WO2016082444A1 - 肽基精氨酸脱亚胺酶1在制备肿瘤临床诊断试剂中的应用 - Google Patents

肽基精氨酸脱亚胺酶1在制备肿瘤临床诊断试剂中的应用 Download PDF

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WO2016082444A1
WO2016082444A1 PCT/CN2015/077352 CN2015077352W WO2016082444A1 WO 2016082444 A1 WO2016082444 A1 WO 2016082444A1 CN 2015077352 W CN2015077352 W CN 2015077352W WO 2016082444 A1 WO2016082444 A1 WO 2016082444A1
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blood
tumor
reagent
cancer
solution
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常晓天
邢艳秋
马芳
杨冬霞
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山东创新药物研发有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

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  • the invention relates to the use of peptidyl arginine deiminase 1 as a tumor blood marker in preparing tumor clinical blood diagnostic reagents.
  • Peptidylarginine deiminase is an enzyme in human tissues.
  • PAD Peptidylarginine deiminase
  • PADI Peptidylarginine deiminase
  • PAD/PADIl 2, 3, 4, and 6
  • These enzymes are encoded by a cluster of genes located on the lp36 region of the human chromosome and have different tissue distributions. It can perform post-translational modification of other tissue proteins in the presence of calcium ions.
  • This enzyme catalyzes the conversion of arginine to citrulline by catalyzing the amino group of arginine in the polypeptide chain to a carbonyl group.
  • Citrulline is an unnatural amino acid.
  • citrullination The process by which arginine is converted to citrulline in a PAD-catalyzed polypeptide is referred to as citrullination.
  • citrulline is an important post-translational modification of proteins as important as phosphorylation, acetylation, glycosylation, methylation, and ubiquitination.
  • PAD4 peptidyl arginine deiminase 4, or PADI4
  • PADI4 peptidyl arginine deiminase 4, or PADI4
  • PAD1 peptidyl arginine deiminase 1, Peptidylarginine deiminase1, or PADI1
  • PAD4 although they are peptide-based arginine deiminase, have different tissue distributions, and there is currently no PAD1 as tumor blood. Relevant reports of markers.
  • the present invention has obtained a new tumor blood marker, peptidyl arginine deiminase 1 (PAD1/PADI1), which can be used for clinical diagnosis of tumors.
  • PADI1 peptidyl arginine deiminase 1
  • the present invention has found for the first time that the expression level of PAD1 in the serum of patients with malignant tumors such as liver cancer, colorectal cancer, breast cancer, esophagus and gastric cardia cancer is compared with the serum of patients with benign tumors, serum of chronic inflammation patients and normal human serum.
  • PAD1 can be expressed in the serum of many malignant tumor patients at the same time, which is relatively rare in the tumor markers currently used (currently, the sensitivity and specificity of tumor blood markers are not ideal, and there are more than 20 clinical serum markers in clinical practice.
  • the vast majority can only be used to detect a tumor with poor sensitivity and specificity. Usually, only joint detection can be used to provide meaningful data for clinical diagnosis.
  • the present invention introduces the expression level of PAD1 in blood with known tumor marker levels. Comparing the results, it was found that the positive rate and specificity of PAD1 tumor detection were no less than CEA, and the broad-spectrum was higher than CA125, CA199, PSA, CA242 and so on.
  • PAD1 has higher specificity as a tumor serum marker.
  • the present invention has developed a tumor diagnosis kit and a detection method with wide diagnostic range, high sensitivity and high specificity, which can be widely used in clinical practice. Preliminary tumor investigation and health screening provide a reliable basis for clinical diagnosis.
  • an anti-peptidyl arginine deiminase 1 antibody is prepared by a conventional method, and a qualitative or quantitative method for detecting peptidyl arginine deiminase 1 and a kit are established.
  • a blood diagnostic reagent for diagnosing tumors comprising a peptidyl arginine deiminase 1 antibody, an HRP-IgG antibody, and a conventional reagent in a blood diagnostic reagent.
