WO2016127276A1 - 尿液甲胎蛋白的应用 - Google Patents

尿液甲胎蛋白的应用 Download PDF

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WO2016127276A1
WO2016127276A1 PCT/CN2015/000624 CN2015000624W WO2016127276A1 WO 2016127276 A1 WO2016127276 A1 WO 2016127276A1 CN 2015000624 W CN2015000624 W CN 2015000624W WO 2016127276 A1 WO2016127276 A1 WO 2016127276A1
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fetoprotein
urine
alpha
antibody
application
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张曼
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张曼
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the invention relates to a new use of urinary alpha fetoprotein, in particular to the expression of alpha fetoprotein in urine and its application in the detection of urine content.
  • Alpha-fetoprotein is the main component of embryonic plasma protein, and its gene encoding human albumin and vitamin D binding protein belongs to the albumin gene family, which are located on chromosome 4. It is produced by yolk sac and fetal liver. It is mainly composed of fetal liver synthesis after 12 weeks of gestation. The serum concentration during fetal period is very high, and it decreases after birth. From February to March, alpha-fetoprotein is basically replaced by albumin. More difficult to check out.
  • Alpha-fetoprotein has many important physiological functions, the most basic of which is the transport function. Alpha-fetoprotein can bind estrogen, fatty acid, bilirubin, Cu2+ and Ni2+. One of the most important functions is to transport fatty acids. Both fetal and embryonic alpha-fetoprotein can bind 2 to 3 fatty acid molecules. Part is unsaturated fatty acids. In addition, alpha-fetoprotein is involved in cell proliferation, regulation of metabolism, and interaction between macrophages and T lymphocytes. The role of alpha-fetoprotein in the immune response is immunosuppression, mainly manifested by inhibition of the maternal immune response to embryonic development and tumor patients' immune response to tumors.
  • Alpha-fetoprotein can also be synthesized in hepatocellular carcinoma, embryonic tumors, and some extrahepatic tumors. Therefore, alpha-fetoprotein is associated with primary liver cancer, gastric cancer, lung cancer, pancreatic cancer, cholangiocarcinoma, and testicular tumor. It can also be elevated in patients with partial hepatitis (15% to 58%) and cirrhosis (11% to 47%).
  • alpha-fetoprotein Due to the special source of alpha-fetoprotein, it is difficult to detect it in the serum of normal adults. This only indicates that the serum concentration is too low, exceeding the minimum detection limit of the general test, so that it cannot be detected.
  • our research has confirmed that a part of alpha-fetoprotein is found in the urine of normal people. Therefore, normal people can still synthesize a certain amount of alpha-fetoprotein, which plays a certain role in normal physiological metabolism, and can also be used in urine. A certain amount of a fragment of alpha-fetoprotein is stably present.
  • Urine is the ultrafiltrate of blood, which is the terminal metabolite produced by the body.
  • the composition change is a specific manifestation of certain disease states, and the collection of urine specimens is non-invasive, multi-volume, patient compliance, and not
  • the advantage of requiring the help of medical personnel, in summary, the present invention aims to apply the advantages of urine specimens, and provides a simple kit for detecting alpha-fetoprotein fragments in urine.
  • amino acid sequence of the urine alpha-fetoprotein is as shown in SEQ ID NO: 1:
  • the preparation is a urine alpha-fetoprotein detection kit.
  • the kit is an antibody antigen reaction.
  • the antigen-antibody reaction is coated or labeled with a urine alpha-fetoprotein or polypeptide and an antibody thereof in a solid phase or liquid phase carrier.
  • the inventors first collected random urine specimens of normal physical examination, took the supernatant after centrifugation, and purified and separated urine specimens using weak cation exchange magnetic beads. 1 ⁇ l of sample with 10 ⁇ l of substrate (0.3% ⁇ -cyano-4- After mixing hydroxycinnamic acid and HCCA), 1 ⁇ l was spotted on an Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target plate, and the sample was ionized and subjected to mass spectrometry. Data in the range of 1000-10000 Da was collected to obtain different mass loads. A mass spectrometric peptide map composed of protein peaks.
