WO2016127278A1 - Application of urine complement component 3 (c3) protein - Google Patents
Application of urine complement component 3 (c3) protein Download PDFInfo
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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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- 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
An application of a urine complement component 3 (C3) protein to noninvasive detection of urine. Complement component 3 (C3) protein segments can be stably expressed in urine of a normal person. The advantage of acquiring a urine specimen noninvasively is played, the urine complement component (C3) protein is detected by using the urine specimen, the bottleneck problem of urinalysis existing all the time, namely how to eliminate urine volume influences in urine protein/polypeptide detection, is solved, the defects that creatinine serves as an internal reference for related detection of urine are overcome, and a simpler and more convenient detection method is provided for disease diagnosis.
Description
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。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.
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。Currently, the most widely used type of specimen in clinical routine testing is blood specimens. However, 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.
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。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. Compared with blood, urine protein is relatively simple and easy to analyze, and has great advantages in the diagnosis of diseases by body fluids. The 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. In addition to containing a large amount of protein, 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.
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。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. During the synthesis of complement C3, when the single-stranded precursor is transferred to the endoplasmic reticulum, the 22 amino acid guide peptide is cleaved, and the strongly basic linker peptide is discarded, and the cells are secreted in the form of two chains. The complement C3 mature protein contains 1663 amino acids with a relative molecular weight of 180 kDa. In
Among the various components of complement, 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. In addition, 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.
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。However, the amount of urine is easily affected by the amount of drinking water and some other physiological factors. Therefore, 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. At present, the effects of creatinine, urinary permeation or urine specific gravity on the calculation of urine volume are used in the study. The most widely used is urinary creatinine, but these methods are not only affected by age, sex, body fatness, mental state, physiological state, disease state and The impact of diet, etc., and the lack of consistency in different reports and limited cross-over studies, the best way to explore corrected urine output is to address the key to quantitative detection of proteins and peptides in urine.
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。In order to apply urine testing to clinical work, 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.
发明内容Summary of the invention
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。It is an object of the present invention to provide a use of a urine complement C3 protein (Complement Component 3) for the preparation of a urine-related examination.
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:Preferably, the amino acid sequence of the urine complement C3 protein is as shown in SEQ ID NO: 1:
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。Preferably, the preparation is a urine complement C3 protein detection kit. The kit is an antibody antigen reaction.
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。Preferably, 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.
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。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 μl of the sample with 10 μl of the substrate (0.3% α-cyano-4-hydroxycinnamic acid, HCCA), 1 μl 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. All mass spectra were analyzed using ClinProTools 2.1 analysis software to screen for highly expressed high-abundance protein peptides. The inventors then identified these screened protein polypeptides by liquid chromatography tandem mass spectrometry and searched for the complement C3 (Complement Component 3) protein in the International Protein Index (IPI human v3.45fasta with 71983entries) database.
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。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.
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。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.
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。The above and other objects, features, and advantages of the present invention will become more apparent and understood by the appended claims appended claims
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。Figure 1 is the average value of all points in the normal physical examination specimens between 1000 and 10000.
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。Figure 2 is a scatter plot of the point of mass-to-charge ratio of 1726.8 expressed in 30 normal medical specimens.
图3是补体C3蛋白的质谱图。Figure 3 is a mass spectrum of the complement C3 protein.
实施例1尿液标本的收集与处理 Example 1 Collection and processing of urine specimens
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。30 normal physical examinations (Beijing Metropolitan University Hospital, Beijing Medical University Hospital) were collected to randomly clean the middle urine samples, centrifuged within 1500 rpm (1500 rpm, 5 min), and the supernatant was retained. After storage, the refrigerator was stored at -80 °C.
实施例2磁珠纯化和分离尿液标本中的多肽
Example 2 Purification and Separation of Peptides from Urine Specimens by Magnetic Beads
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。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. Add 10 ul of MB-WCX and 10 ul of magnetic bead binding buffer to the sample tube. Mix the sample gun up and down to avoid foaming. Add 5 ul of urine supernatant to the sample tube, mix well and let stand for 1 minute on the magnetic stand. 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. Add 100 ul of magnetic bead washing buffer to the sample tube, mix well, and then leave the sample tube on the magnetic stand for 1 minute. The magnetic beads are attached to the wall and separated from the suspended liquid. The suspended liquid is removed by a sample gun. Repeat 3 times and discard the suspension. Add 5 ul of magnetic bead elution buffer to the sample tube and repeatedly pipette 10 times or more to mix the magnetic beads and the elution buffer to avoid foaming. The sample tube was placed on a magnetic stand and allowed to stand for 2 min to separate the magnetic beads from the suspension. The supernatant (eluent) was transferred to a new 0.5 ml sample tube labeled. Add 5 ul of Stabilization Buffer and carefully pipette and mix.
