the most commonly used method for clinical detection of kidney tissue lesions and typing. Immunofluorescence is also used to detect multiple antibody deposits in kidney tissue.
  • H & E staining and general immunofluorescence cannot specifically confirm the deposition and distribution of anti-dsDNA antibodies in kidney tissue. Therefore, it is very important to provide a simple and fast detection method that can diagnose kidney disease and analyze anti-dsDNA antibodies in kidney slices for the diagnosis and treatment of lupus nephritis and lupus erythematosus.
  • the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a DNA immunoadsorbent that uses nano-selenium as an adsorbent carrier. And its preparation method and application
  • the second object of the present invention is to provide a method for preparing the DNA immunosorbent.
  • the third object of the present invention is to provide the application of the DNA immunosorbent in the preparation of blood purification preparations.
  • the fourth object of the present invention is to provide the application of the above-mentioned DNA immunosorbent in the preparation of a medicament for preventing, treating or controlling diseases caused by an increase in anti-dsDNA antibodies.
  • the fifth object of the present invention is to provide an anti-dsDNA antibody detection reagent for fluorescent immunostaining containing the DNA immunosorbent.
  • the sixth object of the present invention is to provide an anti-dsDNA antibody detection reagent for fluorescent immunostaining and a preparation method thereof.
  • a seventh object of the present invention is to provide a method for detecting kidney slices for non-diagnostic purposes using the above-mentioned anti-dsDNA antibody detection reagent.
  • a DNA immunosorbent includes nano-selenium as a carrier for the adsorbent and DNA immobilized on the nano-selenium.
  • the inventor of the present invention has found that nano-selenium is used as a DNA immunosorbent obtained by loading DNA, which has the advantages of good adsorption performance and better safety performance.
  • the amount of the DNA and nano-selenium is calculated according to the ratio of 0.1-5 mg: 1 ⁇ mol; more preferably 0.1-2 mg: 1 ⁇ mol.
  • the DNA is double-stranded DNA combined with an anti-dsDNA antibody; the DNA immunosorbent is DNA-SeNPs.
  • the invention also relates to a method for preparing the DNA immunosorbent: the adsorbent carrier nano-selenium solution is mixed with a DNA buffer solution to perform a DNA grafting reaction, and after the reaction is completed, the resulting product is purified to obtain nano-selenium as an adsorbent Carrier DNA immunosorbent.
  • the temperature of the reaction is 0-10 ° C; more preferably 3-5 ° C; most preferably 4 ° C.
  • the reaction time is 0.5-24 hours.
  • the concentration of the nano-selenium solution is 1-10 mM; more preferably 2-5 mM.
  • the concentration of the DNA solution is 1-10 mg / mL; more preferably 1-5 mg / mL.
  • the pH of the buffer is 7-9; more preferably 7.2-8.2.
  • the buffer is preferably Tris-HCl buffer.
  • the concentration of the Tris-HCl buffer solution is preferably 0.01 to 0.1M; more preferably 0.01 to 0.05M.
  • the purification method is dialysis using a dialysis bag.
  • the specification of the dialysis bag is preferably 6000-8000 kDa; the dialysis time is preferably 12-48h.
  • the nano-selenium is mixed with the inorganic selenium solution and the reducing agent solution in a molar ratio of 1: 3 to 10, and is at 0 to 10 ° C (preferably 3 to 5 ° C; more preferably 4 ° C ) Under the conditions of the reaction.
  • the amount of the inorganic selenium and the reducing agent is preferably according to the excess of the reducing agent, so that the inorganic selenium can be fully reduced.
  • the inorganic selenium is selected from one or at least two of selenite, selenite or selenium dioxide.
  • the reducing agent is selected from one or at least two of vitamin C, sodium borohydride, mercaptoethanol, reduced glutathione, glucose, or sodium thiosulfate pentahydrate.
  • the mixing method is preferably: mixing the inorganic selenium solution and the reducing agent solution; more preferably by mixing the inorganic selenium solution into the reducing agent solution, or the reducing agent solution into the inorganic selenium solution .
  • the concentration of the inorganic selenium solution is preferably 5 to 15 mM; more preferably 10 mM.
  • the concentration of the reducing agent solution is preferably 30-50 mM; more preferably 40 mM.
  • DNA immunosorbent in the preparation of blood purification preparations and the preparation of drugs for preventing, treating or controlling diseases caused by the increase of anti-dsDNA antibodies are also within the scope of the present invention.
  • the disease caused by the increase in anti-dsDNA antibody is systemic lupus erythematosus.
  • the invention also relates to an anti-dsDNA antibody detection reagent for fluorescent immunostaining, which is obtained by labeling a fluorescent display agent on the DNA immunosorbent.
  • the inventors of the present invention found that the use of DNA immunosorbent for immunofluorescence can qualitatively analyze anti-dsDNA antibodies deposited in kidney tissue, specifically confirm the deposition and distribution of anti-dsDNA antibodies in kidney tissue, and the method is simple and easy to operate.
  • the invention also relates to the preparation method of the above anti-dsDNA antibody detection reagent, which is prepared by the following steps:
  • the inorganic selenium described in step (1) is preferably one or at least two of selenite, selenite and selenium dioxide.
  • the selenite is one or at least two of sodium selenite (Na 2 SeO 3 ), potassium selenite (K 2 SeO 3 ) and zinc selenite (ZnSeO 3 ).
  • the concentration of the inorganic selenium solution described in step (1) is preferably 5-15 mM; more preferably 10 mM.
  • the reducing agent in step (2) is preferably one or at least two of vitamin C, sodium borohydride, mercaptoethanol, reduced glutathione, glucose, and sodium thiosulfate pentahydrate.
  • the solution concentration of the reducing agent in step (2) is preferably 30-50 mM; more preferably 40 mM.
  • the amount of the reducing agent and the inorganic selenium in step (2) is appropriate according to the excess of the reducing agent, so that the inorganic selenium can be fully reduced; more preferably, the molar ratio is calculated as 3 to 10: 1.
  • the DNA used in step (3) for binding to the anti-dsDNA antibody is preferably double-stranded DNA.
  • the concentration of the DNA solution for binding to the anti-dsDNA antibody described in step (3) is preferably 1 to 10 mg / mL; more preferably 1 to 5 mg / mL.
  • the solvent in the DNA solution for binding to the anti-dsDNA antibody described in step (3) is preferably a buffer solution with a pH value of 7 to 9; more preferably a buffer solution with a pH value of 7.2 to 8.2.
  • the buffer is preferably Tris-HCl buffer.
  • the concentration of the Tris-HCl buffer solution is preferably 0.01 to 0.1M; more preferably 0.01 to 0.05M.
  • the temperature of the reaction described in step (3) is preferably 0 to 10 ° C; more preferably 3 to 5 ° C; most preferably 4 ° C.
  • the purification method in step (3) is dialysis using a dialysis bag; the specification of the dialysis bag is preferably 6000-8000 kDa; and the dialysis time is preferably 12-48h.
  • the invention also relates to a method for detecting a kidney slice for non-diagnostic purposes using the above-mentioned anti-dsDNA antibody detection reagent, which includes the following steps:
  • Antigen repair Take 12mL of 50 ⁇ EDTA antigen repair solution, dilute to 600mL with ultrapure water, preheat at 95 ⁇ 110 °C for 1min, put in slices, heat at 95 ⁇ 100 °C for 1min, and heat at 60 ⁇ 70 °C for 2min (4 ⁇ 5 times), cooling, PBS buffer; washing;
  • Blocking remove excess liquid, add bovine serum protein solution with a concentration of 5% by mass, and block for 30 minutes;
  • the present invention has the following beneficial effects:
  • the carrier nano selenium (SeNPs) in the DNA immunosorbent provided by the present invention is a beneficial substance that can supplement the human selenium element, remove excess active oxygen free radicals in the body, and has high safety. Therefore, the immunosorbent of the present invention does not require pre-flushing, saves resources, and improves the safety of the use of the immunosorbent.