  • Conventional reagents in the blood diagnostic reagent include carbonate buffer, PBST wash, blocking solution, color developing solution, and stop solution.
  • the qualitative detection method is an indirect ELISA method, and the principle is to use an enzyme-labeled anti-antibody (anti-human immunoglobulin antibody) to detect a test antibody bound to a solid phase antigen, as follows:
  • the blood sample to be tested is diluted 10 times with carbonate buffer (pH 9.6), added to a blank ELISA plate, and incubated at 37 ° C for 2 h;
  • PBST wash solution refers to PBS solution plus Tween-20, the pH of PBS solution is 7.4, the concentration of Tween-20 is 0.1%, volume percentage
  • the antibodies referred to above are prepared by a conventional method.
  • the various reagents referred to above are conventional reagents which are known in the art.
  • the above detection principle is also used to prepare a series of standard standards, and then a standard curve of the standard concentration and the OD 450 nm value or the OD 630 nm value is drawn, and a linear regression equation is obtained, and then the blood sample to be detected is obtained.
  • the OD 450 nm value or the OD 630 nm value is substituted into a standard curve to determine the concentration in the sample.
  • the PAD1 is detected by double antibody sandwich ELISA, and the steps are as follows:
  • PAD1 monoclonal antibody 1 was diluted to 1 ⁇ g/mL with a coating solution (pH 9.6 carbonate buffer), and added to a blank plate, 100 ⁇ L/well, at 4 ° C saturated humidity. Place overnight;
  • each well was added blocking solution (0.5% BSA, mass percentage) 200 ⁇ L / well, incubated at 37 ° C for 1 h;
  • Color development Add coloring solution A and B sequentially to each well [developer A (containing hydrogen peroxide) as hydrogen donor (DH2), substrate B is developer B (including TMB, 3, 3) ',5,5'-tetramethylbenzidine)] 50 ⁇ L each (including blank control), shake and mix, and color at room temperature for 10 minutes;
  • developer A containing hydrogen peroxide
  • substrate B is developer B (including TMB, 3, 3) ',5,5'-tetramethylbenzidine)
  • Termination 50 ⁇ L of each stop solution (including blank control) was added to each well, and the reaction was stopped by shaking and shaking;
  • the qualitative detection method can also be colloidal gold method
  • the detection principle is: using double antibody sandwich method and colloidal gold immunotechnology, pre-adding anti-PAD1 antibody colloidal gold conjugate on colloidal gold pad, detecting in nitrocellulose membrane Line and control lines were coated with anti-PAD1 antibodies, respectively.
  • the PAD1 in the sample can form a complex with the gold standard anti-PAD1 antibody, and the complex is moved forward along the test paper, and then combined with the pre-coated antibody of the detection line to form a ""
  • the gold-labeled antibody-PAD1-antibody” complex is agglutinated for color development.
  • the nitrocellulose membrane was coated with a quality control line as a control at the same time, so it was positive when a red quality control line and a red reaction line appeared.
  • a quality control line When there is no PAD1 antigen in the specimen to be examined, only one red quality control line is judged to be negative. As a quality control, a red quality control line will appear regardless of whether the result is positive or negative. If no red line (or only reaction line) appears, the test is invalid.
  • the specific detection method is:
  • sample samples are collected intravenously according to the conventional method; the collected samples are tested within five days, and should be stored at 4 ° C, and stored at -20 ° C for more than five days to avoid repeated freezing and thawing; if the sample If turbidity or sedimentation occurs, it should be centrifuged or filtered and clarified before testing); open the inner packaging and take out the test strip.
  • Test strip Immerse the end of the test strip marked with MAX in the sample for 30 seconds (note: the liquid surface should not pass the MAX line) and take it out on the table.
  • Test card In the sample hole marked with “S” on the test card, add 4 drops of sample (about 100 ⁇ l) with a dropper.
