WO2016127277A1 - 尿液载脂蛋白c-ii的应用 - Google Patents

尿液载脂蛋白c-ii的应用 Download PDF

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WO2016127277A1
WO2016127277A1 PCT/CN2015/000625 CN2015000625W WO2016127277A1 WO 2016127277 A1 WO2016127277 A1 WO 2016127277A1 CN 2015000625 W CN2015000625 W CN 2015000625W WO 2016127277 A1 WO2016127277 A1 WO 2016127277A1
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urine
apolipoprotein
apo
antibody
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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
    • 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

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  • the invention relates to a new use of urine apolipoprotein C-II, in particular to the expression of apolipoprotein C-II in urine and its application in urine content detection.
  • the Apo C-II gene is clustered with the ApoC-I and ApoE genes and is located in the q13 region of the long arm of chromosome 19.
  • the Apo C-II gene is 3320 bp in length and contains 4 exons and 3 introns.
  • ApoC-II is a single-chain polypeptide consisting of 79 amino acid residues. It contains no cysteine and serine. There are two polymorphisms. The isoelectric points are 4.86 and 4.69, respectively. The ⁇ -helix in the secondary structure is about Accounted for 23%.
  • Apo C-II is one of the structural proteins of CM, VLDL and HDL, accounting for 14%, 7%-10% and 1%-3% of its protein components, respectively.
  • Apo C-II is one of the most important subtypes of the Apo C family. It is a cofactor for LPL and plays an important role in lipid metabolism, which activates lipoprotein lipase (LPL) from a variety of sources.
  • the amino acid residues 55-78 in its structure are the shortest necessary regions to maintain its activation of LPL.
  • the amino acid residue at position 43-50 of the carboxy terminus is a lipid-binding of the a-helical structure.
  • Apo C-II has a variety of biological functions: (1) activation of LPL.
  • Apo C-II is a cofactor for LPL that activates LPL from multiple sources.
  • the mechanism of activation may be that LP L is usually present in the peripheral circulation and binds to heparin-like molecules and attaches to the blood vessels.
  • CM chylomicrons
  • VLDL very low-density lipoprotein
  • LPL interacts with phospholipids on the surface of lipoprotein particles to bind to lipoprotein particles.
  • CM chylomicrons
  • VLDL very low-density lipoprotein
  • Apo C-II can lead to disorders of lipoprotein metabolism.
  • Apo C-II interacts with LPL to alter the structure of LPL, which can catalyze the hydrolysis of triglycerides. Only moderate expression of genes can activate LPL activity. Overexpression or deletion can lead to triglycerideemia.
  • Apo C-II defects can This leads to an increase in plasma TG, VLDL and chylomicron levels and a decrease in LDL, IDL and HDL levels.
  • Apo C-II defective patients are diverse, but most have been identified. Defects in the Apo C-II gene intron splice site can result in low levels of plasma Apo C-II.
  • Apo C-II synthesized or produced by Apo C-II deficiency is thought to be caused by various single amino acid substitutions.
  • Apo C-II gene polymorphism is common in some patients with senile diseases such as coronary heart disease and diabetes.
  • serum Apo C-II content is highest in the early stage of cerebral infarction, and the serum Apo C-II content in the cerebral infarction group gradually decreases with the recovery of the disease.
  • Abnormal levels of Apo C-II are closely related to the occurrence of acute cerebral infarction, and there is a significant correlation with the severity of acute cerebral infarction. Monitoring the level of changes is important for the prevention and improvement of cerebral infarction.
  • Dynamic measurement of changes in serum Apo C-II levels in patients with cerebral infarction contributes to the early diagnosis and prognosis of cerebral infarction, and provides a basis for prevention and changes of the condition of patients with cerebral infarction.
