WO2016127278A1 - 尿液补体c3蛋白的应用 - Google Patents

尿液补体c3蛋白的应用 Download PDF

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WO2016127278A1
WO2016127278A1 PCT/CN2015/000626 CN2015000626W WO2016127278A1 WO 2016127278 A1 WO2016127278 A1 WO 2016127278A1 CN 2015000626 W CN2015000626 W CN 2015000626W WO 2016127278 A1 WO2016127278 A1 WO 2016127278A1
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urine
protein
complement
complement component
detection
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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 urine complement C3 protein, in particular to the normal expression of urine complement C3 protein as an internal reference of a urine test method, and abnormal expression as a biomarker for disease diagnosis.
  • specimens with invasive, lipemia or hemolysis in blood specimens are not detectable for certain items, and collection equipment is often a weapon. This requires not only well-trained and experienced blood collectors, but also blood products themselves that increase occupational exposure. risk. On the other hand, at least 8 hours on an empty stomach increases the risk of hypoglycemia in patients such as pregnant women, the elderly and children. For patients who need frequent and even life-long blood collection, such as diabetes, blood diseases, cancer chemotherapy, etc., it also brings great pain to patients.
  • Urine is the ultrafiltrate of blood. It is the terminal metabolite produced by glomerular filtration, renal tubular and collecting tube reabsorption, excretion and secretion. It is a window reflecting the changes of the body. Its composition is changed by certain diseases.
  • the specific manifestation of the state, and the collection of urine specimens is simple, non-invasive, multi-volume, repeatable multiple times, convenient to store, high patient compliance, and does not require the help of medical personnel.
  • urine protein is relatively simple and easy to analyze, and has great advantages in the diagnosis of diseases by body fluids.
  • composition, quantity and trait changes of urine not only carry various information about the occurrence, development and prognosis of urinary system diseases, but also reflect the overall metabolic state of the body, for studying organ function, evaluating body state, metabolism, and dynamic monitoring. Various information is provided on aspects such as disease progression and judgment of efficacy.
  • urine also contains a greater number of polypeptide fragments. Proteins and peptides with a molecular weight of less than 10 kDa can be freely filtered through the glomerulus. The polypeptide fragments in the urine are likely to be products of proteolysis. Analysis of urine may be more suitable for diagnosing systemic diseases and revealing the pathophysiological characteristics of the disease. It is therefore an ideal material for diagnosing and judging disease progression.
  • Complement C3 is the core protein in the complement system and is mainly produced by hepatocytes and macrophages.
  • the gene encoding complement C3 is located on chromosome 19 and consists of 41 exons.
  • the full length of the cDNA coding sequence is about 5052 bp, which in turn encodes the leader peptide, beta chain and alpha chain.
  • the complement C3 mature protein contains 1663 amino acids with a relative molecular weight of 180 kDa.
  • C3 has the highest serum content; in terms of function, C3 is also at the center, which is the intersection of several activation pathways and the basis of C3b-dependent positive feedback loop; meanwhile, C3 is activated and lysed. After that, the series of fragments and their binding proteins produced are complex and diverse, and play an important role in immune defense, immune regulation and immunopathology.
  • the non-complement function of C3 associated with cell proliferation and differentiation has also been reported, such as the proliferation and establishment of bud base and lens vesicles, involved in the proliferation and growth of B cells in vitro, which may be attributed to the complement component in mitogenic events.
  • Role, C3 may also play a role in early dedifferentiation and even in later differentiation. With the deepening of research, C3 is thought to play a role in tumor formation, signal transduction, and apoptosis. At the same time, it can protect against oxidative stress.