  • PAD1 can be highly expressed in cells of various malignant tumors such as uterine cancer, liver cancer, gastric cancer, esophageal cancer, lung cancer and ovarian cancer as well as in blood. Therefore, PAD1 can be used as a tumor blood marker, and it has been experimentally proved that Its specificity is no less than CEA, and its broad spectrum is higher than CA242, CA125, PSA, CA199 and so on. Although PAD1 is expressed in a variety of tumors, it does not achieve absolute specificity, but it is well known to those skilled in the art that the specificity of clinical detection indicators is not absolutely unique and cannot be denied as one of the means for diagnosing related diseases.
  • PAD1 is feasible as a tumor marker; although PAD1 has non-unique specificity, PAD1 as a tumor blood marker combined with other detection indicators and clinical manifestations is sufficient for detecting tumors (this method is also currently clinically Common way).
  • PAD1 can also be used as a blood marker for the detection of other diseases such as inflammation, such as hepatitis B, general inflammation (with white blood cells, neutral) Granulocyte enlargement, symptoms of decreased lymphocyte ratio), kidney disease (including nephrotic syndrome, renal failure, nephritis), uremia (including uremia and urinary tract infection), can also be used as a clinical diagnosis basis for other diseases such as inflammation .
  • diseases such as hepatitis B, general inflammation (with white blood cells, neutral) Granulocyte enlargement, symptoms of decreased lymphocyte ratio), kidney disease (including nephrotic syndrome, renal failure, nephritis), uremia (including uremia and urinary tract infection), can also be used as a clinical diagnosis basis for other diseases such as inflammation .
  • reagents, methods and the like referred to in the following examples are conventional reagents and methods in the prior art unless otherwise specified.
  • Example 1 Example 1 of PAD1 as a blood marker for tumors
  • PAD1 was used as a tumor blood marker to detect the blood of tumor patients.
  • the number of tumor patients and the types of tumors are shown in Table 1.
  • the detection method is:
  • the blood sample to be tested is diluted 10 times with carbonate buffer (pH 9.6), added to a blank ELISA plate, and incubated at 37 ° C for 2 h;
  • PBST wash solution refers to PBS solution plus Tween-20, the pH of PBS solution is 7.4, the concentration of Tween-20 is 0.1%, volume percentage
  • Results The number of positive cases and positive rate are shown in Table 1.
  • the positive rate of other tumor markers is shown in Table 2 (data that can be disclosed in the prior art). It can be seen from Table 1 and Table 2 that PAD1 is used as a tumor blood marker. , with good specificity, broad spectrum Sex.
  • Tumor category Total number of cases Number of positive cases Positive rate Uterine cancer 654 510 78.0% Liver cancer 537 399 74.3% Gastric cancer 363 219 60.3% Esophageal cancer 252 147 58.3% Lung cancer 387 251 64.8% Ovarian cancer 387 195 50.4% total 2580 1721 66.7%
  • Example 2 Example 1 of PAD1 as a blood marker for tumors
  • the patient's blood was detected by using PAD1 as a tumor blood marker, and the detection method was the same as in Example 1.
  • the detection method is conventional in the prior art
  • the blood sample test data of the tumor blood markers in the prior art is the patient outpatient data of a hospital, and the data can be used as one of the basis for diagnosing whether the patient has a tumor
  • the invention also uses the blood sample test PAD1 of some patients. ).
  • the positive rate of each tumor blood marker was compared, as well as the cross-comparison of PAD1 with each tumor marker.
  • PAD1 can be applied as a tumor blood marker with good specificity.
  • PAD1 as a blood marker for hepatitis B, general inflammation (white blood cells, neutrophils, patients with reduced lymphocyte ratio), kidney disease (including nephrotic syndrome, renal failure, nephritis), uremia (including uremia and urine)
  • the blood of the patient is tested.
  • the number of patients and the types of diseases are shown in Table 4.