  • Some researchers have found that patients with type 2 diabetes and coronary heart disease have higher concentrations of Apo C-II than patients with type 2 diabetes without CHD.
  • Clinical trials have shown that HDL, LDL and cholesterol in patients with chronic hepatitis B and cirrhosis show a significant downward trend.
  • Apo C-II can accurately, directly and quickly reflect the lipid metabolism in various stages of liver disease. Serum Apo C-II levels were positively correlated with bile cholesterol levels in patients with cholesterol stones. Therefore, by detecting changes in Apo C-II and other Apo indicators, it can reflect the changes in lipid metabolism and its role in the genesis of gallstones.
  • amino acid sequence of the urine apolipoprotein C-II is as shown in SEQ ID NO: 1.
  • the preparation is a urine apolipoprotein C-II detection kit.
  • the kit is an antibody antigen reaction.
  • the antigen-antibody reaction is coated with urine apolipoprotein C-II or polypeptide and its antibody Or labeled in a solid or liquid 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 apolipoprotein C-II can stably appear in the urine of a person who is normally examined. Therefore, it is proposed to detect the use of urine apolipoprotein C-II for urine related examination.
  • the invention exerts the advantage of non-invasiveness of urine specimens, and uses a random urine specimen to detect apolipoprotein C-II 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 1735.7 expressed in 30 normal medical specimens.
  • Figure 3 is a mass spectrum of apolipoprotein C-II.
  • 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.
  • 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 apolipoprotein C-II in all urine samples.
  • m/z 1735.7 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 protein apolipoprotein C-II was searched in the database, and the mass spectrum of apolipoprotein C-II is shown in Fig. 3.
  • the antibody and antigen concentrations were determined according to Pierce's BCA Protein Concentration Kit instructions, and then rabbit anti-human apolipoprotein C-II polyclonal antibody (Abcam) was coated with a standard checkerboard method using a coating buffer. Dilute to a concentration of 10.0 ng / ml, 1.0 ng / ml and 0.1 ng / ml, respectively coated on a solid phase ELISA plate and liquid phase magnetic beads, each concentration includes three wales, 4 ° C overnight, wash 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.
  • Murine anti-human apolipoprotein C-II 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.
  • Rabbit anti-human apolipoprotein C-II polyclonal antibody was diluted with a coating buffer, added to a solid phase microplate and liquid phase magnetic beads, and 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 use.
  • the kit is divided into a mouse anti-human apolipoprotein C-II monoclonal antibody, a labeled secondary antibody, and the like.
  • the apolipoprotein C-II 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 murine anti-apolipoprotein C-II monoclonal antibody was diluted 1:2000, 100 ul 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 apolipoprotein C-II was detected, and the results showed that the reagent could detect the concentration of 0.01 ng/ml apolipoprotein C-II, indicating a high detection sensitivity.
  • the above experiments show that the kit of the present invention detects the content of apolipoprotein C-II in urine samples, 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 recombinant apolipoprotein C-II recombinant protein was purified as a standard.
  • the antigen centrifuged urine sample
  • the mouse anti-human apolipoprotein C-II 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 apolipoprotein C-II in the sample.
  • the inventors examined the content of apolipoprotein C-II in 100 normal urine samples, and the accuracy rate was over 97%, which had good specificity.