  • the present invention uses SeNPs as a carrier to modify DNA on its surface, and the prepared nano-selenium-loaded DNA immunosorbent DNA solid loading amount reaches 928 ⁇ g / mL, and the adsorption performance is good.
  • the DNA molecule is fully contacted with the anti-dsDNA antibody to maximize its specific binding ability.
  • the carrier SeNPs in the DNA immunosorbent provided by the present invention also has a certain clearance effect against anti-dsDNA antibodies.
  • DNA immunosorbent is used to detect anti-dsDNA antibodies in kidney slices.
  • DNA-SeNPs can specifically bind to anti-dsDNA antibodies in kidney slices, so it can qualitatively analyze the types of antibodies deposited in kidney tissues. At the same time, the deposition and distribution of anti-dsDNA antibodies in kidney tissue can be confirmed. Therefore, DNA immunosorbent can be used to prepare reagents for the detection and analysis of lupus kidney and lupus erythematosus.
  • the present invention can specifically display the deposition and distribution of kidney tissue anti-dsDNA antibodies, and is simpler and faster.
  • Figure 1 is a characterization diagram of DNA-SeNPs before and after adsorption of anti-dsDNA antibodies in patient plasma; wherein, Figure a is the distribution of nanoparticle size before and after adsorption, Figure b is the potential diagram before and after adsorption, 1 to 4 in Figures a and b DNA-SeNPs after adsorption of plasma of cases No. 61, No. 28, No. 70 and No. 23 respectively; Figure c is the ultraviolet spectrum before and after adsorption, curve 1 is DNA-SeNPs, and curves 2 to 4 are adsorption of No. 19 and 69 respectively DNA-SeNPs after the plasma of cases No. 109 and No. 109; Figure d is the infrared spectrum before and after adsorption, curve 1 is DNA-SeNPs, and curves 2 to 4 are the DNA after adsorption of the plasma of cases 14, 30 and 34 respectively SeNPs.
  • Figure 2 is a transmission electron microscope image of DNA-SeNPs before and after adsorption of anti-dsDNA antibodies in patient plasma; among them, Figure a is a TEM image of DNA-SeNPs, Figure b is a TEM of DNA-SeNPs after adsorption of the plasma of patient No. 17 It is a TEM image of DNA-SeNPs after adsorbing the plasma of case No. 31, and figure d is a TEM image of DNA-SeNPs after adsorbing the plasma of case No. 58.
  • Figure 3 is an atomic force microscope and corresponding thickness analysis diagrams of DNA-SeNPs before and after adsorption of anti-dsDNA antibodies in patient plasma; wherein, Figure a is an AFM diagram (top) and corresponding thickness analysis diagram (bottom) of DNA-SeNPs, Figure b It is the AFM diagram (top) and corresponding thickness analysis diagram (bottom) of DNA-SeNPs after adsorption of the plasma of case No. 32, and Figure c is the AFM diagram (top) and corresponding thickness of DNA-SeNPs after adsorption of the plasma of case 33. Analysis chart (below).
  • Fig. 4 is a graph showing the results of DNA-SeNPs adsorbing anti-dsDNA antibodies, in which figure a is the statistical graph of DNA-SeNPs clearance of anti-dsDNA antibodies in plasma of 128 cases; figure b is DNA-SeNPs against 128 cases The statistical graph of anti-dsDNA antibody titer in plasma after anti-dsDNA antibody clearance in plasma; Figure c is the removal of anti-dsDNA antibody in plasma of three patients with DNA-SeNPs, commercially available product 1 and commercially available product 2 immunosorbent products Rate change chart; Figure d is a graph showing the change of anti-dsDNA antibody titer in the plasma of three patients with the same immunosorbent product of DNA-SeNPs, commercially available product 1 and commercially available product 2 during the clearance of anti-dsDNA antibody in the plasma of the same patient.
  • Fig. 5 is a statistical graph of the clearance rate of anti-dsDNA antibodies in plasma of SeNPs and DNA-SeNPs in three cases.
  • Figure 6 is a comparison of DNA-SeNPs and hematoxylin-eosin staining (H & E) staining of kidney sections.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • SeNPs nano selenium
  • the DNA-SeNPs before and after adsorption of anti-dsDNA antibody were characterized by particle size and potential (Malvern particle size analyzer), ultraviolet spectrophotometer, infrared spectrophotometer, transmission electron microscope (TEM) and atomic force microscope (AFM). The results are shown in Figure 1. -3.
  • the hydrated particle size of the DNA-SeNPs before adsorption is about 126nm.
  • enzyme-linked immunoassay kit QUANTA dsDNA ELISA, 708510, Inova Diagnostics, A Werfen Company
  • anti-dsDNA antibody clearance rate (value of sample before adsorption-value of sample after adsorption) / value of sample before adsorption * 100%.
  • the results are shown in Figures 4a-4b. In most of the 128 cases, the clearance rate of anti-dsDNA antibodies by DNA-SeNPs is greater than 50%, and after DNA-SeNPs adsorb plasma, the titer of anti-dsDNA antibodies in plasma is Significantly reduced.
  • the nano-selenium DNA-loaded immunosorbent provided by the present invention and commercially available immunoadsorbent products are immobilized on carbonized resin ;
  • Commercially available product 2 Selective plasma component adsorber, phenylalanine immobilized polyvinyl alcohol gel
  • the clearance rate of DNA-SeNPs reached 75% at 15 minutes, while the commercially available product 1 was There was basically no removal effect within two hours, and the removal rate of the commercially available product 2 reached 35% in two hours.
  • the change of anti-dsDNA antibody titer in plasma is consistent with the change of clearance rate.
  • the inventors of the present invention tested a total of 53 kidney slices, including kidney tissue slices of normal people, lupus kidney and other kidney disease patients (the detection method is the same as Example 4), and then the H & E staining method (the detection method is the same as Comparative Example 1). Comparing the test results and judging the type of lupus kidney, the accuracy rate is 100%, and the statistical results of the accuracy rate are shown in Table 1. The detection results of H & E staining method and anti-dsDNA antibody detection reagent are shown in Fig. 4.
  • Normal glomerulus H & E staining: the normal glomerular vascular loop under the light microscope is thin and clear, the number of endothelial cells and mesangial cells is normal, and the surrounding renal tubules are also normal; DNA-SeNPs: glomeruli and renal tubular area No obvious fluorescence was seen.
  • LN-I classification H & E staining: the glomeruli are almost normal, and the mesangium is slightly segmental hyperplasia; DNA-SeNPs: a small amount of fluorescence is seen in the mesangial area.
  • LN-II classification H & E staining: moderate mesangial hyperplasia; DNA-SeNPs: glomerular mesangial area see DNA-SeNPs deposition.
  • LN-III classification H & E staining: segmental capillary cell hyperplasia with fewer parenchymal lumens, segmental subendothelial deposits under light microscope; DNA-SeNPs: distribution in glomerular capillary wall segments and Immune deposition of DNA-SeNPs accompanying mesangial deposition.
  • LN-IV classification H & E staining: the lesion is diffuse in biopsy and has segmental endothelial capillary hyperplasia; DNA-SeNPs: glomerular capillary wall segment distribution and DNA-SeNPs accompanying mesangial deposition Immune deposition is diffuse.
  • LN-V classification H & E staining: Glomerular capillary basement membrane diffuse "nail” formation or thickening, capillary loops significantly thickened, but the number of cells did not increase;
  • DNA-SeNPs capillary wall diffused Immune deposition of subcutaneous DNA-SeNPs.
  • kidney diseases HE staining, light microscopy that appears during HE staining of lupus nephritis.
  • the following table also shows a similar situation in patients with other kidney diseases.
  • DNA-SeNPs In other patients with kidney disease, no Se-DNA is deposited.
  • H & E-stained kidney slices can reveal changes in the glomerular tissue structure, and DNA-SeNPs can detect anti-dsDNA antibodies deposited in the glomeruli, clarifying the deposition of anti-dsDNA antibodies in renal tissue and Distribution, and the detection method is simpler and easier to operate than the H & E staining method. Therefore, the DNA immunosorbent DNA-SeNPs can be used to prepare reagents for detection and analysis of anti-dsDNA antibody deposition in kidney sections.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Inorganic Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

