WO2018196544A1 - 检测人外周血EVs-TRPC5含量的试剂盒及方法 - Google Patents

检测人外周血EVs-TRPC5含量的试剂盒及方法 Download PDF

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WO2018196544A1
WO2018196544A1 PCT/CN2018/081053 CN2018081053W WO2018196544A1 WO 2018196544 A1 WO2018196544 A1 WO 2018196544A1 CN 2018081053 W CN2018081053 W CN 2018081053W WO 2018196544 A1 WO2018196544 A1 WO 2018196544A1
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trpc5
tube
magnetic beads
buffer
magnetic
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French (fr)
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马鑫
陆扬帆
黄飚
金坚
杜梦繁
张艺
张珏
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江南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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

Definitions

  • the invention relates to a kit and a method for detecting the content of EVs-TRPC5 in human peripheral blood.
  • Cancer also known as Malignant neoplasm, is a disease caused by a disorder that controls the growth and proliferation of cell growth. In addition to uncontrolled growth of cancer cells, cancer cells locally invade the surrounding normal tissues and even transfer to other parts of the body via the internal circulatory system or lymphatic system. Among various diseases, cancer is the "number one killer.” According to the World Health Organization report, there were approximately 12.7 million new cancer patients in the world in 2008, and 7.6 million died of cancer, especially in developing countries. The number of new cancer cases reached 56%. It is estimated that by 2020, the world will be Add 15 million cancer patients.
  • Breast cancer is a common cancer in women and is a serious threat to women's physical and mental health. According to the latest statistics released by the China Anti-Cancer Association, since the 1990s, the incidence and mortality of breast cancer have continued to rise steadily, the incidence rate has increased by 4.476% per year, and the mortality rate has increased at a rate of 3% per year. Chemotherapy is one of the main treatments for breast cancer.
  • the commonly used drugs are: (i) anthracyclines, taxanes, antimetabolites, anti-microtubules, alkylating agents, miscellaneous, etc.; Anti-estrogen, aromatase inhibitor, etc.; (iii) trastuzumab (anti-Her-2), bevacizumab (anti-VEGF).
  • P-glycoprotein is currently The most important and most important ABC drug transporter, encoded by the multidrug resistance gene mdr1; (2) reduce the drug activity by causing the drug to be catabolized, common enzymes include GST, CYP450, etc.; (3) drug target mutation or Changes in expression levels, etc.; (4) tumor heterogeneity; (5) cancer stem cell resistance.
  • Tumor markers refer to some intracellular substances that can respond to cancerous cells during tumorigenesis and development.
  • MUC1 is a mucin that is a transmembrane glycoprotein when expressed in normal cells. Normally, it is expressed at the apical end of epithelial cells of the breast, gastrointestinal tract and genitourinary tract, and glycosylation is complete. MUC1 normal epithelium provides lubrication and protection, mediates signal transduction and cell adhesion.
  • MUCl Through phosphorylation in breast cancer cell line MCF-7, MUCl binds to Rrb/SOS and is involved in receptor tyrosine kinase-mediated signal transduction, whereas tyrosine phosphorylation is a membrane receptor involved in signal transduction. key step.
  • the expression characteristics of breast cancer MUC1 include: high expression, low abnormal expression; glycosylation, high sialylation; unclear apical positioning, disordered polarity; overexpression of cytoplasm and cell surface, and these molecules will be derived from breast cancer cells Enter the serum.
  • Immunomagnetic beads also known as immunomagnetic microspheres, are spherical, spherical particles of uniform, superparamagnetic and protective shells, essentially composed of carrier microspheres and immunoligands. Its core is paramagnetic particles, the core outer layer encapsulates the polymer, and the outermost layer is the immunological ligand. Immunomagnetic beads technology is an immunological detection and separation technology based on specific antigen-antibody reactions. The antibody-coated magnetic beads are used as a carrier to form an antigen-antibody complex by binding the antibody to a specific antigen in the reaction medium, and the complex is directionally moved under the action of an external magnetic field, thereby achieving the purpose of separating the antigen.
  • TRPC5 is a subtype of transient receptor potential channels (TRP channels) family, a non-selective channel on the cell membrane that is transparent to calcium ions, mainly distributed in the brain, lung, testis and placenta, and mainly Participate in the formation of growth cones and brain development.
  • TRP channels transient receptor potential channels
  • TRPC5 is closely related to multidrug resistance of tumors.
  • TRPC5 is up-regulated in adriamycin-resistant breast cancer cells (MCF-7/ADM) and can indirectly mediate tumorigenesis.
  • MCF-7/ADM adriamycin-resistant breast cancer cells
  • Time-resolved fluoroimmunoassay has been increasingly valued and adopted due to its high specificity, high sensitivity, simple operation, low cost, and is especially suitable for the detection of large quantities of samples.
  • TRFIA Time-resolved fluoroimmunoassay
  • the present applicant provides a kit and method for detecting TRPC5 content in human peripheral blood based on time-resolved fluorescence immunoassay, and the technical scheme of the present invention is as follows:
  • a time-resolved fluorescent immunoassay kit for detecting TRPC5 content including MUC1-coated NHS magnetic beads, porous coated plates, buffer, TRPC5 polypeptide standards, antibodies against TRPC5, tritiated goat anti-rabbit antibodies, washing solutions And enhancer.