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Abstract

一种尿液载脂蛋白C-II ( Apolipoprotein C-II，Apo C-II)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载脂蛋白C-II有较高丰度的表达，因此通过对尿液中载脂蛋白C-II表达量的检测获得对于诊断疾病的信息。

Description

尿液载脂蛋白C-II的应用 技术领域
本发明涉及尿液载脂蛋白C-II的新用途，具体涉及载脂蛋白C-II在尿液中的表达及其在尿液含量检测中的应用。
背景技术
Apo C-II基因与ApoC-I、ApoE基因成簇排列，位于19号染色体长臂q13区。Apo C-II基因长3320bp，含4个外显子和3个内含子。ApoC-II是由79个氨基酸残基组成的单链多肽，不含半胱氨酸和丝氨酸，共有两种多态型，等电点分别为4.86和4.69，其二级结构中α-螺旋约占23％。Apo C-II是CM、VLDL和HDL的结构蛋白之一，分别占其蛋白成份的14％，7％-10％及1％-3％。Apo C-II是Apo C家族中最重要的亚型之一，它是LPL的辅助因子，在脂类代谢中发挥着重要的作用，可激活多种来源的脂蛋白脂肪酶(LPL)。其结构中第55-78位氨基酸残基是维持其对LPL激活作用的最短必需区域。羧基端43-50位氨基酸残基为a螺旋结构的脂质结合。Apo C-II具有多种生物学功能：(1)激活LPL。Apo C-II为LPL的辅助因子，它可激活多种来源的LPL。其激活机制可能是LP L通常存在于外周循环与肝素样分子结合，并附着于血管内上。当LPL接触乳糜微粒(CM)或极低密度脂蛋白(VLDL)时，LPL便同脂蛋白颗粒表面的磷脂发生作用进而结合于脂蛋白颗粒上。(2)维持脂蛋白的稳定性。应用荧光光谱法研究Apo C-II羧基端19-39位氨基酸残基，发现其α-螺旋不仅能介导蛋白质与脂质表面结合，而且它不是自身缔合，而是通过与Apo C-II内其它双极性螺旋及载脂蛋白缔合，从而维持脂蛋白的稳定性。(3)保护血管内皮细胞，在阻止动脉粥样硬化的形成方面发挥重要的作用。
Apo C-II的缺乏或异常能导致脂蛋白代谢紊乱。Apo C-II与LPL发生作用，可以改变LPL的结构，从而可催化水解甘油三酯，只有基因适度表达才可以激活LPL的活性，过度表达或者缺失均会导致甘油三酯血症。有研究发现Apo C-II缺陷可以 导致血浆TG、VLDL及乳糜微粒水平的升高和LDL、IDL和HDL水平的降低。Apo C-II缺陷病人是多种多样，但是大部分已经确定。其Apo C-II基因内含子剪接体位点的缺陷，可以导致血浆Apo C-II的低水平。此外，Apo C-II缺陷合成或者产生的非功能性Apo C-II被认为是由各种单一的氨基酸替换导致的。Apo C-II基因多态性常见于冠心病、糖尿病等一些老年性疾病患者。有研究发现脑梗死组发病初期血清Apo C-II含量最高，随着病情的恢复脑梗死组血清Apo C-II含量呈逐渐降低的趋势。Apo C-II水平异常与急性脑梗死发生密切相关，并与急性脑梗死病情严重程度存在着明显的相关性，监测其水平变化对于脑梗死的预防、病情改善有重要意义。动态测定脑梗死患者血清中Apo C-II水平变化有助于脑梗死的早期诊断及预后推测，为脑梗死患者的预防和病情变化提供一定的依据。有研究表明，高浓度的Apo C-II是CHD的危险因素之一，并且与高浓度的TG相比，Apo C-II浓度的增加是CHD发生更加敏感的指标。有研究者发现与无CHD的2型糖尿病患者相比，2型糖尿病合并冠心病患者体内存在更高浓度的Apo C-II。临床实验表明慢性乙肝、肝硬化患者HDL、LDL、胆固醇均呈显著下降趋势，测定Apo C-II的变化能够准确、直接、快速地反映肝病各个时期的脂质代谢情况。胆固醇结石患者血清Apo C-II水平与胆汁胆固醇含量呈正相关。因此，通过检测Apo C-II等Apo指标的变化可以反映胆固醇结石患者脂质代谢变化及其在胆结石成因中的地位和作用。
发明内容
本发明的目的在于提供一种尿液载脂蛋白C-II在尿液中的表达及其用于尿液含量检测中的应用。
优选地，所述尿液载脂蛋白C-II的氨基酸序列如SEQ ID NO：1所示
Figure PCTCN2015000625-appb-000001
优选地，所述制剂为尿液载脂蛋白C-II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载脂蛋白C-II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)数据库检索得到载脂蛋白C-II(Apolipoprotein C-II，Apo C-II)。
本发明通过研究证实载脂蛋白C-II能够在正常体检的人的尿液中稳定出现。从而提出检测尿液载脂蛋白C-II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载脂蛋白C-II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1735.7的点在30例正常体检标本中表达的散点图。
图3是载脂蛋白C-II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示出载脂蛋白C-II在所有尿标本中的表达，从图中可以看出m/z 1735.7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蛋白载脂蛋白C-II，载脂蛋白C-II的质谱图请参照图3。
表1分析柱梯度洗脱的程序
Figure PCTCN2015000625-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载脂蛋白C-II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。加入鼠抗人载脂蛋白C-II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
将兔抗人载脂蛋白C-II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载脂蛋白C-II单克隆抗体、标记的二抗等。
实施例7 试剂盒灵敏度的检测
将载脂蛋白C-II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将鼠抗载脂蛋白C-II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。检测最低的载脂蛋白C-II量，结果显示，该试剂能检测出0.01ng/ml载脂蛋白C-II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载脂蛋白C-II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，应防止阴道分泌物混入尿液中，常温下1500rpm离心5分钟，取上清待检。
纯化定量的载脂蛋白C-II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。加入鼠抗人载脂蛋白C-II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，计算样本中载脂蛋白C-II的含量。
通过该方法，发明人检测了100例正常体检随机尿中载脂蛋白C-II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. 载脂蛋白C-II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，所述尿液载脂蛋白C-II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，所述制剂为尿液载脂蛋白C-II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，所述反应为尿液载脂蛋白C-II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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