WO2018000381A2 - 改性心磷脂包被的纳米磁珠及其制备方法 - Google Patents

改性心磷脂包被的纳米磁珠及其制备方法 Download PDF

Info

Publication number
WO2018000381A2
WO2018000381A2 PCT/CN2016/088014 CN2016088014W WO2018000381A2 WO 2018000381 A2 WO2018000381 A2 WO 2018000381A2 CN 2016088014 W CN2016088014 W CN 2016088014W WO 2018000381 A2 WO2018000381 A2 WO 2018000381A2
Authority
WO
WIPO (PCT)
Prior art keywords
cardiolipin
modified
modified cardiolipin
biotin derivative
streptavidin
Prior art date
Application number
PCT/CN2016/088014
Other languages
English (en)
French (fr)
Inventor
钱纯亘
邹定标
王刚
黄丕浓
夏福臻
Original Assignee
深圳市亚辉龙生物科技股份有限公司
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
Application filed by 深圳市亚辉龙生物科技股份有限公司 filed Critical 深圳市亚辉龙生物科技股份有限公司
Priority to EP16906781.6A priority Critical patent/EP3480594A4/en
Priority to PCT/CN2016/088014 priority patent/WO2018000381A2/zh
Publication of WO2018000381A2 publication Critical patent/WO2018000381A2/zh