  • the detection method is the same as in the first embodiment.
  • PAD1 can be used as a blood marker to detect hepatitis B and common inflammation (with white blood cells, neutrophils, and decreased lymphocyte ratio).
  • kidney disease including nephrotic syndrome, renal failure, nephritis
  • uremia including uremia and urinary tract infection
  • other diseases the test results can be used as one of the clinical diagnosis.

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Abstract

肽基精氨酸脱亚胺酶1在制备诊断肿瘤的临床诊断试剂或检测物中的应用，所述肿瘤选自子宫癌、肝癌、胃癌、食管癌、卵巢癌、肺癌。具体应用时，以常规方法制备抗肽基精氨酸脱亚胺酶1抗体，建立检测肽基精氨酸脱亚胺酶1的定性或定量方法及配套试剂盒。一种诊断肿瘤的血液诊断试剂，包括肽基精氨酸脱亚胺酶1抗体、HRP-IgG抗体，以及血液诊断试剂中的常规试剂。所述血液诊断试剂中的常规试剂包括碳酸盐缓冲液、PBST洗液、封闭液、显色液和终止液。经实验证明，PAD1可以作为肿瘤血液标志物进行应用，PAD1作为肿瘤标志物具有可行性。

Description

肽基精氨酸脱亚胺酶1在制备肿瘤临床诊断试剂中的应用 技术领域
本发明涉及肽基精氨酸脱亚胺酶1作为肿瘤血液标志物在制备肿瘤临床血液诊断试剂中的应用。
背景技术
肽基精氨酸脱亚胺酶(Peptidylarginine deiminase,PAD或PADI)是在人体组织中一种酶，目前，共发现了五种PAD酶(即PAD/PADIl、2、3、4和6)。这些酶由坐落在人类染色体lp36区域上的一簇基因所编码，并具有不同的组织分布。它可以在钙离子存在的情况下对其它一些组织蛋白进行后翻译修饰(post-translational modification)。这个酶可将多肽链中的精氨酸(arginine)的氨基催化成羰基，从而使精氨酸转化成瓜氨酸(citrulline)。瓜氨酸是一个非自然氨基酸。这个由PAD催化的多肽中精氨酸转化成瓜氨酸的过程被称做瓜氨酸化(citrullination)。蛋白发生瓜氨酸化后由于结构发生变化，导致其酶活性、代谢活性、调节功能和结构功能均发生改变。因此，瓜氨酸化是和磷酸化、乙酰化、糖基化、甲基化、泛素化一样重要的蛋白翻译后修饰方式。
近年来，通过免疫学、细胞生物化学和分子遗传学的研究，PAD4(肽基精氨酸脱亚胺酶4，Peptidylarginine deiminase4，或PADI4)被证明在人类类风湿性关节炎发病过程中起着非常重要的作用，且可作为腺癌标志物，在制备腺癌临床诊断试剂中进行应用。PAD1(肽基精氨酸脱亚胺酶1，Peptidylarginine deiminase1，或PADI1)和PAD4虽然同为肽基精氨酸脱亚胺酶，但具有不同的组织分布，目前并未有关于PAD1作为肿瘤血液标志物的相关报道。
发明内容
针对上述现有技术，本发明通过研究得到了一种新的肿瘤血液标志物——肽基精氨酸脱亚胺酶1(PAD1/PADI1)，可用于肿瘤的临床诊断。