以纳米硒作为吸附剂载体的DNA免疫吸附剂及其制备方法和应用，其DNA固载量大、吸附性能好，且安全性能更佳。在该DNA免疫吸附剂中，DNA分子与抗dsDNA抗体充分接触，可以用于免疫荧光，定性分析肾组织中沉积的抗dsDNA抗体，同时可特异性确认抗dsDNA抗体在肾组织中的沉积和分布，简单易操作，适于制备检测和分析狼疮肾和红斑狼疮的试剂。

Description

一种以纳米硒作为吸附剂载体的DNA免疫吸附剂及其制备方法和应用 技术领域
本发明属于生物医用材料技术领域。更具体地，涉及一种具有谷胱甘肽响应性的纳米载药体系及其制备方法和应用。
背景技术
系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种多系统损害的慢性自身性免疫性疾病，确切病因不明，常引起多器官系统的不可逆性损害，可累及皮肤、浆膜、关节、肾及中枢神经系统等，病理表现为自身抗体及免疫复合物沉积，影响患者的寿命和生活质量。SLE的患病率因人群而异，我国患病以女性多见，尤其是育龄女性。患者血浆中出现以抗核抗体(ANA)、抗双链DNA抗体(抗dsDNA抗体)为代表的多种抗体，其中抗dsDNA抗体是参与SLE发病的主要抗体，其特异性可达90％，是SLE特异性抗体。
早期清除患者血浆中的自身抗体、控制疫病活动、保护肾功能是治疗的关键。免疫吸附疗法是近年来发展起来的新型血液净化技术，它利用吸附材料除去血浆中的致病抗体(如抗dsDNA抗体)，达到治疗疫病的目的。免疫吸附是通过抗原抗体免疫反应或物理化学作用除去致病因子，主要是血浆吸附，从而避免了离心式和一次膜血浆分离大量丢弃血浆的弊端。近年来，免疫吸附越来越多地应用于免疫性疾病的治疗。
其中，应用比较广泛的是DNA免疫吸附，采用DNA免疫吸附疗法可以特异性的吸附和清除抗dsDNA抗体，从而对SLE患者进行有效的治疗。欧洲专利EP0272792A1制备了基于活性炭或者碳化树脂的DNA免疫吸附剂，他们以活性炭或者碳化树脂为载体，以小牛胸腺DNA(下文简称DNA)为配基，利用火棉胶包埋固载小牛胸腺DNA。但是，无论是活性炭还是碳化树脂，均存在碳颗粒易脱落的问题，因而使用之前需要增加时间进行充分预冲至无微粒，从而避免治疗时微粒随血液循环进入人体而导致发生血栓、过敏、刺激等严重的不良反应。此外，DNA采用火棉胶包埋方式固载还存在两个缺点：一是部分DNA会由于包埋不充分，在后续生产过程中可能发生脱落，导致DNA固载量下降；二是部分 被包埋过深的DNA又无法与抗体接触。因此，提供一种吸附性能好，安全性高的DNA免疫吸附剂成为亟待解决的问题。
抗dsDNA抗体，结合临床症状和其它实验检查结果，可辅助诊断系统性红斑狼疮(SLE)以及狼疮肾的分型。在多种结缔组织疾病中，都可以检测到抗核抗体(ANA)，抗核抗体的筛查是一种灵敏的结缔组织疾病检测方法。尽管这种筛查方法对于SLE是一种非常好的检测方法(阴性结果可以排除活动性SLE)，但是这种方法不是一种特异性的检查方法。在SLE患者中，抗dsDNA抗体几乎可以排除其他疾病的可能性，因此该抗体被认为是该疾病的标志。抗dsDNA抗体阳性足以证明SLE；但是，这些抗体的检测阴性并不能在所有的病例当中完全排除SLE。
目前，临床上检测肾组织的病变和分型最常使用的为苏木精—伊红(H&E)染色方法，免疫荧光的方法也被用来检测肾组织中的多种抗体沉积。但是H&E染色和普通的免疫荧光并不能特异性确认抗dsDNA抗体在肾组织中的沉积和分布。因此，提供一种简单快捷，既能诊断肾脏病变又能分析肾切片中抗dsDNA抗体的检测方法对于狼疮肾炎和红斑狼疮的诊断和治疗非常重要。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种以纳米硒作为吸附剂载体的DNA免疫吸附剂。及其制备方法和应用
本发明的第二个目的是提供上述DNA免疫吸附剂的制备方法。
本发明的第三个目的是提供上述DNA免疫吸附剂在制备血液净化制剂中的应用。
本发明的第四个目的是提供上述DNA免疫吸附剂在制备用于预防、治疗或控制抗dsDNA抗体升高导致的疾病的药物中的应用。
本发明的第五个目的是提供一种包含有上述DNA免疫吸附剂的用于荧光免疫染色的抗dsDNA抗体检测试剂。
本发明的第六个目的是提供一种用于荧光免疫染色的抗dsDNA抗体检测试剂及其制备方法。
本发明的第七个目的是提供一种利用上述抗dsDNA抗体检测试剂进行非诊断目的的肾切片检测的方法。
本发明上述目的通过以下技术方案实现：
一种DNA免疫吸附剂，包括作为吸附剂载体的纳米硒和固载在所述纳米硒上的DNA。本发明发明人发现纳米硒用于负载DNA得到的DNA免疫吸附剂，具有吸附性能好，且安全性能更佳的优点。
优选地，所述DNA与纳米硒的用量按0.1～5mg：1μmol配比计算；更优选为0.1～2mg：1μmol。
优选地，所述DNA为与抗dsDNA抗体结合的双链DNA；所述DNA免疫吸附剂为DNA-SeNPs。
本发明还涉及了上述DNA免疫吸附剂的制备方法：将吸附剂载体纳米硒溶液与DNA缓冲溶液混合，进行DNA接枝反应，反应结束后将所得产物纯化，即可得到以纳米硒作为吸附剂载体的DNA免疫吸附剂。
优选地，所述反应的温度为0～10℃；更优选为3～5℃；最优选为4℃。
优选地，所述反应的时间为0.5～24h。
优选地，所述纳米硒溶液的浓度为1～10mM；更优选为2～5mM。
优选地，所述DNA溶液的浓度为1～10mg/mL；更优选为1～5mg/mL。
优选地，所述缓冲液的pH值为7～9；更优选为7.2～8.2。所述缓冲液优选为Tris-HCl缓冲液。所述Tris-HCl缓冲液的浓度优选为0.01～0.1M；更优选为0.01～0.05M。
优选地，所述纯化的方式为使用透析袋透析。所述透析袋的规格优选为6000～8000kDa；所述透析的时间优选为12～48h。