Links

Images

Classifications

    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis

Definitions

  • the invention relates to the field of in vitro detection, in particular to a modified cardiolipin coated nano magnetic bead and a preparation method thereof.
  • An anticardiolipin antibody is an antibody that reacts with a variety of antigenic materials containing a phospholipid structure.
  • the antigen is a negatively charged phospholipid component that is involved in a variety of cell membranes. It is mainly found in patients with antiphospholipid syndrome, various autoimmune diseases and syphilis infections.
  • Antiphospholipid syndrome includes a variety of autoimmune diseases, more common in young people, the ratio of male to female incidence is about 2:8. Patients may have one or more manifestations that may affect multiple systems and organs, including: venous and arterial thrombosis, thrombocytopenia, habitual abortion, cardiomyopathy, heart disease, brain and kidney infarction, and pulmonary hypertension. Malignant APS can be characterized by progressive extensive thrombosis in the short term, resulting in multiple organ failure and even death. It can be secondary to systemic lupus erythematosus or other autoimmune diseases, but it can also occur alone (primary antiphospholipid syndrome).
  • Anti-phospholipid antibodies can also be produced in patients with syphilis.
  • Syphilis is a sexually transmitted disease caused by the spirochete syphilis. More than 5 million people are reported to be infected with syphilis each year, including 30,000 babies born through congenital infections. Syphilis can be lurking and hidden in patients for many years and can lead to a variety of clinical manifestations. Patients in the concealed period of syphilis have no clinical symptoms, and the secondary stage lasts for a lifetime in about two-thirds of untreated patients. Infected persons are not contagious during the concealment period, but children born to mothers in concealed periods may be infected with congenital syphilis.
  • Antiphospholipid antibody assays are widely used in the diagnosis of phospholipid syndrome and in the non-dense spiral test of syphilis. This detection has the advantage of being inexpensive, fast and convenient to perform on a large number of samples. In addition, since the concentration of antiphospholipid antibodies will gradually decrease with the successful treatment of syphilis, the specificity of syphilis infection Antibody Treponema antibodies can persist for years or even lifetimes. Therefore, antiphospholipid antibody test is considered to be a better choice for monitoring syphilis treatment.
  • the traditional method for detecting antiphospholipid antibodies is to apply cardiolipin to a specific solid such as a microplate by physical adsorption, and then combine the antiphospholipid antibodies in the sample to be tested with the cardiolipin attached to the plate. To achieve capture of antiphospholipid antibodies in the sample.
  • the concentration of the antiphospholipid antibody in the sample can be indirectly read by coloring the captured antiphospholipid antibody and measuring the luminescent concentration.
  • the cardiolipin immobilized by physical adsorption is easily dissociated and detached from the solid plate under the influence of external conditions such as solvent and heating, the stability of the test product prepared by the method is poor.
  • a modified cardiolipin coated nano magnetic bead comprising: a modified cardiolipin, a biotin derivative and a streptavidin magnetic bead;
  • the modified cardiolipin is obtained by oxidizing a hydrophobic fatty acid side chain of cardiolipin, the modified cardiolipin containing
  • the structural formula of the biotin derivative is: Wherein -R- is a saturated alkyl chain having 4 to 20 carbon atoms or a polyethylene glycol chain having 2 to 10 carbon atoms, in the modified cardiolipin Forming a -CO-NH- structure with -NH 2 in the biotin derivative to link the modified cardiolipin and the biotin derivative together;
  • streptavidin magnetic beads are nanomagnetic beads bound with streptavidin, the biotin derived And the streptavidin is linked.
  • a method for preparing the modified cardiolipin coated nano magnetic beads comprising the following steps:
  • the cardiolipin and the peroxide are sufficiently reacted in the presence of the first solvent to obtain a modified cardiolipid, wherein the hydrophobic fatty acid side chain of the modified cardiolipin is oxidized, and the modified cardiolipin contains
  • the modified cardiolipin and the biotin derivative are sufficiently reacted in the presence of a second solvent to obtain a biotin derivative-bound modified cardiolipin, wherein the biotin derivative has the structural formula: Wherein -R- is a saturated alkyl chain having 4 to 20 carbon atoms or a polyethylene glycol chain having 2 to 10 carbon atoms, in the modified cardiolipin Forming a -CO-NH- structure with -NH 2 in the biotin derivative to link the modified cardiolipin and the biotin derivative;
  • biotin derivative-bound modified cardiolipin and streptavidin magnetic beads are mixed and fully reacted to obtain modified cardiolipin coated nano magnetic beads, wherein the streptavidin magnetic beads are A nanomagnetic bead that binds streptavidin, the biotin derivative and the streptavidin are linked.
  • the modified cardiolipin-coated nanomagnetic beads directly bond the modified cardiolipin firmly to the biotin derivative through a chemical bond, and the biotin derivative and the streptavidin surface of the streptavidin magnetic beads are linked. Compared with the way of physical adsorption, it is easier to control the amount of modified cardiolipin on the surface of the magnetic beads, and the control of the distance between the amount of modified cardiolipin and the nanomagnetic beads can be achieved by adjusting the length of -R-, which is better. The space in which the amount of modified cardiolipin binds to the antiphospholipid antibody is retained.
  • the modified cardiolipin coated nanomagnetic beads can be directly used for the detection of antiphospholipid antibodies, and compared with traditional physical adsorption.
  • the test product prepared by the method of cardiolipin has high stability.
  • FIG. 1 is a flow chart showing a method of preparing a modified cardiolipin coated nanomagnetic bead according to an embodiment.
  • the modified cardiolipin coated nanomagnetic beads of one embodiment include: a modified cardiolipin, a biotin derivative, and a streptavidin magnetic bead.
  • Cardiolipin is an ester composed of three glycerols, two phosphoric acids, and four long-chain unsaturated alkyl groups.
  • the structure contains two hydrophilic centers and four hydrophobic side chains.
  • the structural formula of cardiolipin is as follows:
  • the modified cardiolipin is obtained by oxidizing the hydrophobic fatty acid side chain of the cardiolipin, and the modified cardiolipin is contained.
  • cardiolipin Since cardiolipin has 4 side chains, 4 side chains can be oxidized at the same time when cardiolipin is modified, so the modified cardiolipin may contain multiple Specifically, the modified cardiolipin may comprise from 1 to 8