  • a time-resolved fluorescence immunoassay method for detecting TRPC5 content by using the above kit comprising the following steps:
  • the TRPC5 polypeptide synthesized by the step (1) is coupled with KLH via a coupling agent to obtain an immunogen
  • the sample to be tested in the step (5) is subjected to competitive inhibition detection analysis, the fluorescence intensity is measured, and the TRPC5 content in the sample is calculated according to the standard curve.
  • the coupling agent in the step (3) is Sulfo-SMCC.
  • the immunogen of the step (3) is immunized to a New Zealand white rabbit, and the antibody is prepared by subcutaneous injection, and purified using an antigen affinity column to obtain a polyclonal antibody against TRPC5 according to the step (4).
  • the solid phase carrier in the step (2) is a 96-well microplate.
  • the buffer in the step (5) is a phosphate buffer.
  • the immunomagnetic beads in the step (5) are NHS magnetic beads.
  • NHS magnetic beads were purchased from Suzhou Beaver Company.
  • the standard curve of the step (6) is the TRPC5 polypeptide synthesized in the step (1) as a standard, and diluted to 0 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 40 ng/mL, 50 ng/ mL, 80 ng/mL, 100 ng/mL, 200 ng/mL, 400 ng/mL, 500 ng/mL, 800 ng/mL, 1000 ng/mL series concentration, 0.1 mol/L pH 7.5 phosphate buffer for competitive inhibition Detection and analysis, the fluorescence value of the test result and the standard concentration were logarithmically log10, linearly fitted as a standard curve.
  • the preparation method of the MUCl antibody coated immunomagnetic beads is:
  • Protein solution preparation take 50-100 ⁇ g The MUC1 protein completely removes the primary amino group-containing substance from the original buffer by dialysis or desalting, and then uses Coupling Buffer to form a protein solution having a concentration of 3.0-5.0 mg/mL, and the prepared protein solution is 2-8. °C save spare;
  • the EP tube is placed in a magnetic separation frame, and the magnetic beads are enriched to remove the supernatant;
  • the EP tube is placed in a magnetic separation frame, enriched with magnetic beads, and the supernatant is removed;
  • the EP tube was vortexed for 15-30 s, placed on a vertical mixer, and reacted at 2-8 ° C overnight;
  • the invention does not need expensive instruments, has low detection cost, simple sample preparation, and can be operated and tested on site.
  • the method has high sensitivity, high accuracy, simple operation, strong specificity, fast detection speed and wide application. It is especially suitable for rapid detection of large quantities of samples.
  • the invention can be detected only by taking blood samples, which greatly reduces the suffering of tumor patients;
  • TRPC5 expression in blood EVs can initially detect the patient's drug resistance at this stage, provide a new reference project, and can be combined with other existing tumor drug resistance diagnosis technology, which is conducive to doctors to propose more accurate Treatment options to get the best results.
  • Figure 1 is a schematic diagram showing the results of clinical detection of samples after pretreatment with MUC1 magnetic beads using indirect TRPC5-TRFIA;
  • Figure 2 is a schematic diagram showing the results of clinical testing of samples without pretreatment with MUC1 magnetic beads using only indirect TRPC5-TRFIA.
  • MUC1antibody was purchased from Abcam, USA; Sulfo-SMCC, KLH, Sulfolink Resin was purchased from Pierce, USA, and Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma, USA.
  • the dialysis bag was purchased from Viskase, USA, and the molecular weight cut-off was 14 kD.
  • the P-10 column was purchased from BioRad Company of the United States, and the NHS magnetic beads were purchased from Beaver Company of Suzhou.
  • the TRPC5 polypeptide is synthesized as a coating, the TRPC5 polypeptide is coupled with KLH (the coupling agent is Sulfo-SMCC) as an immunizing antigen, and the TRPC5 polypeptide is coupled with BSA (the coupling agent is glutaraldehyde) as a detection antigen, and PBS is used.
  • KLH the coupling agent is Sulfo-SMCC
  • BSA the coupling agent is glutaraldehyde
  • PBS is used.
  • the immune antigen was diluted to 1 mg/ml, and stored in a refrigerator at -20 degrees.
  • each antigen was added to 1 ml of Freund's incomplete adjuvant, emulsified, and multiple points (at least 8 points) were injected subcutaneously on the back of the neck, and 2 New Zealand white rabbits were immunized with each antigen.
  • 1 ml of each antigen was added to 1 ml of Freund's incomplete adjuvant, emulsified, and multiple points (at least 8 points) were injected subcutaneously on the back of the neck, and 2 New Zealand white rabbits were immunized with each antigen.
  • the carotid artery was bled, the rabbit was sacrificed, the rabbit blood was placed at 4 degrees overnight, and centrifuged (4 degrees, 10000 rpm) for 30 minutes, and the supernatant was collected.
  • the TRPC5 polypeptide was ligated to the activated Sulfolink Resin to prepare an antigen affinity column, 1 ml of Sulfolink Resin was coupled with 1 mg of TRPC5 polypeptide, the affinity column was equilibrated with 10 column volumes of PBS, and the solution was drained; the rabbit serum was filtered through a 0.45 um filter to pass the antigen. Affinity column, run out of solution, collect flow through, balance 10 times column volume PBS, and drain the solution. Add 5 ml of antibody eluate and collect the eluate in 1 ml of each tube. The collected eluate was tested for absorbance at 280 nm, and the components with absorbance greater than 1.0 were combined, dialyzed against PBS, and identified by dialysis (protein concentration by UV absorption, antibody titer determined by Elisa).