在其中一个优选实施例中，所述纳米硒由无机硒溶液和还原剂溶液按摩尔比1：3～10的比例混合，于0～10℃(优选为3～5℃；更优选为4℃)条件下反应得到。所述无机硒和还原剂的用量按还原剂过量为宜，从而能将无机硒充分还原。
所述无机硒选自亚硒酸、亚硒酸盐或二氧化硒中的一种或至少两种。
所述还原剂选自维生素C、硼氢化钠、巯基乙醇、还原型谷胱甘肽、葡萄糖或五水硫代硫酸钠中的一种或至少两种。
所述混合的方式优选为：将无机硒溶液和还原剂溶液混合；更优选通过如下方式混合：将无机硒溶液滴加到还原剂溶液中，或是将还原剂溶液滴加到无机硒溶液中。
所述无机硒溶液的浓度优选为5～15mM；更优选为10mM。
所述还原剂溶液的浓度优选为30～50mM；更优选为40mM。
上述DNA免疫吸附剂在制备血液净化制剂中的应用、在制备用于预防、治疗或控制抗dsDNA抗体升高导致的疾病的药物中的应用，也在本发明的保护范围之内。
优选地，所述抗dsDNA抗体升高导致的疾病为系统性红斑狼疮。
本发明还涉及了一种用于荧光免疫染色的抗dsDNA抗体检测试剂，是在上述DNA免疫吸附剂上标记荧光显示剂得到。本发明发明人发现将DNA免疫吸附剂用于免疫荧光，可定性分析肾组织中沉积的抗dsDNA抗体，特异性确认抗dsDNA抗体在肾组织中的沉积和分布，方法简单易操作。
本发明还涉及了上述抗dsDNA抗体检测试剂的制备方法，通过如下步骤制备得到：
(1)将无机硒溶液和荧光显示剂溶液混合，得到混合液A；
(2)将混合液A和还原剂混合，反应，得到反应物A；
(3)将用于与抗dsDNA抗体结合的DNA溶液和反应物A混合，反应，纯化，得到用于荧光免疫染色的抗dsDNA抗体检测试剂。
优选地，步骤(1)所述荧光显示剂为香豆素-6；所述荧光显示剂的用量按其与无机硒中硒的量＝5～15mg：1mmol配比计算；更优选为10mg：1mmol。
优选地，步骤(1)中所述的无机硒优选为亚硒酸、亚硒酸盐和二氧化硒中的一种或至少两种。所述的亚硒酸盐为亚硒酸钠(Na 2SeO 3)、亚硒酸钾(K 2SeO 3)和亚硒酸锌(ZnSeO 3)中的一种或至少两种。
优选地，步骤(1)中所述的无机硒溶液的浓度优选为5～15mM；更优选为10mM。
优选地，步骤(2)所述的还原剂优选为维生素C、硼氢化钠、巯基乙醇、还原型谷胱甘肽、葡萄糖和五水硫代硫酸钠中的一种或至少两种。
优选地，步骤(2)所述的还原剂的溶液浓度优选为30～50mM；更优选为40mM。
优选地，步骤(2)所述的还原剂和所述的无机硒的用量按还原剂过量为宜，从而能将无机硒充分还原；更优选为按摩尔比3～10：1配比计算。
优选地，步骤(3)中所述的用于与抗dsDNA抗体结合的DNA优选为双链DNA。
优选地，步骤(3)中所述的用于与抗dsDNA抗体结合的DNA溶液的浓度 优选为1～10mg/mL；更优选为1～5mg/mL。
优选地，步骤(3)中所述的用于与抗dsDNA抗体结合的DNA溶液中的溶剂优选为pH值为7～9的缓冲液；更优选为pH值为7.2～8.2的缓冲液。所述的缓冲液优选为Tris-HCl缓冲液。所述的Tris-HCl缓冲液的浓度优选为0.01～0.1M；更优选为0.01～0.05M。
优选地，步骤(3)中所述的用于与抗dsDNA抗体结合的DNA的用量按其与所述的反应物A中硒的量＝0.1～5mg：1μmol配比计算，更优选按其与所述的反应物A中硒的量＝0.5～2mg：1μmol配比计算。
优选地，步骤(3)中所述的反应的温度优选为0～10℃；更优选为3～5℃；最优选为4℃。
优选地，步骤(3)所述纯化的方式为使用透析袋透析；所述透析袋的规格优选为6000～8000kDa；所述透析的时间优选为12～48h。
本发明还涉及了一种利用上述述抗dsDNA抗体检测试剂进行非诊断目的的肾切片检测的方法，包括如下步骤：
(1)脱蜡至水：将肾石蜡切片依次浸入脱水：二甲苯I 10min，二甲苯II 5min，无水乙醇I 10min，无水乙醇II 5min，95％乙醇I 5min，95％乙醇II 5min，90％乙醇I 5min，90％乙醇II 5min，80％乙醇5min；蒸馏水洗2min；
(2)抗原修复：取12mL 50×EDTA抗原修复液，用超纯水稀释至600mL，95～110℃预热1min，放入切片，95～100℃加热1min，60～70℃加热2min(4～5次)，冷却，PBS缓冲液；洗涤；
(3)封闭：去除多余液体，加入浓度为质量百分比为5％的牛血清蛋白溶液，封闭30min；
(4)孵育：滴加100μL上述用于荧光免疫染色的抗dsDNA抗体检测试剂，孵育2h，倾去液体；
(5)观察：荧光显微镜下观察染色情况。
与现有技术相比，本发明具有以下有益效果：
1、本发明提供的DNA免疫吸附剂中的载体纳米硒(SeNPs)是一种能够补充人体硒元素，清除体内过剩活性氧自由基，且安全性高的有益物质。因此，本发明的免疫吸附剂无需预冲，节约资源，提高了免疫吸附剂使用的安全性。
2、本发明以SeNPs作为载体，将DNA修饰在其表面，制备得到的纳米硒 负载DNA的免疫吸附剂DNA固载量达到了928μg/mL，吸附性能良好。
3、本发明提供的DNA免疫吸附剂中，DNA分子与抗dsDNA抗体充分接触，最大限度发挥其特异性结合能力。
4、本发明提供的DNA免疫吸附剂中的载体SeNPs本身也对抗dsDNA抗体具有一定的清除效果。
5、本发明发明人发现将DNA免疫吸附剂用于检测肾切片中抗dsDNA抗体，DNA-SeNPs可以与肾脏切片中的抗dsDNA抗体特异性结合，因此能定性的分析肾组织中沉积的抗体种类，同时可确认抗dsDNA抗体在肾组织中的沉积和分布，因此，DNA免疫吸附剂可用于制备检测和分析狼疮肾和红斑狼疮的试剂。
6、本发明与临床上检测肾组织的病变和分型常用的苏木精—伊红(H&E)法相比，能特异性显示肾组织抗dsDNA抗体的沉积和分布，且更加简单快捷。
附图说明