  • the modified cardiolipin contains a
  • the structural formula of the modified cardiolipin is as follows:
  • the structural formula of the biotin derivative is:
  • -R- is a saturated alkyl chain having 4 to 20 carbon atoms or a polyethylene glycol chain having 2 to 10 carbon atoms.
  • Modified cardiolipin The -NH 2 in the biotin derivative forms a -CO-NH- structure to link the modified cardiolipin and the biotin derivative.
  • -R- is a saturated alkyl chain containing from 4 to 10 carbon atoms.
  • -R- is -(CH 2 ) 6 -.
  • the size of the cardiolipin molecule is very small, the space that can be modified is seriously insufficient, and modification of the hydrophilic phosphate ester center reduces the affinity of the cardiolipin and the antiphospholipid antibody, and even the antigen activity disappears.
  • the modified cardiolipin coated nanomagnetic beads retain the modified cardiolipin of the cardiolipin hydrophilic phosphate center by modifying the hydrophobic side chain of the cardiolipin.
  • the modified cardiolipin-coated nanomagnetic beads directly bond the modified cardiolipin firmly to the biotin derivative through a chemical bond, and the biotin derivative and the streptavidin surface of the streptavidin magnetic beads are linked. Compared with the way of physical adsorption, it is easier to control the amount of modified cardiolipin on the surface of the magnetic beads, and the control of the distance between the amount of modified cardiolipin and the nanomagnetic beads can be achieved by adjusting the length of -R-, which is better. The space in which the amount of modified cardiolipin binds to the antiphospholipid antibody is retained.
  • the modified cardiolipin coated nano magnetic beads can be directly used for the detection of antiphospholipid antibodies, and has higher stability than the detection products prepared by the conventional physical adsorption of cardiolipin.
  • the modified cardiolipin-coated nanomagnetic beads amplify the detected signal by the biotin-streptavidin amplification effect. Double, greatly improving the detection sensitivity of antiphospholipid antibodies.
  • the preparation method of the above modified cardiolipin coated nano magnetic beads as shown in FIG. 1 comprises the following steps:
  • the fatty acid side chain of cardiolipin is oxidized by reaction of cardiolipin with peroxide.
  • the peroxide is peroxybenzoic acid, m-chloroperoxybenzoic acid, peroxyacetic acid or peroxypropionic acid.
  • the molar ratio of cardiolipin to peroxide is from 1:1 to 8.
  • the first solvent is dichloromethane, chloroform, chloroform, benzene or toluene.
  • S10 also includes an operation for purifying the intermediate product, which can be purified by liquid phase preparative chromatography after extraction with ethyl acetate. After purification, a modified cardiolipin having a purity of about 80% can be obtained.
  • reaction temperature is from 60 ° C to 100 ° C.
  • cardiolipin Since cardiolipin has 4 side chains, 4 side chains can be oxidized at the same time when cardiolipin is modified, so the modified cardiolipin may contain multiple Specifically, the modified cardiolipin may comprise from 1 to 8
  • the modified cardiolipin contains a
  • the structural formula of the modified cardiolipin is as follows:
  • the structural formula of the biotin derivative is:
  • -R- is a saturated alkyl chain having 4 to 20 carbon atoms or a polyethylene glycol chain having 2 to 10 carbon atoms.
  • Modified cardiolipin The -NH 2 in the biotin derivative forms a -CO-NH- structure to link the modified cardiolipin and the biotin derivative.
  • -R- is a saturated alkyl chain containing from 4 to 10 carbon atoms.
  • -R- is -(CH 2 ) 6 -.
  • Biotin derivatives are commercially available.
  • the second solvent is DMSO, DMF, tetrahydrofuran or a phosphate buffer having a pH of 6.5 to 8.5.
  • the molar ratio of the modified cardiolipin to the biotin derivative is from 1:1.5 to 20.
  • streptavidin magnetic beads are nanomagnetic beads bound to streptavidin, the biotin derivative and streptavidin are linked.
  • Streptavidin magnetic beads can be purchased directly, for example, from MagnaBind Corporation (Cat. No. 21344).
  • the concentration of the modified cardiolipin bound by the biotin derivative is 0.1 mg/mL to 1 mg/mL, and the chain
  • the concentration of the mycele magnetic beads is from 5 mg/mL to 15 mg/mL.
  • the preparation method of the modified cardiolipin-coated nano magnetic beads directly bonds the modified cardiolipin firmly to the biotin derivative through a chemical bond, and the biotin derivative and the streptavidin on the surface of the streptavidin magnetic beads Avidin linkage, relative to the physical adsorption method, it is easier to control the amount of modified cardiolipin on the surface of the magnetic beads, and the distance between the modified cardiolipin and the nanomagnetic beads can be achieved by adjusting the length of -R-. Control, better retains the space for the amount of modified cardiolipin to bind to antiphospholipid antibodies.
  • the prepared modified cardiolipin coated nano magnetic beads can be directly used for the detection of antiphospholipid antibodies, and has higher stability than the detection products prepared by the conventional physical adsorption of cardiolipin.
  • the prepared modified cardiolipin-coated nanomagnetic beads amplify the detected signal by the biotin-streptavidin amplification effect. 4 times, greatly improving the detection sensitivity of antiphospholipid antibodies.
  • cardiolipin 0.5 mmol of cardiolipin was dissolved in 1 mL of anhydrous toluene, 1.5 mmol of m-chloroperoxybenzoic acid was added, and the mixture was gradually heated to 110 ° C while stirring, and the temperature was maintained for 72 hours. After cooling the reaction mixture to room temperature, it was poured into 25 mL of ice water, and the solution was extracted three times with 20 mL of ethyl acetate. The combined ethyl acetate was washed once with 10 mL of a saturated sodium chloride solution and dried over anhydrous sodium sulfate for 12 hr. The ethyl acetate was evaporated to dryness under reduced pressure.
  • the residual solid was dissolved in a small amount of methanol, and the insoluble material was filtered off.
  • the solution was purified by preparative liquid chromatography to obtain about 210 mg of a colorless oily liquid, which was modified cardiolipin, MS (ESI + , m/z): 1497.83131.
  • the structural formula of the biotin derivative is: -R- is -(CH 2 ) 6 -.
  • biotin derivative-bound modified cardiolipin prepared in this example can be used for subsequent use without special purification.
  • Example 4 Avidin magnetic beads coated with modified cardiolipin reacted with antiphospholipid samples
  • Table 1 Examples 1 to 3 and control group (physical adsorption method) measure OD values of different samples
  • the avidin magnetic beads coated with the modified cardiolipin prepared in Examples 1 to 3 have a marked improvement in detection sensitivity with respect to the magnetic beads of the conventional physical adsorption method when measuring the antiphospholipid sample.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Endocrinology (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