实施例3尿液标本的点靶与多肽谱图的生成 Example 3 Generation of Point Targets and Peptide Spectra of Urine Specimens
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。After calibrating the instrument with the standard, 1 μl of the eluate was mixed with 10 μl of the substrate (0.3% α-cyano-4-hydroxycinnamic acid, HCCA), and 1 μl of the spot was taken at the Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target. Dry on the plate at room temperature. The sample was ionized by irradiation with a nitrogen laser, mass spectrometry was performed, and data in the range of 1000-10000 Da was collected to obtain a mass spectrum composed of protein peaks of different mass-to-charge ratios. For each MALDI crystallization point, a total of 400 lasers were irradiated (each of the 8 different positions of each crystallization point was irradiated 50 times), and the average value represents one specimen, thereby obtaining a polypeptide map of all samples. The ClinProTools2.1 analysis software was used to analyze the mass spectra of normal control group, type 2 diabetes without complications and comorbidity group and type 2 diabetes mellitus with early kidney injury group, and to screen differential polypeptides. Screening conditions: mass range 1000-10000 Da, signal to noise ratio (S/N) greater than 5, mass drift no more than 0.1%, all mass spectra normalized according to total ion current. Specifically, please refer to 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.
实施例4蛋白多肽的鉴定 Example 4 Identification of Protein Polypeptides
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。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 μl/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, and 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.
表1 分析柱梯度洗脱的程序Table 1 Procedure for analytical column gradient elution
实施例5试剂盒的制备 Example 5 Preparation of the kit
1.包被抗体和酶标抗体工作浓度的选择1. Selection of working concentration of coated antibody and enzyme labeled antibody
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。
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.
2.试剂盒的制备2. Preparation of the kit
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。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.
实施例7试剂盒灵敏度的检测 Example 7 Detection of sensitivity of the kit
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。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.
实施例8试剂盒的特异性、稳定性的检测 Example 8 Detection of specificity and stability of the kit
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。Take a normal medical examination (Beijing Shijitan Hospital affiliated to Capital Medical University) to measure 30-50ml of random urine in the middle of clean, put into a cleaned urinary catheter, women should avoid the menstrual period when taking urine specimens, and prevent vaginal secretions from mixing into the urine. Centrifuge at 1500 rpm for 5 minutes at room temperature, and take the supernatant for examination.
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。Purification of the quantitative complement C3 recombinant protein as a standard, 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.
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。By this method, the inventors examined the content of complement C3 protein in 100 normal urine urine samples with an accuracy of more than 97%, and had good specificity.
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。Two random urines of normal physical examination were taken, and ELISA was performed by the above method. The measurement was performed once a day for 10 times. The coefficient of variation (CV)=S/X×100% (S is the standard deviation, X is flat).
Mean) Calculate the inter-batch and intra-assay coefficient of variation. Finally, the intra- and inter-assay coefficients of variation were 2.81% and 3.26%, respectively, indicating good stability.
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。
While the invention has been described above in terms of a preferred embodiment, it is not intended to limit the invention, and those skilled in the art can make some modifications and improvements without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the claims.
Claims (5)
- 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。The use of urine complement C3 protein or polypeptide for the preparation of non-invasive detection of urine.
- 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。The use according to claim 1, wherein the amino acid sequence of the urine complement C3 protein or polypeptide is as shown in SEQ ID NO: 1.
- 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。The use according to claim 1, wherein the preparation is a urine complement C3 protein or polypeptide detection kit.
- 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。The use according to claim 3, wherein the kit is an antigen-antibody reaction.
- 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。 The use according to claim 4, wherein the reaction is a urine complement C3 protein or polypeptide and an antibody thereof is coated or labeled in a solid phase or a liquid phase carrier.
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