图1是DNA-SeNPs吸附病人血浆中抗dsDNA抗体前后的表征图；其中，图a是吸附前后的纳米粒径大小分布图，图b是吸附前后的电位图，图a和b中1～4分别为吸附61号、28号、70号和23号病例血浆后的DNA-SeNPs；图c是吸附前后的紫外光谱图，曲线1为DNA-SeNPs，曲线2～4分别为吸附19号、69号、109号病例血浆后的DNA-SeNPs；图d是吸附前后的红外光谱图，曲线1为DNA-SeNPs，曲线2～4分别为吸附14号、30号、34号病例血浆后的DNA-SeNPs。
图2是DNA-SeNPs吸附病人血浆中抗dsDNA抗体前后的透射电子显微镜图；其中，图a是DNA-SeNPs的TEM图，图b是吸附17号病例血浆后的DNA-SeNPs的TEM，图c是吸附31号病例血浆后的DNA-SeNPs的TEM图，图d是吸附58号病例血浆后的DNA-SeNPs的TEM图。
图3是DNA-SeNPs吸附病人血浆中抗dsDNA抗体前后的原子力显微镜和相应的厚度分析图；其中，图a是DNA-SeNPs的AFM图(上)及相应的厚度分析图(下)，图b是吸附32号病例血浆后的DNA-SeNPs的AFM图(上)及相应的厚度分析图(下)，图c是吸附33号病例血浆后的DNA-SeNPs的AFM图(上)及相应的厚度分析图(下)。
图4是DNA-SeNPs吸附抗dsDNA抗体的的结果图，其中，图a是DNA-SeNPs对128个病例的血浆中抗dsDNA抗体的清除率统计图；图b是DNA-SeNPs对 128个病例的血浆中抗dsDNA抗体清除后血浆中抗dsDNA抗体滴度的统计图；图c是DNA-SeNPs、市售产品1和市售产品2三种免疫吸附产品对同一病例病人血浆中抗dsDNA抗体的清除率变化图；图d是DNA-SeNPs、市售产品1和市售产品2三种免疫吸附产品对同一病例病人血浆中抗dsDNA抗体清除过程中的血浆中抗dsDNA抗体滴度变化图。
图5是SeNPs与DNA-SeNPs对3个病例的血浆中抗dsDNA抗体的清除率统计图。
图6是DNA-SeNPs和苏木精—伊红染色法(H&E)对肾切片的染色比较图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 DNA-SeNPs纳米粒子的制备
4℃下，将1mL浓度为10mM的Na 2SeO 3溶液放入小烧杯中，再缓慢加入1mL浓度为40mM的维生素C(Vc，阿拉丁公司)溶液，滴加完后加入1mL浓度为5mg/mL的DNA(D4522，Sigma公司)Tris-HCl溶液(0.05M，pH＝7.2)，再用超纯水定容到5mL，4℃反应12h，用透析袋(6000～8000kDa)在超纯水中透析24h，得到DNA-SeNPs纳米粒子。
纳米硒(SeNPs)的制备：4℃下，将1mL浓度为10mM的Na 2SeO 3溶液放入小烧杯中，再缓慢加入1mL浓度为40mM的维生素C，再用超纯水定容到5mL，4℃反应12h，用透析袋(6000～8000kDa)在超纯水中透析24h，得到SeNPs纳米粒子。
实施例2 DNA-SeNPs吸附抗dsDNA抗体前后的表征
分别将150μL实施例1制备的DNA免疫吸附剂加入300μL血浆中(分别来源于128个SLE病例)，室温混匀(20rpm，试管旋转混匀器，TZL-5010，苏州珀西瓦尔实验设备有限公司)吸附2h。通过粒度和电位(马尔文粒度仪)、紫外分光光度计、红外分光光度计、透射电子显微镜(TEM)以及原子力显微镜(AFM)对吸附抗dsDNA抗体前后的DNA-SeNPs进行表征，结果见图1-3。吸附前DNA-SeNPs的水合粒径大约为126nm，吸附4位病人血浆(由中山三院提 供)后，将其离心(12000rpm，10min)，去上清，重悬，检测吸附后DNA-SeNPs的水合粒径分别增加到189nm(病例1：吸附前抗dsDNA抗体的滴度为563)、254nm(病例2：吸附前抗dsDNA抗体的滴度为353)、405nm(病例3：吸附前抗dsDNA抗体的滴度为600)和259nm(病例4：吸附前抗dsDNA抗体的滴度为336)(图1a)。但是，由于抗dsDNA抗体和DNA-SeNPs的表面电位相近，吸附前后DNA-SeNPs的表面电位没有明显的变化(图1b)。吸附前后DNA-SeNPs的紫外可见和红外光谱相一致(图1c和d)。吸附前DNA-SeNPs的TEM图像表现为单分散的、大小约为107nm的球状纳米粒子，且表面可以清晰地看见一层DNA的修饰(图2a)。吸附病人血浆之后，DNA-SeNPs的大小基本没变，但是纳米粒子表面的DNA层变厚了(图2b-d，分别对应病例17、病例31和病例58)。AFM的结果显示吸附后DNA-SeNPs的高度明显的增加了(图3)。以上结果都说明抗dsDNA抗体被吸附至纳米粒子表面。
实施例3 DNA-SeNPs吸附抗dsDNA抗体的性能评价
将实施例2中吸附后的血浆采用酶联免疫试剂盒(QUANTA dsDNA ELISA，708510，Inova Diagnostics，A Werfen Company，检测步骤按说明书操作)对抗dsDNA抗体进行定量检测，最后用酶标仪在450nm和620nm处读取数值。样本数值＝(样本OD 450nm-样本OD 620nm)/(dsDNA ELISA校准品OD 450nm-dsDNA ELISA校准品OD 620nm)*375。样品中抗dsDNA抗体清除率计算公式如下：抗dsDNA抗体清除率＝(吸附前样本数值-吸附后样本数值)/吸附前样本数值*100％。结果如图4a-图4b所示，128个病例的大部分病例中，DNA-SeNPs对抗dsDNA抗体的清除率都大于50％，而且DNA-SeNPs吸附血浆后，血浆中抗dsDNA抗体的滴度有显著地降低。