改性心磷脂包被的纳米磁珠及其制备方法 技术领域：
本发明涉及体外检测领域，尤其涉及一种改性心磷脂包被的纳米磁珠及其制备方法。
背景技术：
抗心磷脂抗体是一种能与多种含有磷脂结构的抗原物质发生反应的抗体，抗原为参与多种细胞膜组成的带负电荷的磷脂成分。临床上主要存在于抗磷脂综合征、各种自身免疫性疾病和梅毒感染患者体内。
抗磷脂综合症(APS)包括多种自身性免疫性疾病，多见于年轻人，男女发病比率约为2∶8。患者可出现一种或多种表现，可累及多个系统、器官，主要有：静脉和动脉血栓形成、血小板减少症、习惯性流产、心肌病、心脏病、大脑和肾脏梗塞、肺高压。恶性APS可表现为短期内进行性广泛血栓形成，造成多器官功能衰竭甚至死亡。可继发于系统性红斑狼疮或者其它自身免疫病，但也可单独出现(原发抗磷脂综合征)。
梅毒患者体内也能产生抗磷脂抗体，梅毒是由螺旋体细菌梅毒密螺旋体引起的性传播疾病。据报道，每年有超过5百万人感染梅毒，其中包括通过先天性传染的3万名婴儿。梅毒可于患者体内潜伏和隐藏许多年，并可导致各种各样的临床表现。其中处于梅毒隐藏期的患者并无临床症状，次阶段在约三分之二未治疗病人中持续终生。感染者在隐藏期不具有传染性，然而处于隐藏期的母亲所生的孩子可能感染先天性梅毒。
抗磷脂抗体检测试验被广泛应用于磷脂综合征诊断以及梅毒的非密螺旋体试验。该检测具有廉价、快速和方便对大量样本执行的优点。此外，由于抗磷脂抗体的浓度会随着梅毒的治疗成功而逐渐降低，而梅毒感染的特异性 抗体密螺旋体抗体会持续数年甚至终生都高。因此抗磷脂抗体检测试验被认为是监测梅毒治疗的更好选择。
传统的检测抗磷脂抗体的主要方法是将心磷脂用物理吸附的方式涂抹在特定的固体如酶标板上面，然后利用附着在酶标板上面的心磷脂与待检测样本中的抗磷脂抗体结合，实现对样本中的抗磷脂抗体的捕获。通过对捕获的抗磷脂抗体显色并测定发光浓度，就能间接的读取样本中的抗磷脂抗体浓度。
由于用物理吸附方式固定的心磷脂在溶剂、加热等外部条件影响下容易从固体板上解离和脱落，因此通过该方法制备的检验产品的稳定性较差。
发明内容：
基于此，有必要提供一种用于检测抗心磷脂抗体的稳定性较好的改性心磷脂包被的纳米磁珠及其制备方法。
一种改性心磷脂包被的纳米磁珠，包括：改性心磷脂、生物素衍生物和链霉亲和素磁珠；
所述改性心磷脂为心磷脂的疏水性脂肪酸侧链被氧化得到，所述改性心磷脂含有
Figure PCTCN2016088014-appb-000001
所述生物素衍生物的结构式为：
Figure PCTCN2016088014-appb-000002
其中，所述-R-为含有4个～20个碳原子的饱和烷基链或含有2个～10个碳原子的聚乙二醇链，所述改性心磷脂中的
Figure PCTCN2016088014-appb-000003
与所述生物素衍生物中的-NH2生成-CO-NH-结构从而将所述改性心磷脂和所述生物素衍生物连接到一起；
所述链霉亲和素磁珠为结合了链霉亲和素的纳米磁珠，所述生物素衍生 物和所述链霉亲和素连接。
一种上述的改性心磷脂包被的纳米磁珠的制备方法，包括如下步骤：
将心磷脂和过氧化物在第一溶剂存在的条件下充分反应，得到改性心磷脂，其中，所述改性心磷脂的疏水性脂肪酸侧链被氧化，所述改性心磷脂含有
Figure PCTCN2016088014-appb-000004
将所述改性心磷脂和生物素衍生物在第二溶剂存在的条件下充分反应，得到生物素衍生物结合的改性心磷脂，其中，所述生物素衍生物的结构式为：
Figure PCTCN2016088014-appb-000005
其中，所述-R-为含有4个～20个碳原子的饱和烷基链或含有2个～10个碳原子的聚乙二醇链，所述改性心磷脂中的
Figure PCTCN2016088014-appb-000006
与所述生物素衍生物中的-NH2生成-CO-NH-结构从而将所述改性心磷脂和所述生物素衍生物连接到一起；以及
将所述生物素衍生物结合的改性心磷脂和链霉亲和素磁珠混合并充分反应，得到改性心磷脂包被的纳米磁珠，其中，所述链霉亲和素磁珠为结合了链霉亲和素的纳米磁珠，所述生物素衍生物和所述链霉亲和素连接。
这种改性心磷脂包被的纳米磁珠通过化学键直接将改性心磷脂牢固与生物素衍生物牢固的连接，生物素衍生物和链霉亲和素磁珠表面的链霉亲和素连接，相对于物理吸附的方式，更加易于控制磁珠表面的改性心磷脂量，并且可以通过调节-R-的长度，实现改性心磷脂量与纳米磁珠之间的距离的控制，更好的保留了改性心磷脂量与抗磷脂抗体结合的空间。这种改性心磷脂包被的纳米磁珠可以直接用于抗磷脂抗体的检测，并且相对于传统物理吸附 心磷脂的方法制备的检测产品，具有较高的稳定性。
附图说明
图1为一实施方式的改性心磷脂包被的纳米磁珠的制备方法的流程图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