  • the present invention confirmed by studies that alpha fetoprotein can stably appear in the urine of a person who is normally examined. Therefore, it is proposed to detect the application of urine protein alpha fetoprotein in urine related examination.
  • the invention exerts the advantage of non-invasiveness of urine specimens, and uses random urine specimens to detect alpha-fetoprotein or polypeptide.
  • Figure 1 is the average value of all points in the normal physical examination specimens between 1000 and 10000.
  • Figure 2 is a scatter plot of the point of mass-to-charge ratio of 1893.6 expressed in 30 normal medical specimens.
  • Figure 3 is a mass spectrum of alpha-fetoprotein.
  • the urine sample was taken out from the -80 ° C refrigerator, recombined at 4 ° C, centrifuged (3000 rpm, 10 min), and the supernatant was taken for use.
  • the weak cation magnetic beads (MB-WCX) were equilibrated at room temperature and the magnetic bead suspension was manually mixed.
  • the magnetic beads are separated from the suspended liquid. Use a sample gun to remove the suspended clear liquid. The tip should avoid contact with the magnetic beads and avoid picking up the magnetic beads.
  • FIG. 1 which shows the average value of all the mass-to-charge ratio points between 1000-10000 Da in 30 urine samples; the peak area as a quantitative standard, and FIG. 2 shows the expression of alpha fetoprotein in all urine samples. It can be seen from the figure that m/z 1893.6 has a peak area greater than 600 in all specimens.
  • the magnetic bead eluate in the sample tube was evaporated to dryness, dissolved in 20 ul of mobile phase A (5% acetonitrile, 0.1% formic acid in water), and transferred to a sample bottle.
  • the injection volume was 18 ul, firstly desalted into the trap column at a rate of 15 ⁇ l/min, and the capture time was 3 min. Then enter the analytical column at a flow rate of 400 nl / min for gradient elution, the elution gradient is 5% B-50% B-80% B-80% B-50% B-5% B (mobile phase B: 95% acetonitrile) , 0.1% aqueous solution of formic acid, see Table 1).
  • the analysis time was 60 min, the column temperature was 35 ° C, and all eluted components were analyzed by mass spectrometry.
  • Nano ion source spray voltage 1.8kV; mass spectrometry mode for data dependence and dynamic exclusion, scan range 400-2000m/z; first-order scan (MS) using Obitrap, resolution set to 100000; CID and secondary scan using LTQ; A single isotope of the strongest 10 ions was selected as the parent ion in the MS spectrum for MS/MS (single charge exclusion, not as parent ion).
  • the mass spectrometry scan time was 60 min. Sequest TM search was performed using the data analysis software Bioworks Browser 3.3.1 SP1.
  • the search database is International Protein Index (IPI human v3.45 fasta with 71983entries).
  • the mother ion error was set to 100 ppm
  • the fragment ion error was set to 1 Da
  • the digestion method was non-enzymatically cut
  • the variable modification was methionine oxidation.
  • the search result parameters are set to deltacn ⁇ 0.10, two charges Xcorr 2.6, three charges Xcorr 3.1, and three charges above Xcorr 3.5.
  • the protein alpha-fetoprotein is retrieved in the database, and the mass spectrum of the alpha-fetoprotein is shown in Figure 3.
  • the antibody and antigen concentrations were determined according to Pierce's BCA Protein Concentration Kit instructions, and then the rabbit anti-human alpha-fetoprotein polyclonal antibody (Abcam) was diluted to a concentration using a standard checkerboard assay. 10.0 ng/ml, 1.0 ng/ml, and 0.1 ng/ml were coated on solid phase ELISA plates and liquid phase magnetic beads, respectively, each concentration consisting of three wales, overnight at 4 ° C, and washed 3 times. A strong positive antigen solution was added to one of the transverse coated wells, a weak positive antigen solution was added to the other row, and a negative control was added to the third row. Incubate for 2 hours at 37 ° C and wash 3 times.
  • a murine anti-human alpha fetoprotein monoclonal antibody (Abeam) was added, incubated at 37 ° C for 1 hour, and washed 3 times.