  • Protein solution preparation take 50 ⁇ g The MUC1 protein completely removes the primary amino group-containing substance from the original buffer by dialysis or desalting, and then uses Coupling Buffer to prepare a protein solution with a concentration of ⁇ 3.0 mg/mL, and the prepared protein solution is stored at 4 ° C. .
  • the EP tube was vortexed for 15 s, placed on a vertical mixer, and reacted overnight at 4 °C.
  • Magnetic frame adsorbs magnetic beads and washes once with PBS
  • the TRPC5 polypeptide was diluted to a coating solution of 0.19 ⁇ g/L with 50 mmol/L Na2CO3-NaHCO3 pH 9.6 buffer, 100 ⁇ L of each well of a 96-well coating plate, and left at 4 ° C overnight. The coating solution was discarded, washed twice, and 150 ⁇ L of the above buffer containing 3 g/L of BSA was added and allowed to stand at room temperature for 2 hours. Discard the blocking solution, vacuum dry, and seal the strips and store at -20 °C.
  • TRPC5 standard solution preparation Dilute the TRPC5 standard to 10 ng/mL, 20 ng/mL, 40 ng/mL, 50 ng/mL, 80 ng/mL, 100 ng/mL, 200 ng/mL, 400 ng/mL, 500 ng/mL , 800 ng / mL, 1000 ng / mL series concentration, the dilution is 0.1 mol / L pH 7.5 phosphate buffer;
  • b. buffer 8mmol / L NaCl, 0.2% OVA, 50 ⁇ mol / L diethylene triamine pentaacetic acid (DTPA), 0.1mL / LTweeen - 80 and 0.1% by mass of NaN3 50mmol / L Tris-HCl pH 7.8;
  • DTPA diethylene triamine pentaacetic acid
  • washing liquid 14.5mmol / L NaCl, 0.2mL / L Tweeen-80 and 0.2% by mass of NaN3 50mmol / LTris-HCl pH 7.8;
  • Multi-channel pipettes are recommended if the sample size is large.
  • TRPC5 polypeptide Take the coated plate with the original (TRPC5 polypeptide), add 50 ⁇ L of TRPC5 standard or the treated sample to the respective wells, add 50 ⁇ L of anti-TRPC5 antibody diluted in buffer, and shake at 25 °C. Hour, the washing solution was washed 4 times, diluted with buffer solution of 100 ⁇ L Eu3+-goat anti-rabbit antibody, shaken at 25 ° C for 1 hour, washed 6 times, and added with 200 ⁇ L of the enhancement solution for 5 minutes, the fluorescence intensity cps was measured, calculated according to the standard curve The content of TRPC5 in the sample.
  • the coated plate was coated with the original (TRPC5 polypeptide), 50 ⁇ L of TRPC5 standard or patient serum was added to the respective wells, and 50 ⁇ L of anti-TRPC5 antibody diluted in buffer was added, and shaken at 25 ° C for 1 hour. Wash the washing solution 4 times, and dilute 100 ⁇ L of Eu3+-goat anti-rabbit antibody in buffer, shake at 25 °C for 1 hour, wash the washing solution 6 times, add 200 ⁇ L of the enhancement solution for 5 minutes, measure the fluorescence intensity cps, and calculate the sample according to the standard curve.
  • the TRPC5 content The TRPC5 content.
  • Peripheral blood was collected from the procoagulant serum tube, and the supernatant was collected after centrifugation at 1000-2000 r/min for 5-20 min; the NHS magnetic beads coated with MUC1 antibody were incubated with the supernatant for 1-1.5 h, and 50 ⁇ L was added to the buffer.
  • the magnetic beads have non-specific adsorption, and the effect of the non-specific adsorption on the results is not negligible;
  • the basis of the assay is the labeling of the immune response.
  • the porous coated plate coated with TRPC5 polypeptide is added to the test sample to the respective microwells, and then the anti-TRPC5 antibody is added to shake the reaction.
  • the TRPC5 in the free sample competes with the TRPC5 polypeptide on the microplate for anti-TRPC5 antibody, and the washing solution Washed, unligated TRPC5 antibody was removed in the washing step.
  • the Eu3+-goat anti-rabbit antibody was added, subjected to a labeled immunological reaction, and washed with a washing solution, and the unconjugated Eu3+-goat anti-rabbit antibody was removed in the washing step after the reaction.
  • the enhancement liquid is shaken, a strong fluorescence is emitted under the excitation of the ultraviolet lamp, and the fluorescence intensity cps is measured by a time-resolving fluorometer.
  • the fluorescence intensity is inversely proportional to the concentration in the sample, and the amount of TRPC5 in the sample can be determined by comparing the standard curve. .