一实施方式的改性心磷脂包被的纳米磁珠，包括：改性心磷脂、生物素衍生物和链霉亲和素磁珠。
心磷脂是由3个甘油、2个磷酸以及4个长链不饱和烷基构成的酯类物质，该结构含有2个亲水性中心以及4条疏水性侧链。心磷脂的结构式如下：
Figure PCTCN2016088014-appb-000007
改性心磷脂为心磷脂的疏水性脂肪酸侧链被氧化得到，改性心磷脂含有
Figure PCTCN2016088014-appb-000008
由于心磷脂有4条侧链，心磷脂改性时4条侧链可以同时被氧化，因此该改性心磷脂可能包含多个
Figure PCTCN2016088014-appb-000009
具体来说，该改性心磷脂可以包含1个～8个
Figure PCTCN2016088014-appb-000010
优选的，改性心磷脂含有一个
Figure PCTCN2016088014-appb-000011
特别优选的，改性心磷脂的结构式如下：
Figure PCTCN2016088014-appb-000012
生物素衍生物的结构式为：
Figure PCTCN2016088014-appb-000013
其中，-R-为含有4个～20个碳原子的饱和烷基链或含有2个～10个碳原子的聚乙二醇链。
改性心磷脂中的
Figure PCTCN2016088014-appb-000014
与生物素衍生物中的-NH2生成-CO-NH-结构从而将改性心磷脂和生物素衍生物连接到一起。
优选的，-R-为含有4个～10个碳原子的饱和烷基链。
特别优选的，-R-为-(CH2)6-。
由于心磷脂分子的尺寸非常小，可改造的空间严重不足，对亲水性的磷酸酯中心进行修饰会降低心磷脂与抗磷脂抗体的亲和性，甚至使抗原活性消失。
这种改性心磷脂包被的纳米磁珠通过对心磷脂疏水性侧链进行改造，保留了心磷脂亲水性磷酸酯中心的改性心磷脂。
这种改性心磷脂包被的纳米磁珠通过化学键直接将改性心磷脂牢固与生物素衍生物牢固的连接，生物素衍生物和链霉亲和素磁珠表面的链霉亲和素连接，相对于物理吸附的方式，更加易于控制磁珠表面的改性心磷脂量，并且可以通过调节-R-的长度，实现改性心磷脂量与纳米磁珠之间的距离的控制，更好的保留了改性心磷脂量与抗磷脂抗体结合的空间。这种改性心磷脂包被的纳米磁珠可以直接用于抗磷脂抗体的检测，并且相对于传统物理吸附心磷脂的方法制备的检测产品，具有较高的稳定性。
此外，由于每个链霉亲和素能与4个生物素紧密结合，这种改性心磷脂包被的纳米磁珠通过生物素-链霉亲和素放大效应，使被检测信号放大了4倍，极大的提高了抗磷脂抗体的检测灵敏度。
如图1所示的上述改性心磷脂包被的纳米磁珠的制备方法，包括如下步骤：
S10、将心磷脂和过氧化物在第一溶剂存在的条件下充分反应，得到改性心磷脂。
通过心磷脂与过氧化物反应，对心磷脂的脂肪酸侧链进行氧化。
过氧化物为过氧苯甲酸、间氯过氧苯甲酸、过氧乙酸或过氧丙酸。
心磷脂和过氧化物的摩尔比为1∶1～8。
第一溶剂为二氯甲烷、三氯甲烷、氯仿、苯或甲苯。
S10还包括对中间产物进行纯化的操作，纯化可以为乙酸乙酯萃取后经过液相制备色谱纯化。纯化后可以得到纯度约80％的改性心磷脂。
S10中，反应温度为60℃～100℃。
由于心磷脂有4条侧链，心磷脂改性时4条侧链可以同时被氧化，因此该改性心磷脂可能包含多个
Figure PCTCN2016088014-appb-000015
具体来说，改性心磷脂可以包含1个～8个
Figure PCTCN2016088014-appb-000016
优选的，改性心磷脂含有一个
Figure PCTCN2016088014-appb-000017
特别优选的，改性心磷脂的结构式如下：
Figure PCTCN2016088014-appb-000018
S20、将S10得到的改性心磷脂和生物素衍生物在第二溶剂存在的条件下充分反应，得到生物素衍生物结合的改性心磷脂。
生物素衍生物的结构式为：
Figure PCTCN2016088014-appb-000019
其中，-R-为含有4个～20个碳原子的饱和烷基链或含有2个～10个碳原子的聚乙二醇链。
改性心磷脂中的
Figure PCTCN2016088014-appb-000020
与生物素衍生物中的-NH2生成-CO-NH-结构从而将改性心磷脂和生物素衍生物连接到一起。
优选的，-R-为含有4个～10个碳原子的饱和烷基链。
特别优选的，-R-为-(CH2)6-。
生物素衍生物可以直接购买得到。
S20中，第二溶剂为DMSO、DMF、四氢呋喃或pH为6.5～8.5的磷酸盐缓冲液。
S20中，改性心磷脂与生物素衍生物的摩尔比为1∶1.5～20。
S30、将S20得到的生物素衍生物结合的改性心磷脂和链霉亲和素磁珠混合并充分反应，得到改性心磷脂包被的纳米磁珠。