  • the labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, washed 4 times, the substrate was added, and the mixture was allowed to stand at room temperature for 20 minutes in the dark, and the stop solution was added for reading. Choose the optimal concentration of coated antibody.
  • the rabbit anti-human alpha-fetoprotein polyclonal antibody was diluted with a coating buffer, added to a solid phase microplate and liquid phase magnetic beads, and gently shaken overnight at 4 °C.
  • the uncoated liquid was poured out, washed 3 times, and a blocking solution was added to prevent non-specific binding sites, incubated at 37 ° C for 1 hour, and washed 3 times. Store at 4 ° C for later use.
  • the kit is divided into a mouse anti-human alpha fetoprotein monoclonal antibody, a labeled secondary antibody, and the like.
  • the alpha-fetoprotein recombinant protein (OriGene, Germany) was diluted with PBS to 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 10 ng/ml, 2 ng/ml, 0.5 ng/ml, 0.05 ng/ml, 0.01. Ng/ml, 0 ng/ml, 100 ul per well was added to the above coated ELISA plate and the liquid magnetic beads, incubated at 37 ° C for 2 hours, and washed 3 times.
  • the mouse anti-alphafetoprotein monoclonal antibody was diluted 1:2000, 100 ul was added per well, incubated at 37 ° C for 1 hour, and washed 3 times.
  • the labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, and washed 3 times.
  • the substrate was added to room temperature for 15 minutes, and the stop solution was added for reading.
  • the lowest amount of alpha-fetoprotein was detected.
  • the results showed that the reagent could detect the concentration of 0.01 ng/ml alpha-fetoprotein, indicating a higher detection sensitivity.
  • the above experiments show that the kit of the present invention detects the content of alpha-fetoprotein in the urine sample, and has high sensitivity.