本发明是通过以下技术方案实现的：
本发明通过实验研究首次发现，与良性肿瘤患者血清、慢性炎症患者血清和正常人血清相比，PAD1在肝癌、结/直肠癌、乳腺癌、食管及贲门癌等恶性肿瘤患者血清中的表达水平明显增高。PAD1能同时在多种恶性肿瘤患者血清中表达，这在目前使用的肿瘤标记物中是比较少的(目前肿瘤血液标记物敏感性和特异性不理想，临床现有肿瘤血清标记物达20余种，但绝大多数往往只能用于检测一种肿瘤，敏感性和特异性差，通常只能采用联合检测才能为临床诊断提供有意义的数据)。本发明将PAD1在血液中的表达水平与已知肿瘤标记物水平进 行比较，发现PAD1肿瘤检测阳性率和特异性不亚于CEA，广谱性高于CA125、CA199、PSA、CA242等。
基于以上发现，PAD1作为肿瘤血清标记物应用具有更高的特异性，为此，本发明研发了诊断范围广、敏感性强、特异性高的肿瘤诊断试剂盒及检测方法，可广泛用于临床肿瘤初步调查和健康普查，为临床诊断提供可靠依据。
具体地，以常规方法制备抗肽基精氨酸脱亚胺酶1抗体，建立检测肽基精氨酸脱亚胺酶1的定性或定量方法及配套试剂盒。
一种诊断肿瘤的血液诊断试剂，包括肽基精氨酸脱亚胺酶1抗体、HRP-IgG抗体，以及血液诊断试剂中的常规试剂。
所述血液诊断试剂中的常规试剂包括碳酸盐缓冲液、PBST洗液、封闭液、显色液和终止液。
进一步地，定性的检测方法为间接ELISA方法，其原理为利用酶标记的抗抗体(抗人免疫球蛋白抗体)以检测与固相抗原结合的受检抗体，具体方式如下：
(1)将待检测血样用碳酸盐缓冲液(pH9.6)稀释10倍后，加至空白酶标板中，37℃温育2h；
(2)弃去孔内液体，PBST洗液洗板三次(所述PBST洗液是指PBS溶液加上Tween-20，PBS溶液的pH为7.4，Tween-20的浓度为0.1％，体积百分数)；
(3)每孔加封闭液(0.5％BSA，质量百分数)200μL/孔，37℃温育1h；
(4)弃去孔内液体，PBST洗液洗板三次；
(5)每组加PAD1抗体稀释液(1:2000稀释)100μL/孔，37℃温育1h；
(6)弃去孔内液体，PBST洗液洗板三次；
(7)每孔加HRP-IgG抗体稀释液(1:3000稀释)100μL，37℃温育1h；
(8)弃去孔内液体，PBST洗液洗板五次；
(9)每孔加显色液A、B【显色剂A(含过氧化氢)为供氢体(DH2)，底物B就是显色剂B(含TMB，3,3',5,5'-四甲基联苯胺)】各50μL，37℃显色10min。
(10)每孔加50μL终止液(2M H2SO4溶液)终止显色，振匀，用酶标仪检测OD450nm值；
(11)计算：以阴性血清(正常人血清)为对照，检测阴性血清OD450nm均值(是以多份阴性血清为对照检测出来再平均的值)；将OD450nm值代入公式P/N值＝待测血样OD450nm值/阴性血清OD450nm均值，计算得到待检测血样的P/N值；
(12)判断：若待检测血样的P/N值大于或等于2.1，则该待检测血样的检测结果为阳性，该待检测血样所属的患者有可能患有肿瘤，可作为临床诊断的依据之一。
上述所涉及的抗体，为通过常规方法制备得到。上述所涉及的各种试剂，均为现有技术中已有的常规试剂。
同理，进行定量检测时，也采用上述检测原理，配制系列浓度的标准品，然后绘制标准品浓度与OD450nm值或OD630nm值的标准曲线，并得到线性回归方程，然后将待检测血样的OD450nm值或OD630nm值代入标准曲线，求得样品中的浓度。
进一步地，定量检测时，采用双抗体夹心ELISA检测PAD1，步骤如下：