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Abstract

一种检测人外周血中TRPC5含量的时间分辨荧光免疫检测试剂盒及检测方法：包被有TRPC5多肽的多孔包被板加入测试样品到各自的微孔中，再加入抗TRPC5抗体，振荡反应，游离样品中的TRPC5与微孔板上的TRPC5多肽竞争抗TRPC5抗体，洗涤液洗涤；加入Eu3+-羊抗兔抗体，进行标记免疫反应，再用洗涤液洗涤；加增强液振荡后，在紫外灯的激发下发射很强的荧光，用时间分辨荧光仪测定其荧光强度cps，荧光强度与样品中的浓度成反比，对照标准曲线即可确定样品中TRPC5的量。本发明根据血液EVs中TRPC5的表达的高低，可显示出肿瘤细胞耐药转变的情况，具有检测方法简单、速度快、灵敏度高、成本低的优点。

Description

检测人外周血EVs-TRPC5含量的试剂盒及方法 技术领域
本发明涉及检测人外周血EVs-TRPC5含量的试剂盒及方法。
 背景技术
癌症(Cancer)，亦称恶性肿瘤(Malignant neoplasm)，为由控制细胞生长增殖机制失常而引起的疾病。癌细胞除了生长失控外，还会局部侵入周遭正常组织甚至经由体内循环系统或淋巴系统转移到身体其他部分。在各种疾病中，癌症可谓是“头号杀手”。世界卫生组织报告显示，2008年全世界约有1270万癌症新增患者，760万死于癌症，尤其在发展中国家，癌症新增例数达56％，据推测到2020年前，全球每年将新增1500万癌症患者。
乳腺癌是女性常见的癌症，严重威胁女性身心健康。中国抗癌协会公布的最新统计数字显示，从20世纪90年代以来，乳腺癌发病率和死亡率均持续稳定上升，发病率以每年4.476％的速度增长，死亡率以每年3％的速度增长。化学药物治疗是乳腺癌的主要治疗手段之一，目前临床常用药物有：(i)蒽环类，紫杉类，抗代谢类，抗微管类，烷化剂，杂类等；(ii)抗雌激素，芳香化酶抑制剂等；(iii)曲妥珠单抗(抗Her-2)、贝伐单抗(抗VEGF)。
但是，据美国癌症协会报道，90％以上肿瘤患者死于不同程度的化疗耐药。对化疗药物产生耐药已经成为肿瘤治疗的一大难题。经过近几十年的研究，目前发现的耐药机制有(1)细胞膜ABC药物转运蛋白表达增强，减少药物摄入及增加药物排出，P-糖蛋白(P-glycoprotein，P-gp)是目前研究最多也是最为重要的ABC药物转运蛋白，由多药耐药基因mdr1编码；(2)通过使药物发生异化降低药物活性，常见的酶有GST、CYP450等；(3)药物作用靶点突变或表达水平变化等；(4)肿瘤异质性；(5)肿瘤干细胞耐药等。
目前，早期诊断肿瘤的最新、最有效的方法是通过验血寻找肿瘤标记物。肿瘤标志物指的是在肿瘤发生、发展的过程中，能反应细胞癌变的一些细胞内物质。临床上通过对各种肿瘤标志物的检测，可以对相应肿瘤做早期诊断、检测疗效和肿瘤复发状况。因此，在深入研究耐药机制的基础上，开发肿瘤耐药分子诊断指标、方法，实现试剂化，达到耐药早期、动态监测，在现阶段对于乳腺癌耐药的诊疗至关重要，也为优化药物结构设计、研发耐药逆转药物提供有益借鉴，因而具有重大而现实的社会价值。
MUC1是一种粘蛋白，在正常细胞表达时是一种跨膜糖蛋白，正常情况下，在乳腺、胃肠道及泌尿生殖道的上皮细胞顶端表达，糖基化完全。MUC1度正常上皮起润滑和保护作用、介导信号转导和细胞黏附。
在乳腺癌细胞株MCF-7中通过磷酸化，MUC1能结合Rrb/SOS，参与受体酪氨酸激酶介导的信号转导，而酪氨酸磷酸化是膜受体参与信号转导的一个关键步骤。乳腺癌MUC1的表达特点包括：高表达、异常表达低；糖基化、高唾液酸化；顶端定位不清，极性混乱；细胞浆和细胞表面都有过度表达，并且这些分子会从乳腺癌细胞进入血清。有研究者检测基底样乳腺癌患者，发现高表达MUC1，大约有92％的患者能检测出过表达的MUC1。
免疫磁珠，也称免疫磁性微球，是一种均匀、具有超顺磁性及保护性壳的球形小粒子，基本上由载体微球和免疫配基结合而成。其核心为顺磁性粒子，核心外层包裹聚合物，最外层是免疫配基。免疫磁珠技术，是一种以特异的抗原抗体反应为基础的免疫学检测和分离技术。它是以抗体包被的磁珠为载体，通过抗体与反应介质中特异性抗原结合，形成抗原—抗体复合物，此复合物在外加磁场的作用下发生定向移动，从而达到分离抗原的目的。
TRPC5是瞬时受体电位通道(transient receptor potential channels，TRP通道)家族的一个亚型，它是细胞膜上能通透钙离子的非选择性通道，主要分布于脑、肺、睾丸和胎盘，并主要参与生长锥的形成和脑的发育。本研究室研究发现，瞬时受体电位通道TRPC5与肿瘤多药耐药密切相关，TRPC5在耐阿霉素的乳腺癌细胞(MCF-7/ADM)中发生明显上调，并能间接介导肿瘤产生多药耐药的P-gp蛋白在多药耐药肿瘤细胞(如MCF-7/ADM)中的表达水平。本申请人针对该性质申请了相关专利，专利名称为《TRPC5作为药物靶点在逆转肿瘤多药耐药中的应用》(专利号：201210318389.5)《EVs-TRPC5在检测乳腺癌耐药程度中的应用》(授权公告号：CN103954770B)。