  • the concentration of markers related to urine diseases depends not only on the excretion rate of the marker but also on the urine flow.
  • 24h urine is the gold standard for assessing urine markers, but the collection of such specimens is cumbersome and the patient's compliance is poor.
  • the effects of creatinine, urinary permeation or urine specific gravity on the calculation of urine volume are used in the study.
  • the present invention first screens for high abundance protein polypeptides that are stably present in urine.
  • the urine protein polypeptide can be stabilized at a certain level when the body is in a normal physiological state, and an increase or decrease in its level is associated with certain diseases.
  • a urine complement C3 protein Complement Component 3
  • amino acid sequence of the urine complement C3 protein is as shown in SEQ ID NO: 1:
  • the preparation is a urine complement C3 protein detection kit.
  • the kit is an antibody antigen reaction.
  • the antigen-antibody reaction is coated or labeled with a urine complement C3 protein 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. After mixing 1 ⁇ l of the sample with 10 ⁇ l of the substrate (0.3% ⁇ -cyano-4-hydroxycinnamic acid, HCCA), 1 ⁇ l of the spot was taken on an Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target plate, and the sample was ionized. Mass spectrometry analysis was performed to acquire data in the range of 1000-10000 Da, and a mass spectrometric peptide map composed of protein peaks of different mass-to-charge ratios was obtained.
  • Anchorchip Autoflex MALDI TOF, Bruker-Dalton
  • the present invention confirmed by studies that the complement C3 protein can stably appear in the urine of a person who is normally examined. Therefore, it is proposed to detect the use of urine complement C3 protein for urine related examination.
  • the invention exerts the advantage of obtaining non-invasive urine specimens, and uses the random urine specimen to detect the complement C3 protein 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 1726.8 expressed in 30 normal medical specimens.
  • Figure 3 is a mass spectrum of the complement C3 protein.
  • 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 complement C3 in all urine samples. It can be seen from the figure that m/z 1726.8 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 the International Protein Index (IPI human v3.45fasta 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 complement C3 protein was searched in the database, and the mass spectrum of the complement C3 protein is shown in Fig. 3.
  • the antibody and antigen concentrations were determined according to Pierce's BCA Protein Concentration Kit instructions, and then the rabbit anti-human complement C3 protein 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.
  • Murine anti-human complement C3 monoclonal antibody (Abcam) 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 complement C3 protein 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 complement C3 monoclonal antibody, a labeled secondary antibody, and the like.
  • Complement C3 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-complement C3 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 complement C3 was detected.
  • the results showed that the reagent could detect the concentration of 0.01 ng/ml complement C3 protein, indicating a higher detection sensitivity.
  • the above experiments show that the kit of the present invention detects the content of complement C3 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 antigen (centrifuged urine sample) was diluted 1:3, added to the previously coated enzyme plate, incubated at 37 ° C for 2 hours, washed away unbound The antigen, blotting the residual liquid.
  • the mouse anti-human complement C3 protein monoclonal antibody was added and incubated at 37 ° C for 1 hour, the unbound antibody was washed away, and the residual liquid was blotted.
  • the labeled secondary antibody was added, incubated at 37 ° C for 30 minutes, washed 4 times, and the residual liquid was blotted dry. Add the chromogenic substrate, leave it at room temperature for 10 minutes, stop the reaction by adding a stop solution, and read the microplate reader to calculate the content of complement C3 protein in the sample.