(1)包被：将PAD1单克隆抗体1用包被液(pH9.6的碳酸盐缓冲液)稀释成1μg/mL，加至空白酶标板中，100μL/孔，4℃饱和湿度下放置过夜；
(2)洗板：弃去孔内液体，每孔加入PBST洗液300μL(所述PBST洗液是指PBS溶液加上Tween-20，PBS溶液的pH为7.4，Tween-20的浓度0.1％，体积百分数)，浸泡15秒，甩弃液体。连续洗板三次；
(3)封闭：每孔加封闭液(0.5％BSA，质量百分数)200μL/孔，37℃温育1h；
(4)洗板三次，同步骤(2)；
(5)设定：留一孔作空白对照，暂不加任何液体；另设PAD1标准品7孔，各加100μL标准品；
(6)加样：将待检测血样用PBST稀释10倍后，按顺序加至反应孔中，室温孵育1.5h；
(7)洗板三次，同步骤(2)；
(8)加酶：每个孔加入100μLHRP标记的PAD1单克隆抗体稀释液(1/3000)(不包括空白对照)，室温孵育45min；
(9)洗板三次，同步骤(2)；
(10)显色：每孔依次加入显色液A、B【显色剂A(含过氧化氢)为供氢体(DH2)，底物B就是显色剂B(含TMB，3,3',5,5'-四甲基联苯胺)】各50μL(包括空白对照)，震荡混匀，室温避光显色10分钟；
(11)终止：每孔加入终止液各50μL(包括空白对照)，震荡混匀终止反应；
(12)测定：用空白对照孔调零，并于30分钟内用酶标仪单波长450nm测定各孔OD值；也可用双波长450nm/630nm测定各孔OD值；
(13)计算：以系列标准品浓度值的对数值为横坐标(X轴)，以标准品OD值的对数值为纵坐标(Y轴)，建立(log-log)标准曲线，计算待测样本的PAD1含量。
另外，定性的检测方法也可为胶体金法，其检测原理为：采用双抗体夹心法和胶体金免疫技术，在胶体金垫上预加入抗PAD1抗体胶体金结合物，在硝酸纤维素膜的检测线和对照线上分别包被有抗PAD1抗体。当进行检测时，如为阳性样本，样本中的PAD1可与金标抗PAD1抗体结合形成复合物，由于层析作用复合物沿试纸向前移动，再与检测线预包被的抗体结合形成“金标抗体-PAD1-抗体”复合物而凝集显色。硝酸纤维膜上同时包被有一条质控线作为对照，故当出现一条红色质控线和一条红色反应线时判为阳性。当待检标本中无PAD1抗原时，只出现一条红色质控线判为阴性。作为质控，不管结果为阳性或阴性，都会出现一条红色质控线。若无红线(或只有反应线)出现，则检测无效。具体的检测方法为：
(1)用洁净的容器收集少量样本(血清样本按常规方法静脉采集；采集的样本在五天内检测，需放置4℃保存，五天以上需放置-20℃保存，避免反复冻融；如果样本出现浑浊或沉淀，应离心或过滤澄清后再检测)；打开内包装，取出检测试纸。
(2)依据产品型号进行操作：
①检测条：将检测条标有MAX字样的一端浸入样本中30秒(注意：液面不要没过MAX线)取出平放于台面上。
②检测卡：在检测卡标有“S”字样的加样孔中，用滴管滴加4滴样本(约100μl)。
(3)待检测条质控线(C线)出现后计时，20分钟内观察结果，20分钟以后观察结果无效。
检验结果的解释：
(1)阴性结果：仅出现质控线(C线)。
(2)阳性结果：出现两条线，即质控线(C线)和检测线(T线)。
(3)无效结果：无线出现，或仅出现检测线(T线)，须重新检测。