 技术问题
时间分辨荧光免疫分析(time-resolved fluoroimmunoassay,TRFIA)由于其特异性强、灵敏度高、操作简单、廉价，且特别适于大批量样品的检测等优点而越来越被人们所重视和采用。目前还没有针对TRPC5检测的时间荧光免疫分析法的专利和文献报道。
 技术解决方案
针对现有技术存在的上述问题，本申请人提供了一种基于时间分辨荧光免疫分析检测人外周血中TRPC5含量的试剂盒及方法，本发明的技术方案如下：
一种检测TRPC5含量的时间分辨荧光免疫分析试剂盒，包括MUC1包被的NHS磁珠、多孔包被板、缓冲液、TRPC5多肽标准品、TRPC5的抗体、铕标记的羊抗兔抗体、洗涤液及增强液。
一种利用上述试剂盒检测TRPC5含量的时间分辨荧光免疫分析方法，包括以下步骤：
(1)合成TRPC5多肽；
(2)用步骤(1)合成的所述TRPC5多肽作为包被原包被固相载体；
(3)用步骤(1)合成的所述TRPC5多肽通过偶联剂与KLH偶联得到免疫原；
(4)用步骤(3)得到的所述免疫原制备抗TRPC5的多克隆抗体；
(5)用促凝血清管采集新鲜血液，1000-2000r/min离心5-20min后收集上清液，用MUC1抗体包被的免疫磁珠与所述上清液混合后，经25-37℃震荡孵育0.5-2h或经2-8℃反应过夜，磁力架吸附磁珠，用缓冲液冲洗并重悬，得到待测样品；
(6)将步骤(5)所述待测样品进行竞争抑制检测分析，测量荧光强度，对照标准曲线计算样品中的TRPC5含量。
进一步的，步骤(3)所述偶联剂为Sulfo-SMCC。
进一步的，步骤(3)所述的免疫原免疫新西兰白兔，通过皮下多点注射制备抗体，使用抗原亲和柱进行纯化，获得步骤(4)所述的抗TRPC5的多克隆抗体。
进一步的，步骤(2)所述固相载体为96孔微孔板。
进一步的，步骤(5)所述缓冲液为磷酸盐缓冲液。
进一步的，步骤(5)所述免疫磁珠为NHS磁珠。
进一步的，所述NHS磁珠购自苏州Beaver公司。
进一步的，步骤(6)所述标准曲线为步骤(1)合成的所述TRPC5多肽作为标准品，稀释成为0ng/mL，5ng/mL，10ng/mL，20ng/mL，40ng/mL，50ng/mL，80ng/mL，100ng/mL，200ng/mL，400ng/mL，500ng/mL，800ng/mL，1000ng/mL系列浓度，稀释液为0.1mol/L pH7.5磷酸盐缓冲液，进行竞争抑制检测分析，将检测结果荧光值与标准品浓度取对数即log10，线性拟合，作为标准曲线。
进一步的，所述MUC1抗体包被的免疫磁珠制备方法为：
a.蛋白溶液配制：取50-100μg MUC1蛋白通过透析或者脱盐的方法彻底除去原有buffer里含伯胺基的物质，然后再用Coupling Buffer配成浓度为3.0-5.0mg/mL的蛋白溶液，将配制好的蛋白溶液于2-8℃保存备用；
b.取200-500μL 20％的磁珠悬浮液于EP管中；
c.将EP管置于磁性分离架内，富集磁珠，去除上清液；
d.加1mL 2-8℃的Washing Buffer A于离心管中，涡旋15-30s，使磁珠混合均匀；
e.将EP管置于磁性分离架内，富集磁珠，去除上清液；
f.加200-500μL蛋白溶液于EP管中，涡旋15-30s，使其混合均匀；
g.将EP管涡旋15-30s，置于垂直混合仪上，2-8℃反应过夜；
h.采用磁性分离架富集磁珠，保存流穿液；
i.加0.5-1mL Blocking Buffer于EP管中，涡旋15-30s，将EP管置于磁性分离架内，富集磁珠，弃上清液；
j.加0.5-1mL Blocking Buffer于EP管中，涡旋15-30s，将EP管置于垂直混合仪中室温反应2-4h；
k.将EP管置于磁性分离架内，富集磁珠，弃上清液；
l.加0.5-1mL超纯水于EP管中，充分混合，用磁力架富集磁珠，弃上清液；
m.加0.5-1mL Storage Buffer于EP管中，充分混合，用磁力架富集磁珠，弃上清液；重复该操作2次；
n.加入0.5-1mL Storage Buffer于EP管中，充分混合，2-8℃保存备用。
 有益效果
1、本发明不需要昂贵的仪器，检测成本低，样品前处理简单，现场即可操作检测，同时，该方法灵敏度高，准确度高，操作简便，特异性强，检测速度快，应用广泛，特别适用于大批样品的快速检测。
2、本发明只需抽取血液样本便可检测，极大程度地减轻了肿瘤病人的痛苦；
3、通过血液EVs中TRPC5表达的高低，可以初步检测出病人在该阶段的耐药情况，提供新的参考项目，同时可联合其它已有的肿瘤耐药诊断技术，有利于医生提出更加准确的治疗方案，以获得最佳疗效。
 附图说明
图1为样品经过MUC1磁珠预处理，后使用间接TRPC5-TRFIA方法临床检测结果示意图；
图2为样品未经过MUC1磁珠预处理，仅使用间接TRPC5-TRFIA方法临床检测结果示意图。
 本发明的实施方式
实施例中所使用的材料来源或制备方法如下：
1、主要试剂及耗材