分别取1mL实施例1制备的DNA-SeNPs和40mg市售DNA免疫吸附剂中各加入同1位SLE病人的血浆2mL，室温震荡(20rpm，试管旋转混匀器，TZL-5010，苏州珀西瓦尔实验设备有限公司)吸附2h，每隔15min取样一次，将吸附前后的血浆采用酶联免疫试剂盒对抗dsDNA抗体进行定量检测，最后用酶标仪在450nm和620nm处读取数值。结果如图4c-图4d所示，可见，本发明提供的纳米硒负载DNA的免疫吸附剂与市售的免疫吸附产品(市售产品1：DNA免疫吸附柱，碳化树脂上固定小牛胸腺DNA；市售产品2：选择性血浆成分吸附器，苯基丙氨酸固定化聚乙烯醇凝胶)进行比较，DNA-SeNPs在15min的时 候清除率就达到了75％，而市售产品1在两小时之内基本没有清除效果，市售产品2在两小时的时候清除率达到了35％。血浆中抗dsDNA抗体滴度的变化与清除率的变化相一致。
分别将150μL SeNPs(实施例1制备)和150μL DNA-SeNPs加入300μL血浆中(来源于3个SLE病例，重复数为3)，室温20rpm混匀，吸附2h，以上述方法检测吸附后的血浆中抗dsDNA抗体清除率。结果如图5所示，单独的SeNPs对抗dsDNA抗体也具有一定的清除效果。
实施例4 DNA-SeNPs检测肾切片中抗dsDNA抗体沉积的性能评价
(1)将肾石蜡切片浸入以下溶液依次脱蜡至水：二甲苯I 10min，二甲苯II 5min，无水乙醇I 10min，无水乙醇II 5min，95％乙醇I 5min，95％乙醇II 5min，90％乙醇I 5min，90％乙醇II 5min，80％乙醇5min，经蒸馏水洗2min。
(2)抗原修复：取12mL 50×EDTA抗原修复液(主要由EDTA、Tris等组成，pH＝8.0，市购得到)用超纯水稀释到600mL，在微波炉(格兰仕，P70D20P-TD(W0))高火下预热1min，放入切片，高火加热1min，中火加热2min(4～5次)，冷却，PBS缓冲液(0.01M，pH＝7.4)洗一次。
(3)封闭：甩干多余液体，加入浓度为质量百分比5％的牛血清蛋白溶液(溶质为BSA，溶剂为0.01M，pH＝7.4的PBS)，封闭30min。
(4)孵育：滴加100μL香豆素6标记的DNA-SeNPs(实施例1制备得到)孵育2h，倾去液体。
(5)观察：在荧光显微镜(Life Technologies，EVOS FL auto)下观察染色情况，拍照。
对比例1苏木精—伊红染色法(H&E)
(1)将肾石蜡切片放入以下溶液依次脱蜡至水：二甲苯I 10min，二甲苯II 5min，无水乙醇I 10min，无水乙醇II 5min，95％乙醇I 5min，95％乙醇II 5min，90％乙醇I 5min，90％乙醇II 5min，80％乙醇5min，经蒸馏水洗2min。
(2)苏木素染色10min，水洗2min，1％盐酸乙醇(75％乙醇配制)分化3～5s，流水冲洗10min。
(3)伊红染色2min，蒸馏水洗数秒。
(4)切片脱水脱蜡：80％酒精3-5s，90％酒精I 3-5s，90％酒精II 5min，95％酒精I 5min，95％酒精II 5min，100％酒精I 5min，100％酒精II 10min，二甲 苯I5min，二甲苯II 10min。
(5)中性树胶封片。
(6)荧光显微镜(Life Technologies,EVOS FL auto)下观察染色情况，拍照，并将结果与实施例4的结果进行比较。
比较实施例
本发明发明人共检测了53例肾切片，包括正常人、狼疮肾以及其他肾脏疾病患者的肾组织切片(检测方法同实施例4)，然后与H&E染色法(检测方法同对比例1)的检测结果相比较，判断狼疮肾的分型，其正确率为100％，准确率统计结果见表1。H&E染色法和抗dsDNA抗体检测试剂的检测结果如图4所示。
正常肾小球：H&E染色：光镜下的正常的肾小球血管袢薄而清晰，内皮细胞和系膜细胞数目正常，周围的肾小管也正常；DNA-SeNPs：肾小球及肾小管区域未见明显荧光。
LN-I分型：H&E染色：肾小球几乎正常，系膜轻度节段性增生；DNA-SeNPs：肾小球系膜区域见少量荧光。
LN-II分型：H&E染色：系膜中度增生；DNA-SeNPs：肾小球系膜区域见DNA-SeNPs沉积。
LN-III分型：H&E染色：节段性毛细血管内细胞增生伴实质管腔较少，光镜下节段性内皮下沉积物；DNA-SeNPs：在肾小球毛细血管壁节段分布和伴随系膜沉积的DNA-SeNPs免疫沉积。
LN-IV分型：H&E染色：病变在活检组织检查中呈弥漫性，具有节段性内皮毛细血管增生；DNA-SeNPs：肾小球毛细血管壁节段分布和伴随系膜沉积的DNA-SeNPs免疫沉积呈弥漫性。
LN-V分型：H&E染色：肾小球毛细血管基底膜弥漫性“钉突”形成或增厚，毛细血管袢显著增厚，但细胞数目不增加；DNA-SeNPs：毛细血管壁中弥漫上皮下DNA-SeNPs免疫沉积。
其他肾病：HE染色，狼疮肾炎的HE染色时出现的光镜下表现在其他肾病患者中也可以出现类似的情况，此时结合Se-DNA染色结果，可以明确有无狼疮的特异性抗体抗dsDNA抗体的沉积，从而明确光镜下的改变是否由于抗dsDNA抗体引起，帮助诊断狼疮性肾炎。DNA-SeNPs：在其他肾病患者中，未见Se-DNA 的沉积。
综上所述，H&E染色的肾切片可以看出肾小球中组织结构的改变，而DNA-SeNPs可以检测到肾小球中沉积的抗dsDNA抗体，明确抗dsDNA抗体在肾组织中的沉积和分布，且检测的方法比H&E染色的方法更加简单易操作。因此，可以将DNA免疫吸附剂DNA-SeNPs用于制备检测和分析肾切片中抗dsDNA抗体沉积的试剂。
表1
Figure PCTCN2019115559-appb-000001
申请人声明，以上具体实施方式为便于理解本发明而说明的较佳实施例，但本发明并不局限于上述实施例，即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (16)