  • the minimum detection limit of the sample is 0.01 ng/ml, and the recovery rate is 90% ⁇ 13%.
  • the kit requires fewer instruments and requires only a microplate reader, an oscillator, a centrifuge, a pipette, etc., and the cost is low.
  • the inventors examined the content of alpha-fetoprotein in 100 normal urine urine samples with an accuracy of more than 97%, and had good specificity.

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Abstract

一种尿液甲胎蛋白(Alpha-fetoprotein，AFP)的应用，具体为尿液甲胎蛋白在尿液中的表达及其用于尿液含量检测中的应用。通过研究证实尿液中甲胎蛋白有较高丰度的表达，因此通过对尿液中甲胎蛋白表达量的检测获得对于疾病诊断的信息。发挥尿液标本获取无创保存方便的优势，利用尿液标本检测甲胎蛋白。

Description

尿液甲胎蛋白的应用 技术领域
本发明涉及尿液甲胎蛋白的新用途，具体涉及甲胎蛋白在尿液中的表达及其在尿液含量检测中的应用。
背景技术
甲胎蛋白是胚胎期血浆蛋白的主要成分，其编码基因与人血白蛋白、维生素D结合蛋白的基因同属白蛋白基因家族，都位于第4号染色体上。由卵黄囊和胎肝产生，大约妊娠12周后以胎肝合成为主，胎儿时期血清浓度非常高，出生后则下降，至生后2～3月甲胎蛋白基本被白蛋白替代，血液中较难检出。
甲胎蛋白有很多重要的生理功能，最基本的就是运输功能。甲胎蛋白能够结合***、脂肪酸、胆红素、Cu2+及Ni2+，其中最主要的功能之一就是运输脂肪酸，不管是肝脏还是胚胎来源的甲胎蛋白都可以结合2～3个脂肪酸分子，大部分是不饱和脂肪酸。此外，甲胎蛋白还参与细胞的增殖、代谢的调控以及巨噬细胞和T淋巴细胞之间的相互作用。甲胎蛋白在免疫应答中的作用是免疫抑制，主要表现为抑制母体对胚胎发育的免疫应答以及肿瘤患者对肿瘤的免疫应答。在肝细胞癌、胚胎性肿瘤、及部分肝外肿瘤也可合成甲胎蛋白，所以，甲胎蛋白与原发性肝癌、胃肿瘤、肺癌、胰腺癌和胆管癌、睾丸肿瘤等相关。在部分肝炎(15％～58％)、肝硬化(11％～47％)的患者中也可有升高。
由于甲胎蛋白的来源特殊，在正常成年人的血清中很难检出，这只是说明血清浓度太低，超过了一般检测的最低检测限，以至于检测不出。但是我们的研究证实在正常人的尿液中发现了甲胎蛋白的片段，所以，正常人还是能合成一定量的甲胎蛋白，在正常的生理代谢中发挥一定的作用，尿液中也能稳定出现一定量的甲胎蛋白的片段。
尿液是血液的超滤液，是机体产生的终末代谢产物，其组成的改变是某些疾病状态的特异表现，并且尿液标本的收集具有无创、量多、患者依从性高，且不需要医护人员的帮助的优势，综上所述，本发明旨在应用尿液标本的优势，提供一种简便的检测尿液甲胎蛋白片段的试剂盒。
发明内容
本发明的目的在于提供一种尿液甲胎蛋白在尿液中的表达及其用于尿液含量检测中的应用。
优选地，所述尿液甲胎蛋白的氨基酸序列如SEQ ID NO：1所示：
Figure PCTCN2015000624-appb-000001
优选地，所述制剂为尿液甲胎蛋白检测试剂盒。所述试剂盒为抗体抗原反应。
优选地，所述抗原抗体反应由尿液甲胎蛋白或多肽以及其抗体被包被或标记在固相或液相载体。
发明人首先收集了正常体检的随机尿液标本，离心后取上清，利用弱阳离子交换磁珠纯化和分离尿液标本。将1μl标本与10μl基质(0.3％的α-氰基-4- 羟基肉桂酸，HCCA)混匀后，取1μl点在Anchorchip(Autoflex MALDI TOF，Bruker-Dalton)靶板上，标本离子化后进行质谱分析，采集1000-10000Da范围内的数据，获得由不同质荷比的蛋白峰构成的质谱多肽图。应用ClinProTools2.1分析软件所有质谱图进行分析，筛选出稳定表达的高丰度蛋白多肽。然后发明人利用液相色谱串联质谱仪对这些筛选出的蛋白多肽进行鉴定，在International Protein Index(IPI human v3.45fasta with 71983entries)数据库检索得到甲胎蛋白(Alpha-fetoprotein，甲胎蛋白蛋白)蛋白。