MUC1antibody购自美国Abcam公司；Sulfo-SMCC、KLH、Sulfolink Resin购自美国Pierce公司，福氏完全佐剂、福氏不完全佐剂购自美国Sigma公司。透析袋购自美国Viskase公司，截留分子量14kD，P-10柱购自美国BioRad公司，NHS磁珠购自苏州Beaver公司。
2、实验仪器
磁力搅拌器(上海梅颖浦)，旋转混合器(上海强运)，紫外/可见光分光光度计(上海奥普勒)，高速离心机(日立)。
实施例：
1、免疫原与包被原制备
合成TRPC5多肽作为包被原，TRPC5多肽与KLH偶联(偶联剂为Sulfo-SMCC)作为免疫抗原，TRPC5多肽与BSA偶联(偶联剂为戊二醛)作为检测抗原,，用PBS将免疫抗原分别稀释为1mg/ml，分装冻存于-20度冰箱。
2、多克隆抗体制备
2.1动物免疫
第1天，每种抗原取1ml抗原加入1ml福氏完全佐剂，乳化(检验乳化程度：将一滴乳化抗原液滴入生理盐水中，若不散开，表明已达到要求)，颈背部皮下多点(至少8点)注射，每种抗原免疫2只新西兰白兔。第15天，每种抗原取1ml抗原加入1ml福氏不完全佐剂，乳化，颈背部皮下多点(至少8点)注射,每种抗原免疫2只新西兰白兔。第29天，每种抗原取1ml抗原加入1ml福氏不完全佐剂，乳化，颈背部皮下多点(至少8点)注射,每种抗原免疫2只新西兰白兔。第43天，每种抗原取1ml抗原加入1ml福氏不完全佐剂，乳化，颈背部皮下多点(至少8点)注射,每种抗原免疫2只新西兰白兔。第53天，颈动脉取血，将兔子处死，兔血在4度放置过夜，离心(4度，10000rpm)30分钟，收集上清液。
2.2多克隆抗体的纯化
TRPC5多肽连接到活化的Sulfolink Resin上，制备抗原亲和柱，1ml Sulfolink Resin偶联1mgTRPC5多肽，亲和柱用10倍柱体积PBS平衡，流尽溶液；兔血清经0.45um滤膜过滤，过抗原亲和柱，流尽溶液，收集流穿，10倍柱体积PBS平衡，流尽溶液。加入5ml抗体洗脱液，分管收集洗脱液，每管1ml。收集的洗脱液检测280nm处的吸光度，吸光度大于1.0的组分合并，对PBS透析，透析后的抗体鉴定(紫外吸收法检测蛋白浓度，Elisa确定抗体效价)。
3、MUC1包被磁珠方法
a.蛋白溶液配制：取50μg MUC1蛋白通过透析或者脱盐的方法彻底除去原有buffer里含伯胺基的物质，然后再用Coupling Buffer配成浓度为≥3.0mg/mL的蛋白溶液，将配制好的蛋白溶液于4℃保存备用。
b.取500μL 20％的磁珠悬浮液于1.5mL EP管中。
c.将EP管置于磁性分离架内，富集磁珠，去除上清液。
d.加1mL 2～8℃的Washing Buffer A于离心管中，涡旋15s，使磁珠混合均匀。
e.将EP管置于磁性分离架内，富集磁珠，去除上清液。
f.加200μL蛋白溶液于EP管中，涡旋30s，使其混合均。
g.将EP管涡旋15s，置于垂直混合仪上，4℃反应过夜。
h.采用磁性分离架富集磁珠，保存流穿液。
i.加1mL Blocking Buffer于EP管中，涡旋30s，将EP管置于磁性分离架内，富集磁珠，弃上清液。
j.加1mL Blocking Buffer于EP管中，涡旋30s，将EP管置于垂直混合仪中室温反应2h。
k.将EP管置于磁性分离架内，富集磁珠，弃上清液。
l.加1mL超纯水于EP管中，充分混合，用磁力架富集磁珠，弃上清液。
m.加1mL Storage Buffer于EP管中，充分混合，用磁力架富集磁珠，弃上清液。重复该操作2次。
n.加入1mL Storage Buffer于EP管中，充分混合，4℃保存备用。
*4、样品预处理方法(必要步骤)
a.用促凝血清管采集人外周血，1000r/min离心5min后收集上清液；
b.用包有MUC1抗体的NHS磁珠与上清液震荡孵育1h；
c.磁力架吸附磁珠，PBS洗一次；
d.用PBS重悬作为待测样品。
5、制备试剂盒和检测样品
5.1Eu3+-羊抗兔抗体制备
取溶解于50mmol/L PBS pH7.0的5g/L羊抗兔抗体1～2mL，经PD-10柱转换缓冲条件，洗脱液为含0.155mmol/L NaCl的50mmol/L Na2CO3-NaHCO3pH8.5缓冲液。收集蛋白峰，经紫外吸收分析定量(1.46A280-0.74A260)，用上述洗脱液稀释兔抗鼠抗体至2g/L。取500～1000μL稀释后的羊抗兔抗体加入含0.2～0.4mg的Eu3+-N2-[p-异氰酸-苄基]-二乙烯三胺四乙酸(Eu3+-DTTA)的小瓶中，30℃磁力搅拌反应20小时。反应液经用80mmol/LTris-HCl pH7.8缓冲液平衡的SepharoseCL-6B柱(1×40cm)层析，A280监测收集蛋白峰，稀释分装备用。
5.2包被板固相抗原制备
将TRPC5多肽用50mmol/L Na2CO3-NaHCO3pH9.6缓冲液稀释至0.19μg/L的包被液，96孔包被板各孔加100μL，4℃放置过夜。弃去包被液，冲洗2次，加150μL含3g/L BSA的上述缓冲液封闭，室温放置2h。弃去封闭液，真空抽干，板条密封后置-20℃冷冻保存。