链霉亲和素磁珠为结合了链霉亲和素的纳米磁珠，生物素衍生物和链霉亲和素连接。
链霉亲和素磁珠可以为直接购买，例如，购自MagnaBind公司(货号为21344)。
将生物素衍生物结合的改性心磷脂和链霉亲和素磁珠混合并充分反应的操作中，生物素衍生物结合的改性心磷脂的浓度为0.1mg/mL～1mg/mL，链霉亲和素磁珠的浓度为5mg/mL～15mg/mL。
这种改性心磷脂包被的纳米磁珠的制备方法，通过化学键直接将改性心磷脂牢固与生物素衍生物牢固的连接，生物素衍生物和链霉亲和素磁珠表面的链霉亲和素连接，相对于物理吸附的方式，更加易于控制磁珠表面的改性心磷脂量，并且可以通过调节-R-的长度，实现改性心磷脂量与纳米磁珠之间的距离的控制，更好的保留了改性心磷脂量与抗磷脂抗体结合的空间。制得的改性心磷脂包被的纳米磁珠可以直接用于抗磷脂抗体的检测，并且相对于传统物理吸附心磷脂的方法制备的检测产品，具有较高的稳定性。
此外，由于每个链霉亲和素能与4个生物素紧密结合，制得的改性心磷脂包被的纳米磁珠通过生物素-链霉亲和素放大效应，使被检测信号放大了4倍，极大的提高了抗磷脂抗体的检测灵敏度。
以下为具体实施例。
实施例1：改性心磷脂的制备
将0.5mmol心磷脂溶于1mL无水甲苯中，加入1.5mmol的间氯过氧苯甲 酸，搅拌中逐渐加热升温至110℃，并维持该温度反应72小时。将反应液冷却至室温后，倒入25mL冰水中，再用20mL乙酸乙酯萃取溶液3次，合并乙酸乙酯。将合并的乙酸乙酯溶用10mL饱和氯化钠溶液洗涤1次后，加入无水硫酸钠干燥12小时。液负压下蒸干乙酸乙酯，残余的固体用少量甲醇溶解，过滤掉不溶物质，溶液用制备液相色谱纯化，得到约210mg无色油状液体即为改性心磷脂，MS(ESI+，m/z)：1479.83131。
实施例2：生物素衍生物结合的改性心磷脂的制备
搅拌下往4mL无水DMF溶液中依次加入200mg环乙氧基改性心磷脂、110mg末端氨基化的生物素衍生物，室温下搅拌反应10小时。减压下蒸干反应液，残余固体用5mL乙酸乙酯溶解，过滤不溶物，并将所得的乙酸乙酯溶解蒸干，得到的白色固体即生物素衍生物结合的改性心磷脂。
本实施例中，生物素衍生物的结构式为：
Figure PCTCN2016088014-appb-000021
-R-为-(CH2)6-。
本实施例制备的生物素衍生物结合的改性心磷脂不需要经过特别纯化即可用于后续的使用。
实施例3：包被有改性心磷脂的链霉亲和素磁珠的制备
取100μL含有0.2mg/mL的生物素衍生物结合的改性心磷脂的磷酸盐缓冲液，加入100μL含有10mg/mL的链霉亲和素磁珠，混匀后37℃孵育10分钟。磁分离后用1mL 30mM Tris缓冲液复溶，复溶所得的混合液震荡后再次磁分离，并将所得的固体用5mL 30mM Tris缓冲液复溶，得到浓度为0.02mg/mL的包被有改性心磷脂的链霉亲和素磁珠溶液。
实施例4：包被有改性心磷脂的亲和素磁珠与抗磷脂样本反应
分别取200μL实施例1～3中制备的包被有改性心磷脂的亲和素磁珠溶液，加入5μL抗磷脂抗体血清样本并于37℃孵育30分钟。磁分离后将包被有改性心磷脂的链霉亲和素磁珠复溶至200μL，加入辣根过氧化物酶标记的二抗，37℃孵育30分钟后依次清洗、加入TMB底物液并孵育10分钟，再加入100μL终止液，10分钟内酶标仪上读取OD值，得到样本的发光信号值。
分别取三个心磷脂阳性血清样本和三个心磷脂阴性血清样本，并且以传统的物理吸附法为对照，对比测得的OD值，得到下表1。
表1：实施例1～3和对照组(物理吸附法)测量不同样本的OD值
Figure PCTCN2016088014-appb-000022
由表1可以看出，实施例1～3制备的包被有改性心磷脂的亲和素磁珠与物理吸附法吸附的磁珠(对照组)在测试抗磷脂抗体样本时的发光信号比较，实施例1～3制备的包被有改性心磷脂的亲和素磁珠在测量阴性样本时发光信号值较对照组显著低(降低了1倍～4倍)，同时实施例1～3制备的包被有改性心磷脂的亲和素磁珠在测量阳性样本时发光信号较对照组有极大的提高(提高了3～10倍)。
由此说明，实施例1～3制备的包被有改性心磷脂的亲和素磁珠在测量抗磷脂样本时，相对于传统的物理吸附法的磁珠，检测灵敏度具有明显提升。
以上所述实施例仅表达了本发明的一种或几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是， 对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种改性心磷脂包被的纳米磁珠，其特征在于，包括：改性心磷脂、生物素衍生物和链霉亲和素磁珠；
    所述改性心磷脂为心磷脂的疏水性脂肪酸侧链被氧化得到，所述改性心磷脂含有
    Figure PCTCN2016088014-appb-100001
    所述生物素衍生物的结构式为：