本发明通过研究证实甲胎蛋白能够在正常体检的人的尿液中稳定出现。从而提出检测尿液蛋白甲胎蛋白可用于尿液相关检查中的应用。
本发明发挥尿液标本获取无创的优势，利用随机尿标本检测甲胎蛋白或者多肽。
为让本发明的上述和其它目的、特征和优点能更明显易懂，下文特举较佳实施例，并配合附图，作详细说明如下。
附图说明
图1是质荷比为1000-10000之间所有点在30例正常体检标本中的平均值。
图2是质荷比为1893.6的点在30例正常体检标本中表达的散点图。
图3是甲胎蛋白的质谱图。
具体实施方式
实施例1尿液标本的收集与处理
收集30例正常体检(首都医科大学附属北京世纪坛医院体检中心)随机清洁中段尿液标本，2h内离心(1500rpm，5min)，保留上清。分装后-80℃冰箱冻存。
实施例2磁珠纯化和分离尿液标本中的多肽
从-80℃冰箱取出尿液标本，4℃复融，离心(3000rpm，10min)后取上清备用。室温下平衡弱阳离子磁珠(MB-WCX)，并手动混匀磁珠悬浮液。在样品管中加入10ul MB-WCX和10ul磁珠结合缓冲液，加样枪上下吹打混匀，避免起泡。向样品管中加入5ul尿液上清，充分混匀后磁力架上静置1分钟，磁珠与悬浮的液体分离。用加样枪除去悬浮的清澈液体，枪头应避免接触到磁珠，避免吸走磁珠。在样品管中加入100ul磁珠清洗缓冲液，充分混匀后将样品管在磁力架上静置1分钟，磁珠贴壁，与悬浮的液体分离，用加样枪除去悬浮的液体。 重复3次，弃去悬浮液。在样品管中加入5ul磁珠洗脱缓冲液，反复吸打10次以上，使磁珠和洗脱缓冲液混匀，避免起泡。将样品管放置于磁力架上，静置2min，使磁珠与悬浮液充分分离，将上清液(洗脱液)移入已标记的新的0.5ml样品管。加入5ul稳定缓冲液，加样枪小心吹打混匀。
实施例3尿液标本的点靶与多肽谱图的生成
用标准品校正仪器后，将1μl洗脱液与10μl基质(0.3％的α-氰基-4-羟基肉桂酸，HCCA)混匀，取1μl点在Anchorchip(Autoflex MALDI TOF，Bruker-Dalton)靶板上，室温干燥。通过氮激光器照射使标本离子化后进行质谱分析，采集1000-10000Da范围内的数据，获得由不同质荷比的蛋白峰构成的质谱图。对于每一个MALDI结晶点来说，共照射400次激光(每个结晶点的8个不同的位置各照射50次)，平均值代表一个标本，从而得到所有样本的多肽图谱。应用ClinProTools2.1分析软件对正常对照组、2型糖尿病无并发症及合并症组和2型糖尿病合并早期肾损伤组的质谱图进行分析，筛选差异性多肽。筛选条件：质量范围1000-10000Da，信噪比(S/N)大于5，质量飘移不超过0.1％，所有质谱图根据总离子流进行归一化。具体请参照图1，其示30例尿液标本中1000-10000Da之间所有质荷比的点的平均值；峰面积作为定量的标准，图2示出甲胎蛋白在所有尿标本中的表达，从图中可以看出m/z 1893.6在所有标本中的峰面积都大于600。
实施例4蛋白多肽的鉴定
将样品管中磁珠洗脱液旋转蒸干，加入20ul流动相A(5％乙腈，0.1％甲酸的水溶液)溶解，转移至进样瓶中。进样体积18ul，首先以15μl/min的速度进入捕集柱脱盐，捕集时间3min。然后以400nl/min的流速进入分析柱进行梯度洗脱，洗脱梯度为5％B-50％B-80％B-80％B-50％B-5％B(流动相B：95％乙腈，0.1％甲酸的水溶液，见表1)。分析时间60min，色谱柱温度35℃，所有洗脱成分进入质谱仪分析。Nano离子源，喷雾电压1.8kV；质谱模式为数据依赖及动态排除，扫描范围400-2000m/z；一级扫描(MS)使用Obitrap，分辨率设定为100000；CID及二级扫描使用LTQ；在MS谱图中选取强度最强的10个离子的单一同位素作为母离子进行MS/MS(单电荷排除，不作为母离子)。质谱扫描时间60min。应用数据分析软件BioworksBrowser 3.3.1 SP1进行SequestTM检索。检索数据库为International Protein Index(IPI human v3.45 fasta with 71983entries)。母离子 误差设定为100ppm，碎片离子误差设为1Da，酶切方式为非酶切，可变修饰为甲硫氨酸氧化。检索结果参数设定为deltacn≥0.10，两电荷Xcorr 2.6，三电荷Xcorr 3.1，三电荷以上Xcorr 3.5。在数据库中检索得到蛋白甲胎蛋白，甲胎蛋白蛋白的质谱图请参照图3。