5.3试剂的配制
a.TRPC5标准品溶液配制：将TRPC5标准品，稀释成为10ng/mL，20ng/mL，40ng/mL，50ng/mL，80ng/mL，100ng/mL，200ng/mL，400ng/mL，500ng/mL，800ng/mL，1000ng/mL系列浓度，稀释液为0.1mol/L pH7.5磷酸盐缓冲液；
b.缓冲液：8mmol/L NaCl、0.2％OVA、50μmol/L二乙烯三胺五乙酸(DTPA)、0.1mL/LTweeen-80及0.1质量％NaN3的50mmol/L Tris-HCl pH7.8；
c.洗涤液为：14.5mmol/L NaCl、0.2mL/L Tweeen-80及0.2质量％NaN3的50mmol/LTris-HCl pH7.8；
d.增强液的配制：由15μmolβ-萘甲酰三氟丙酮、50μmol三正辛基氧化膦及1mL曲拉通X-100加入pH3.2邻苯二甲酸氢钾缓冲液中，再定容至1L配制而成。
5.4测定之前注意事项：
a.使用之前将所有试剂回升至室温(18-30℃)。
b.使用之后立即将所有试剂放回2-8℃。
c.如果样品量大建议使用多通道移液器。
d.取出需用数量的微孔板及框架，将不用的微孔板放进原锡箔袋中并且与提供的干燥剂一起重新密封，保存于2-8℃。
5.5具体检测步骤如下：
取包被有包被原(TRPC5多肽)的多孔包被板，加入50μL的TRPC5标准品或处理好的样品到各自的微孔中，加50μL以缓冲液稀释的抗TRPC5抗体，25℃振荡1小时，洗涤液洗4次，加以缓冲液稀释的100μL Eu3+-羊抗兔抗体，25℃振荡1小时，洗涤液洗6次，加200μL增强液振荡5分钟后测量荧光强度cps，根据标准曲线计算样品中的TRPC5含量。
6间接TRPC5-TRFIA方法临床应用
收集临床血清样本，乳腺癌化疗耐药21例，化疗尚可21例，正常人12例，使用间接 TRPC5-TRFIA方法检测乳腺癌来源TRPC5含量，结果见图1。对照组与化疗尚可组比较无明显差异(p>0.6)，而化疗耐药组与化疗尚可组差异有统计学意义(p<0.0001)。
对比例：
1、未进行样品预处理的间接TRPC5—TRFIA方法临床应用
1.1操作步骤
取包被有包被原(TRPC5多肽)的多孔包被板，加入50μL的TRPC5标准品或病人血清到各自的微孔中，加50μL以缓冲液稀释的抗TRPC5抗体，25℃振荡1小时，洗涤液洗4次，加以缓冲液稀释的100μL Eu3+-羊抗兔抗体，25℃振荡1小时，洗涤液洗6次，加200μL增强液振荡 5分钟后测量荧光强度cps，根据标准曲线计算样品中的TRPC5含量。
1.2实验结果
收集临床血清样本，乳腺癌化疗耐药21例，化疗尚可21例，正常人12例，使用间接TRPC5-TRFIA方法检测乳腺癌来源TRPC5含量，结果见图2。化疗耐药组与化疗尚可组比较无明显差异(p>0.1)。
2、使用MUC1磁珠与TRPC5夹心法检测
2.1操作步骤
用促凝血清管采集人外周血，1000-2000r/min离心5-20min后收集上清液；用包有 MUC1抗体的NHS磁珠与上清液震荡孵育1-1.5h，加50μL以缓冲液稀释的抗TRPC5抗体，25-37℃振荡1-1.5小时，磁性分离，洗涤液洗4次，加以缓冲液稀释的100μL Eu3+-羊抗兔抗体，25-37℃振荡1-1.5小时，磁性分离，洗涤液洗6次，加200μL增强液振荡5分钟后测量荧光强度cps，根据标准曲线计算样品中的TRPC5含量。
2.1存在问题
(1)磁珠存在非特异性吸附，且该非特异性吸附对结果的影响不可忽略；
(2)磁珠为纯手工操作，造成批内、批间差异(CV)较大，影响实验结果。
本发明测定方法：测定的基础是标记免疫反应。包被有TRPC5多肽的多孔包被板，加入测试样品到各自的微孔中，再加入抗TRPC5抗体，振荡反应，游离样品中的TRPC5与微孔板上的TRPC5多肽竞争抗TRPC5抗体，洗涤液洗涤，没有连接的TRPC5抗体在洗涤步骤中被除去。加入Eu3+-羊抗兔抗体，进行标记免疫反应，再用洗涤液洗涤，反应后没有连接的Eu3+-羊抗兔抗体在洗涤步骤中被除去。加增强液振荡后，在紫外灯的激发下发射很强的荧光，用时间分辨荧光仪测定其荧光强度cps，荧光强度与样品中的浓度成反比，对照标准曲线即可确定样品中TRPC5的量。
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
上面结合附图对本发明专利进行了示例性的描述，显然本发明专利的实现并不受上述方式的限制，只要采用了本发明专利的方法构思和技术方案进行的各种改进，或未经改进将本发明专利的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (10)

  1. 一种检测TRPC5含量的时间分辨荧光免疫分析试剂盒，其特征在于：包括MUC1包被的NHS磁珠、多孔包被板、缓冲液、TRPC5多肽标准品、TRPC5的抗体、铕标记的羊抗兔抗体、洗涤液及增强液。