    Figure PCTCN2016088014-appb-100002
    其中，所述-R-为含有4个～20个碳原子的饱和烷基链或含有2个～10个碳原子的聚乙二醇链，所述改性心磷脂中的
    Figure PCTCN2016088014-appb-100003
    与所述生物素衍生物中的-NH2生成-CO-NH-结构从而将所述改性心磷脂和所述生物素衍生物连接到一起；
    所述链霉亲和素磁珠为结合了链霉亲和素的纳米磁珠，所述生物素衍生物和所述链霉亲和素连接。
  2. 根据权利要求1所述的改性心磷脂包被的纳米磁珠，其特征在于，所述-R-为含有4个～10个碳原子的饱和烷基链。
  3. 根据权利要求1所述的改性心磷脂包被的纳米磁珠，其特征在于，所述改性心磷脂含有一个
    Figure PCTCN2016088014-appb-100004
  4. 根据权利要求3所述的改性心磷脂包被的纳米磁珠，其特征在于，所述改性心磷脂的结构式如下：
    Figure PCTCN2016088014-appb-100005
  5. 一种权利要求1～4中任一项所述的改性心磷脂包被的纳米磁珠的制备方法，其特征在于，包括如下步骤：
    将心磷脂和过氧化物在第一溶剂存在的条件下充分反应，得到改性心磷脂，其中，所述改性心磷脂的疏水性脂肪酸侧链被氧化，所述改性心磷脂含有
    Figure PCTCN2016088014-appb-100006
    将所述改性心磷脂和生物素衍生物在第二溶剂存在的条件下充分反应，得到生物素衍生物结合的改性心磷脂，其中，所述生物素衍生物的结构式为：
    Figure PCTCN2016088014-appb-100007
    其中，所述-R-为含有4个～20个碳原子的饱和烷基链 或含有2个～10个碳原子的聚乙二醇链，所述改性心磷脂中的
    Figure PCTCN2016088014-appb-100008
    与所述生物素衍生物中的-NH2生成-CO-NH-结构从而将所述改性心磷脂和所述生物素衍生物连接到一起；以及
    将所述生物素衍生物结合的改性心磷脂和链霉亲和素磁珠混合并充分反应，得到改性心磷脂包被的纳米磁珠，其中，所述链霉亲和素磁珠为结合了链霉亲和素的纳米磁珠，所述生物素衍生物和所述链霉亲和素连接。
  6. 根据权利要求5所述的改性心磷脂包被的纳米磁珠的制备方法，其特征在于，所述将心磷脂和过氧化物在第一溶剂存在的条件下充分反应的操作中，所述过氧化物为过氧苯甲酸、间氯过氧苯甲酸、过氧乙酸或过氧丙酸，所述心磷脂和所述过氧化物的摩尔比为1∶1～8。
  7. 根据权利要求5所述的改性心磷脂包被的纳米磁珠的制备方法，其特征在于，所述将心磷脂和过氧化物在第一溶剂存在的条件下充分反应的操作中，所述第一溶剂为二氯甲烷、三氯甲烷、氯仿、苯或甲苯。
  8. 根据权利要求5所述的改性心磷脂包被的纳米磁珠的制备方法，其特征在于，所述将所述改性心磷脂和生物素衍生物在第二溶剂存在的条件下充分反应的操作中，所述第二溶剂为DMSO、DMF、四氢呋喃或pH为6.5～8.5的磷酸盐缓冲液。
  9. 根据权利要求5所述的改性心磷脂包被的纳米磁珠的制备方法，其特征在于，所述将所述改性心磷脂和生物素衍生物在第二溶剂存在的条件下充分反应的操作中，所述改性心磷脂与所述生物素衍生物的摩尔比为1∶1.5～20。
  10. 根据权利要求5所述的改性心磷脂包被的纳米磁珠的制备方法，其特征在于，所述将所述生物素衍生物结合的改性心磷脂和链霉亲和素磁珠混合并充分反应的操作中，所述生物素衍生物结合的改性心磷脂的浓度为0.1mg/mL～1mg/mL，所述链霉亲和素磁珠的浓度为5mg/mL～15mg/mL。
PCT/CN2016/088014 2016-06-30 2016-06-30 改性心磷脂包被的纳米磁珠及其制备方法 WO2018000381A2 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP16906781.6A EP3480594A4 (en) 2016-06-30 2016-06-30 MODIFIED CARDIOLIPIN COATED MAGNETIC NANO BALLS AND PRODUCTION METHOD THEREFOR
PCT/CN2016/088014 WO2018000381A2 (zh) 2016-06-30 2016-06-30 改性心磷脂包被的纳米磁珠及其制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/088014 WO2018000381A2 (zh) 2016-06-30 2016-06-30 改性心磷脂包被的纳米磁珠及其制备方法