  1. 一种DNA免疫吸附剂，其特征在于，包括作为吸附剂载体的纳米硒和固载在所述纳米硒上的DNA。
  2. 根据权利要求1所述DNA免疫吸附剂，其特征在于，所述DNA与纳米硒的用量按0.1～5mg：1μmol配比计算。
  3. 根据权利要求1或2所述DNA免疫吸附剂，其特征在于，所述DNA为与抗dsDNA抗体结合的双链DNA；所述DNA免疫吸附剂为DNA-SeNPs。
  4. 权利要求1～3任一所述DNA免疫吸附剂的制备方法，其特征在于，将吸附剂载体纳米硒溶液与DNA缓冲溶液混合，进行DNA接枝反应，反应结束后将所得产物纯化，即可得到以纳米硒作为吸附剂载体的DNA免疫吸附剂。
  5. 根据权利要求4所述制备方法，其特征在于，所述反应的温度为0～10℃；所述反应的时间为0.5～24h。
  6. 根据权利要求5所述制备方法，其特征在于，所述纳米硒溶液的浓度为1～10mM；所述DNA溶液的浓度为1～10mg/mL；所述缓冲液的pH值为7～9；所述纯化的方式为使用透析袋透析。
  7. 根据权利要求6所述制备方法，其特征在于，所述纳米硒溶液的浓度为2～5mM；所述DNA溶液的浓度为1～5mg/mL；所述缓冲液的pH值为7.2～8.2；所述透析袋的规格为6000～8000kDa；所述透析的时间为12～48h。
  8. 根据权利要求4所述制备方法，其特征在于，所述纳米硒由无机硒溶液和还原剂溶液按摩尔比1：3～10的比例混合，于0～10℃条件下反应得到。
  9. 根据权利要求8所述制备方法，其特征在于，所述无机硒选自亚硒酸、亚硒酸盐或二氧化硒中的一种或至少两种；所述还原剂选自维生素C、硼氢化钠、巯基乙醇、还原型谷胱甘肽、葡萄糖或五水硫代硫酸钠中的一种或至少两种。
  10. 权利要求1～3任一所述DNA免疫吸附剂或权利要求4～9任一所述制备方法制备得到的DNA免疫吸附剂在制备血液净化制剂中的应用。
  11. 权利要求1～3任一所述DNA免疫吸附剂或权利要求4～9任一所述制备方法制备得到的DNA免疫吸附剂在制备用于预防、治疗或控制抗dsDNA抗体升高导致的疾病的药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述抗dsDNA抗体升高导致的疾病为系统性红斑狼疮。
  13. 一种用于荧光免疫染色的抗dsDNA抗体检测试剂，其特征在于，是在权利要求1～3任一所述DNA免疫吸附剂或在权利要求4～9任一所述制备方法制备得到的DNA免疫吸附剂上标记荧光显示剂得到。
  14. 根据权利要求13所述抗dsDNA抗体检测试剂，其特征在于，通过如下步骤制备得到：
    (1)将无机硒溶液和荧光显示剂溶液混合，得到混合液A；
    (2)将混合液A和还原剂混合，反应，得到反应物A；
    (3)将用于与抗dsDNA抗体结合的DNA溶液和反应物A混合，反应，纯化，得到用于荧光免疫染色的抗dsDNA抗体检测试剂。
  15. 根据权利要求14所述抗dsDNA抗体检测试剂，其特征在于，所述荧光显示剂为香豆素-6；所述荧光显示剂的用量按其与无机硒中硒的量＝5～15mg：1mmol配比计算；所述纯化的方式为使用透析袋透析；所述透析袋的规格为6000～8000kDa；所述透析的时间为12～48h。
  16. 一种利用权利要求13～15任一项所述抗dsDNA抗体检测试剂进行非诊断目的的肾切片检测的方法，其特征在于，包括如下步骤：
    (1)脱蜡至水：将肾石蜡切片依次浸入脱水：二甲苯I 10min，二甲苯II 5min，无水乙醇I 10min，无水乙醇II 5min，95％乙醇I 5min，95％乙醇II 5min，90％乙醇I 5min，90％乙醇II 5min，80％乙醇5min；蒸馏水洗2min；
    (2)抗原修复：取12mL 50×EDTA抗原修复液，用超纯水稀释至600mL，95～110℃预热1min，放入切片，95～100℃加热1min，60～70℃加热2min(4～5次)，冷却，PBS缓冲液；洗涤；
    (3)封闭：去除多余液体，加入浓度为质量百分比为5％的牛血清蛋白溶液，封闭30min；
    (4)孵育：滴加100μL上述用于荧光免疫染色的抗dsDNA抗体检测试剂，孵育2h，倾去液体；
    (5)观察：荧光显微镜下观察染色情况。
PCT/CN2019/115559 2018-11-05 2019-11-05 一种以纳米硒作为吸附剂载体的dna免疫吸附剂及其制备方法和应用 WO2020093989A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201811305882.7 2018-11-05
CN201811305882.7A CN109317083B (zh) 2018-11-05 2018-11-05 纳米硒在制备dna免疫吸附剂中的应用
CN201811305886.5A CN109521193B (zh) 2018-11-05 2018-11-05 DNA免疫吸附剂在制备抗dsDNA抗体检测试剂中的应用
CN201811305886.5 2018-11-05