  2. 一种利用权利要求1所述的试剂盒检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于包括以下步骤：
    (1)合成TRPC5多肽；
    (2)用步骤(1)合成的所述TRPC5多肽作为包被原包被固相载体；
    (3)用步骤(1)合成的所述TRPC5多肽通过偶联剂与KLH偶联得到免疫原；
    (4)用步骤(3)得到的所述免疫原制备抗TRPC5的多克隆抗体；
    (5)用促凝血清管采集新鲜血液，1000-2000r/min离心5-20min后收集上清液，用MUC1抗体包被的免疫磁珠与所述上清液混合后，经25-37℃震荡孵育0.5-2h或经2-8℃反应过夜，磁力架吸附磁珠，用缓冲液冲洗并重悬，得到待测样品；
    (6)将步骤(5)所述待测样品进行竞争抑制检测分析，测量荧光强度，对照标准曲线计算样品中的TRPC5含量。
  3. 根据权利要求2所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于：步骤(3)所述偶联剂为Sulfo-SMCC。
  4. 根据权利要求2所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于：步骤(3)所述的免疫原免疫新西兰白兔，通过皮下多点注射制备抗体，使用抗原亲和柱进行纯化，获得步骤(4)所述的抗TRPC5的多克隆抗体。
  5. 根据权利要求2所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于：步骤(2)所述固相载体为96孔微孔板。
  6. 根据权利要求2所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于：步骤(5)所述缓冲液为磷酸盐缓冲液。
  7. 根据权利要求2所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于：步骤(5)所述免疫磁珠为NHS磁珠。
  8. 根据权利要求7所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于：所述NHS磁珠购自苏州Beaver公司。
  9. 根据权利要求2所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于：步骤(6)所述标准曲线为步骤(1)合成的所述TRPC5多肽作为标准品，稀释成为0ng/mL， 5ng/mL，10ng/mL，20ng/mL，40ng/mL，50ng/mL，80ng/mL，100ng/mL，200ng/mL，400ng/mL，500ng/mL，800ng/mL，1000ng/mL系列浓度，稀释液为0.1mol/L pH7.5磷酸盐缓冲液，进行竞争抑制检测分析，将检测结果荧光值与标准品浓度取对数即log10，线性拟合，作为标准曲线。
  10. 根据权利要求2～9所述的一种检测TRPC5含量的时间分辨荧光免疫分析方法，其特征在于，所述MUC1抗体包被的免疫磁珠制备方法为：
    a.蛋白溶液配制：取50-100μg MUC1蛋白通过透析或者脱盐的方法彻底除去原有 buffer里含伯胺基的物质，然后再用Coupling Buffer配成浓度为3.0-5.0mg/mL的蛋白溶液，将配制好的蛋白溶液于2-8℃保存备用；
    b.取200-500μL 20％的磁珠悬浮液于EP管中；
    c.将EP管置于磁性分离架内，富集磁珠，去除上清液；
    d.加1mL 2-8℃的Washing Buffer A于离心管中，涡旋15-30s，使磁珠混合均匀；
    e.将EP管置于磁性分离架内，富集磁珠，去除上清液；
    f.加200-500μL蛋白溶液于EP管中，涡旋15-30s，使其混合均匀；
    g.将EP管涡旋15-30s，置于垂直混合仪上，2-8℃反应过夜；
    h.采用磁性分离架富集磁珠，保存流穿液；
    i.加0.5-1mL Blocking Buffer于EP管中，涡旋15-30s，将EP管置于磁性分离架内，富集磁珠，弃上清液；
    j.加0.5-1mL Blocking Buffer于EP管中，涡旋15-30s，将EP管置于垂直混合仪中室温反应2-4h；
    k.将EP管置于磁性分离架内，富集磁珠，弃上清液；
    l.加0.5-1mL超纯水于EP管中，充分混合，用磁力架富集磁珠，弃上清液；
    m.加0.5-1mL Storage Buffer于EP管中，充分混合，用磁力架富集磁珠，弃上清液；重 复该操作2次；
    n.加入0.5-1mL Storage Buffer于EP管中，充分混合，2-8℃保存备用。
PCT/CN2018/081053 2017-04-28 2018-03-29 检测人外周血EVs-TRPC5含量的试剂盒及方法 WO2018196544A1 (zh)

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