Publications (1)

Publication Number Publication Date
WO2018000381A2 true WO2018000381A2 (zh) 2018-01-04

Family

ID=60785781

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/088014 WO2018000381A2 (zh) 2016-06-30 2016-06-30 改性心磷脂包被的纳米磁珠及其制备方法

Country Status (2)

Country Link
EP (1) EP3480594A4 (zh)
WO (1) WO2018000381A2 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114950375A (zh) * 2022-06-02 2022-08-30 杭州隆基生物技术有限公司 链霉亲和素琼脂糖磁珠及其制备方法和运用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04503865A (ja) * 1989-12-27 1992-07-09 バクスター、ダイアグノスティックス、インコーポレイテッド カルジオリピン、フォスファチジルコリン及びコレステロールを固相に固定化する方法及びイムノアッセイ
GB0104057D0 (en) * 2001-02-20 2001-04-04 Babraham Inst Antiphospholipid antibody syndrome
BRPI0618713B8 (pt) * 2005-11-18 2021-07-27 The Government Of The United State Of America Representado Por The Secretary Of The Dept Of Health A cardiolipina modificada e seus usos

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114950375A (zh) * 2022-06-02 2022-08-30 杭州隆基生物技术有限公司 链霉亲和素琼脂糖磁珠及其制备方法和运用
CN114950375B (zh) * 2022-06-02 2024-04-30 杭州隆基生物技术有限公司 链霉亲和素琼脂糖磁珠及其制备方法和运用

Also Published As

Publication number Publication date
EP3480594A4 (en) 2020-03-11
EP3480594A2 (en) 2019-05-08

Similar Documents

Publication Publication Date Title
JP3844358B2 (ja) 新しい官能化親水性アクリジニウムエステル
JP2604993B2 (ja) 新規なビオチン化試薬
EP0322926B2 (en) Assays utilizing improved chemiluminescent esters, thioesters and amides
JP4068137B2 (ja) ビオチニル化した化学発光性標識、結合体、測定法及び測定法キット
CN107652343B (zh) 化合物、缀合物、试剂盒及其在检测雌醇中的用途
JPH09504505A (ja) プテリン誘導体およびその製法と利用法
WO2018000381A2 (zh) 改性心磷脂包被的纳米磁珠及其制备方法
WO2018000364A1 (zh) 改性心磷脂包被的纳米磁珠及其制备方法
CN107652344B (zh) 化合物、缀合物、试剂盒及其在检测睾酮或其类似物中的用途
CN109239368A (zh) 测定孕酮的方法及试剂盒
WO2018000339A1 (zh) 改性心磷脂包被的纳米磁珠及其制备方法
CN110872344B (zh) 一种氯霉素完全抗原及其制备方法与应用
WO2012066003A1 (en) Compound for the covalent attachment of the chemiluminescent probe n-(4-aminobutyl)-n-ethylisoluminol (abei) to target molecules and uses thereof
CN106124783B (zh) 改性心磷脂包被的纳米磁珠及其制备方法
CN106198956B (zh) 改性心磷脂包被的纳米磁珠及其制备方法
KR101159011B1 (ko) 햅텐 화합물 및 항체
CN106198957B (zh) 改性心磷脂包被的纳米磁珠及其制备方法
CN114195821B (zh) 一种心磷脂衍生物及其制备方法和应用
JPS63246382A (ja) ビオチニル試薬及びそれを用いるビオチニル化法
CN118240220A (zh) 聚醚胺化合物及其制备方法和封闭剂
JPS6061597A (ja) 新規ステロイド化合物，その製造方法，及びその化合物からなる免疫化学的測定方法によるエストリオ−ル−３−サルフエ−ト−１６α−グルクロナイド測定用抗原
JPH06148193A (ja) 抗リン脂質抗体結合用担体及び該担体を用いた免疫学的測定法
JPH05255263A (ja) アクリジウム化合物を用いた物質の測定法
US4436828A (en) Reactive asymmetrical dicarboxylic acid esters and reagents for the investigation of cardiac glycosides
CN107652345B (zh) 化合物、缀合物、试剂盒及其用途

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: 16906781

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2016906781

Country of ref document: EP

Effective date: 20190130