表1分析柱梯度洗脱的程序
Figure PCTCN2015000624-appb-000002
实施例5试剂盒的制备
1.包被抗体和酶标抗体工作浓度的选择
按照Pierce公司的BCA蛋白浓度测定试剂盒说明书操作，测定抗体及抗原的浓度，然后采用标准的棋盘测定方法，用包被缓冲液将兔抗人甲胎蛋白多克隆抗体(Abcam公司)稀释至浓度为10.0ng/ml、1.0ng/ml和0.1ng/ml，分别在固相ELISA板和液相磁珠上包被，每个浓度包括三个纵行，4℃过夜，洗涤3次。在其中一个横行的包被孔中加入强阳性抗原液，另一横行中加入弱阳性抗原液，第三行加入阴性对照。37℃孵育2小时，洗涤3次。加入鼠抗人甲胎蛋白单克隆抗体(Abcam公司)，37℃孵育1小时，洗涤3次。加入标记的二抗，37℃孵育30分钟，洗涤4次，加入底物，室温避光放置20分钟，加入中止液，读数。选择包被抗体最佳浓度。
2.试剂盒的制备
将兔抗人甲胎蛋白多克隆抗体用包被缓冲液进行稀释，将其加入到固相微孔板和液相磁珠中，4℃包被轻摇过夜。倒去未包被的液体，洗涤3遍，加入阻滞液阻止非特异性结合位点，37℃孵育1小时，洗涤3次。放入4℃保存备用。试剂盒分装加入鼠抗人甲胎蛋白单克隆抗体、标记的二抗等。
实施例7试剂盒灵敏度的检测
将甲胎蛋白重组蛋白(德国OriGene公司)用PBS稀释成200ng/ml、100ng/ml、50ng/ml、25ng/ml、10ng/ml、2ng/ml、0.5ng/ml、0.05ng/ml、0.01ng/ml、0ng/ml，每孔100ul加入到上述包被好的酶标板中和液相磁珠中，37℃孵育2小时，洗涤3遍。按1∶2000将鼠抗甲胎蛋白单克隆抗体稀释，每孔加入100ul，37℃孵育1小时，洗3遍。加入标记二抗，37℃孵育30分钟，洗3遍。加入底物室温放置15分钟，加入终止液，读数。检测最低的甲胎蛋白量，结果显示，该试剂能检测出0.01ng/ml甲胎蛋白的浓度，说明具有较高的检测灵敏度。经上述实验表明本发明的试剂盒检测尿液样本中甲胎蛋白的含量，具有很高的灵敏度，样本的最低检测限0.01ng/ml，回收率为90％±13％。本试剂盒所需仪器较少，只需要酶标仪、振荡器、离心机、移液器等，所需成本低。
实施例8试剂盒的特异性、稳定性的检测
取正常体检(首都医科大学附属北京世纪坛医院)待测清洁中段随机尿30-50ml，装入清洁的尿管，女性留取尿标本时应避开经期，应防止***分泌物混入尿液中，常温下1500rpm离心5分钟，取上清待检。
纯化定量的甲胎蛋白重组蛋白作为标准品，将抗原(离心后的尿标本)按1∶3稀释后，加入到前面已经包被好的酶标板中，37℃孵育2小时，洗去未结合的抗原，吸干残余液体。加入鼠抗人甲胎蛋白单克隆抗体37℃孵育1小时，洗去未结合的抗体，吸干残余液体。加入标记的二抗，37℃孵育30分钟，洗涤4次，吸干残余液体。加入显色底物，室温放置10分钟，加入终止液终止反应，酶标仪读数，计算样本中甲胎蛋白的含量。
通过该方法，发明人检测了100例正常体检随机尿中甲胎蛋白含量，准确率达到97％以上，具有良好的特异性。
分别取两位正常体检的随机尿，利用上述方法进行了ELISA测定，每天测定一次，共重复10次，按公式变异系数(CV)＝S/X×100％(S为标准差，X为平均值)计算批间及批内变异系数。最终得到批内与批间变异系数分别为2.81％和3.26％，说明稳定性好。
虽然本发明已以较佳实施例披露如上，然其并非用以限定本发明，任何所属技术领域的技术人员，在不脱离本发明的精神和范围内，当可作些许的更动与改进，因此本发明的保护范围当视权利要求所界定者为准。

Claims (5)

  1. 甲胎蛋白及其多肽在尿液中的表达及其在尿液含量检测中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述尿液甲胎蛋白的氨基酸序列如SEQ ID NO：1所示。
  3. 根据权利要求1所述的应用，其特征在于，所述制剂为尿液甲胎蛋白或多肽检测试剂盒。
  4. 根据权利要求3所述的应用，其特征在于，所述试剂盒为抗原抗体反应。
  5. 根据权利要求4所述的应用，其特征在于，所述反应为尿液甲胎蛋白或多肽以及其抗体被包被或标记在固相或液相载体。
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