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Abstract

一种尿液补体C3蛋白(Complement Component 3)在制备用于尿液无创检测的应用。在正常人的尿液中补体C3蛋白片段能够稳定表达。发挥尿液标本获取无创优势，利用尿液标本检测尿液补体C3蛋白，解决一直以来尿液检查的瓶颈问题即尿蛋白/多肽检测如何排除尿量影响的检测方法，弥补肌酐作为尿液相关检测内参的不足，为疾病的诊断提供更为简单、方便的检测方法。

Description

尿液补体C3蛋白的应用 技术领域
本发明涉及尿液补体C3蛋白的新用途，具体涉及尿液补体C3蛋白的正常表达作为尿液检查方法的内参，异常表达作为疾病诊断的生物标志物。
背景技术
目前，在临床常规检测中，利用最多的标本类型是血液标本。但是，血液标本获取有创、出现脂血或溶血的标本对某些项目无法检测、采集设备多为利器，这不仅需要训练有素、经验丰富的采血者，而且血制品本身增加了职业暴露的风险。另一方面，空腹至少8个小时给孕妇、老人以及儿童这样的患者增加了低血糖的风险。对于需要经常反复甚至终生采血的疾病如糖尿病、血液病、肿瘤化疗等也给患者带来极大的痛苦。
尿液是血液的超滤液，是经肾小球滤过、肾小管和集合管重吸收、***及分泌产生的终末代谢产物，是反映机体变化的窗口，其组成的改变是某些疾病状态的特异表现，并且尿液标本收集简单、无创、量多、可多次重复、保存方便、患者依从性高，且不需要医护人员的帮助。与血液相比，尿蛋白构成相对简单、易于分析，在体液诊断疾病方面具有很大优势。尿液的组成、数量与性状的变化不仅携带有泌尿***疾病发生、发展及预后的各种信息，也反映了机体的整体代谢状态，为研究脏器功能、评价机体状态、代谢情况、动态监测疾病进展和判断疗效等方面提供了各种信息。尿液中除了含有大量蛋白质之外，也包含数量更多的多肽片断。分子量小于10kDa的蛋白质和多肽可以自由滤过肾小球，尿液中的多肽片断很可能是蛋白酶解的产物，分析尿液可能更适合于诊断***性的疾病，揭示疾病的病理生理学特征。因此它是诊断和判断疾病分级进展的理想物质。
补体C3是补体***中的核心蛋白，主要由肝细胞和巨噬细胞产生。编码补体C3的基因位于19号染色体，由41个外显子组成，其cDNA编码序列全长约为5052bp，依次编码前导肽、β链和α链。补体C3在合成的过程中，其单链前体转到内质网时切去22个氨基酸的引导肽，舍弃强碱性的连接肽，以两链的形式分泌出细胞。补体C3成熟蛋白含1663个氨基酸，相对分子量为180kDa。在 补体各种成分中，C3的血清含量最高；在功能上，C3亦居于中心地位，它既是几条激活途径的交汇，又是C3b依赖性阳性反馈环路的基础；同时，C3经活化和裂解后，其产生的系列片段及其结合蛋白复杂而多样，在免疫防御、免疫调控以及免疫病理中发挥重要作用。此外，C3与细胞增殖分化相关的非补体功能也有报道，如参与芽基和晶状体泡的增殖和确立，参与体外B细胞的增殖和生长，这可能归功于补体成分在促有丝***事件中所发挥的作用，C3可能在早期的去分化甚至对后期的分化过程中也起作用。随着研究的深入，C3被认为在肿瘤形成、信号转导、细胞凋亡中也发挥作用。同时，在抗氧化应激时能起到保护作用。
但是，尿量容易受饮水量和一些其他的生理因素的影响，所以，尿液疾病相关标志物的浓度不仅取决于标志物的***率，而且取决于尿流量。24h尿是评估尿液标志物的金标准，但是这种标本收集方式繁琐，而且病人的依从性差。目前研究中应用肌酐、尿渗量或者尿比重等计算校正尿量的影响，应用最为广泛的是尿肌酐，但是这些方法不仅受年龄、性别、体型胖瘦、精神状态、生理状态、疾病状态和饮食等的影响，而且在不同的报告中缺乏一致性并且限制了交叉对比研究，所以，探索校正尿量的最佳方法是解决尿液中蛋白和多肽定量检测的关键。
为了把尿液检查应用到临床工作中，本发明首先筛选出了在尿液中稳定出现的高丰度蛋白多肽。在机体处于正常的生理状态时，此尿液蛋白多肽能够稳定在一定水平，其水平的升高或降低与某些特定的疾病相关。
发明内容
本发明的目的在于提供一种尿液补体C3蛋白(Complement Component 3)在制备用于尿液相关检查中的应用。
优选地，所述尿液补体C3蛋白的氨基酸序列如SEQ ID NO：1所示：
Figure PCTCN2015000626-appb-000001
Figure PCTCN2015000626-appb-000002
Figure PCTCN2015000626-appb-000003
优选地，所述制剂为尿液补体C3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补体C3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)数据库检索得到补体C3(Complement Component 3，补体C3蛋白)蛋白。
本发明通过研究证实补体C3蛋白能够在正常体检的人的尿液中稳定出现。从而提出检测尿液补体C3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补体C3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1726.8的点在30例正常体检标本中表达的散点图。
图3是补体C3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示出补体C3在所有尿标本中的表达，从图中可以看出m/z 1726.8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 3.3.1SP1进行SequestTM检索。检索数据库为International Protein Index(IPI human v3.45fasta 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补体C3蛋白，补体C3蛋白的质谱图请参照图3。
表1 分析柱梯度洗脱的程序
Figure PCTCN2015000626-appb-000004
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补体C3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。加入鼠抗人补体C3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
将兔抗人补体C3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补体C3单克隆抗体、标记的二抗等。
实施例7试剂盒灵敏度的检测
将补体C3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将鼠抗补体C3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。检测最低的补体C3量，结果显示，该试剂能检测出0.01ng/ml补体C3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补体C3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。
纯化定量的补体C3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。加入鼠抗人补体C3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，计算样本中补体C3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补体C3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. 尿液补体C3蛋白或多肽在制备用于尿液无创检测中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述尿液补体C3蛋白或多肽的氨基酸序列如SEQ ID NO：1所示。
  3. 根据权利要求1所述的应用，其特征在于，所述制剂为尿液补体C3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，所述反应为尿液补体C3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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