Publications (1)

Publication Number Publication Date
WO2020093989A1 true WO2020093989A1 (zh) 2020-05-14

Family

ID=70611127

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/115559 WO2020093989A1 (zh) 2018-11-05 2019-11-05 一种以纳米硒作为吸附剂载体的dna免疫吸附剂及其制备方法和应用

Country Status (1)

Country Link
WO (1) WO2020093989A1 (zh)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1199643A (zh) * 1998-06-08 1998-11-25 南开大学 球形纤维素dna免疫吸附剂
WO2005050224A2 (en) * 2003-11-13 2005-06-02 Epitome Biosystems Inc. Small molecule and peptide arrays and uses thereof
CN101165491A (zh) * 2006-10-19 2008-04-23 陕西西大北美基因股份有限公司 以金磁微粒为载体检测抗双链dna抗体的方法
CN102631683A (zh) * 2012-03-31 2012-08-15 暨南大学 功能化纳米硒在抑制肿瘤血管生成和抗肿瘤药物中的应用
CN105126784A (zh) * 2015-07-31 2015-12-09 珠海健帆生物科技股份有限公司 吸附剂及其制备方法、用于血液灌流的吸附装置
CN105289538A (zh) * 2015-11-04 2016-02-03 珠海健帆生物科技股份有限公司 Dna免疫吸附剂及其制备方法
CN109317083A (zh) * 2018-11-05 2019-02-12 暨南大学 纳米硒在制备dna免疫吸附剂中的应用
CN109521193A (zh) * 2018-11-05 2019-03-26 暨南大学 DNA免疫吸附剂在制备抗dsDNA抗体检测试剂中的应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1199643A (zh) * 1998-06-08 1998-11-25 南开大学 球形纤维素dna免疫吸附剂
WO2005050224A2 (en) * 2003-11-13 2005-06-02 Epitome Biosystems Inc. Small molecule and peptide arrays and uses thereof
CN101165491A (zh) * 2006-10-19 2008-04-23 陕西西大北美基因股份有限公司 以金磁微粒为载体检测抗双链dna抗体的方法
CN102631683A (zh) * 2012-03-31 2012-08-15 暨南大学 功能化纳米硒在抑制肿瘤血管生成和抗肿瘤药物中的应用
CN105126784A (zh) * 2015-07-31 2015-12-09 珠海健帆生物科技股份有限公司 吸附剂及其制备方法、用于血液灌流的吸附装置
CN105289538A (zh) * 2015-11-04 2016-02-03 珠海健帆生物科技股份有限公司 Dna免疫吸附剂及其制备方法
CN109317083A (zh) * 2018-11-05 2019-02-12 暨南大学 纳米硒在制备dna免疫吸附剂中的应用
CN109521193A (zh) * 2018-11-05 2019-03-26 暨南大学 DNA免疫吸附剂在制备抗dsDNA抗体检测试剂中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JING, JING: "The Preparation andApplication of Selenium Nanoparticles in Lateral Flow Immunochromatographic Strip", CHINA MASTER'S THESES FULL-TEXT DATABASE, 15 June 2018 (2018-06-15) *

Similar Documents

Publication Publication Date Title
CN106018792B (zh) 一种胃功能检测用免疫层析试剂盒及其制备方法
JP3969722B2 (ja) カンジダの検出
WO2019148753A1 (zh) 一种thsd7a抗体的检测试纸条及检测方法
KR20110128606A (ko) 자성 나노입자-플래티늄 나노입자-다공성 탄소 복합체 및 그 제조방법
CN111175505B (zh) 一种p53自身抗体检测试剂盒及其应用
WO1988000702A1 (en) Cancer screening test
WO2023202271A1 (zh) 一种多项病原体联合检测装置及其制备方法
Idil et al. Concanavalin A immobilized magnetic poly (glycidyl methacrylate) beads for prostate specific antigen binding
WO2020093989A1 (zh) 一种以纳米硒作为吸附剂载体的dna免疫吸附剂及其制备方法和应用
CN109317083B (zh) 纳米硒在制备dna免疫吸附剂中的应用
WO2012130143A1 (zh) 一种抗体组合物及其应用
CN211347643U (zh) 一种纯化尿蛋白的装置
CN109289760B (zh) 二氧化硅纳米粒子在dna免疫吸附剂中的应用
CN109521193B (zh) DNA免疫吸附剂在制备抗dsDNA抗体检测试剂中的应用
JPS6089755A (ja) 診断用試薬及びその製造法並びにそれを用いる診断法
Fan et al. Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies
CN103604920A (zh) 一种诊断卵巢癌的磁性纳米探针的制备方法及检测方法
CN110988359A (zh) Au@HIF-1α aptamer@Au纳米酶的制备及在隐匿型冠心病检测中的应用
JP7081861B1 (ja) ＬＩＰに基づいてＵＭＯＤを修飾した尿中のＮｅｕ５Ｇｃを識別する尿路上皮癌の検査用キット、およびその製造方法
WO2016082444A1 (zh) 肽基精氨酸脱亚胺酶1在制备肿瘤临床诊断试剂中的应用
CN109521194B (zh) DNA免疫吸附剂在制备抗ds-DNA抗体检测试剂中的应用
CN111551740B (zh) 幽门螺杆菌尿素酶IgG和IgM抗体联合检测装置及制备方法
CN114539401A (zh) 一种磁微粒标记方法及检测试剂盒
CN110779788A (zh) 一种纯化尿蛋白的装置及方法
WO2016061861A1 (zh) 一种自身免疫性抗原阳性血清的纯化制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19882526

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19882526

Country of ref document: EP

Kind code of ref document: A1