本发明首次发现PAD1可在子宫癌、肝癌、胃癌、食管癌、肺癌和卵巢癌等多种恶性肿瘤的细胞中以及血液中高表达，因此，PAD1可以作为肿瘤血液标志物进行应用，经实验证明，其特异性不亚于CEA，广谱性高于CA242、CA125、PSA、CA199等。尽管PAD1在多种肿瘤中表达，达不到绝对的特异性，但是，所属领域技术人员公知，临床检测指标的特异性的非绝对唯一并不能否定检测指标作为诊断相关疾病的手段之一的可行性，例如医生经常将几种检测指标结合临床表现作出最后诊断。因此，PAD1作为肿瘤标志物具有可行性；尽管PAD1具有非唯一的特异性，但将PAD1作为肿瘤血液标志物结合其它检测指标及临床表现也足以用于检测肿瘤(这种方式也是目前临床上的常见方式)。另外，本发明还发现，PAD1还可作为血液标志物应用于炎症等其他疾病的检测，如乙肝、普通炎症(具有白细胞、中性 粒细胞增多，淋巴细胞比率降低的症状)、肾病(包括肾病综合征、肾衰竭、肾炎)、尿毒症(包括尿毒症和尿路感染)，也可作为炎症等其它疾病的临床诊断依据之一。
具体实施方式
下面结合实施例对本发明作进一步的说明。
下述实施例中所涉及的试剂、方法等，若无特别说明，均为现有技术中的常规试剂、方法。
实施例1PAD1作为肿瘤血液标志物的检测实例1
以PAD1作为肿瘤血液标志物，对肿瘤患者的血液进行检测，肿瘤患者的例数以及所患肿瘤的种类详见表1。
检测方法为：
(1)将待检测血样用碳酸盐缓冲液(pH9.6)稀释10倍后，加至空白酶标板中，37℃温育2h；
(2)弃去孔内液体，PBST洗液洗板三次(所述PBST洗液是指PBS溶液加上Tween-20，PBS溶液的pH为7.4，Tween-20的浓度为0.1％，体积百分数)；
(3)每孔加封闭液(0.5％BSA，质量百分数)200μL/孔，37℃温育1h；
(4)弃去孔内液体，PBST洗液洗板三次；
(5)每组加PAD1抗体稀释液(1:2000稀释)100μL/孔，37℃温育1h；
(6)弃去孔内液体，PBST洗液洗板三次；
(7)每孔加HRP-IgG抗体稀释液(1:3000稀释)100μL，37℃温育1h；
(8)弃去孔内液体，PBST洗液洗板五次；
(9)每孔加显色液A、B【显色剂A(含过氧化氢)为供氢体(DH2)，底物B就是显色剂B(含TMB，3,3',5,5'-四甲基联苯胺)】各50μL，37℃显色10min。
(10)每孔加50μL终止液(2MH2SO4溶液)终止显色，振匀，用酶标仪检测OD450nm值；
(11)计算：以阴性血清(正常人血清)为对照，检测阴性血清OD450nm均值(是以多份阴性血清为对照检测出来再平均的值)；将OD450nm值代入公式P/N值＝待测血样OD450nm值/阴性血清OD450nm均值，计算得到待检测血样的P/N值；
(12)判断：若待检测血样的P/N值大于或等于2.1，则该待检测血样的检测结果为阳性。
结果：阳性例数及阳性率见表1，其它肿瘤标志物的阳性率见表2(为现有技术中能公开的数据)，通过表1、表2可以看出，PAD1作为肿瘤血液标志物，具有良好的特异性，广谱 性。
表1
肿瘤类别 总例数 阳性例数 阳性率
子宫癌 654 510 78.0％
肝癌 537 399 74.3％
胃癌 363 219 60.3％
食管癌 252 147 58.3％
肺癌 387 251 64.8％
卵巢癌 387 195 50.4％
总计 2580 1721 66.7％
表2
肿瘤类别 PAD1 CEA CA199 CA125 CA242
子宫癌 78.0％ - - - -
肝癌 74.3％ 62％～75％ 65％ - -
肺癌 64.8％ 56％～80％   44％ 50％
胃癌 60.3％ 60％～90％ 50％ 47％ -
食管癌 58.3％ - - - 62％
卵巢癌 50.4％ - 35％ 61.4％ -
实施例2 PAD1作为肿瘤血液标志物的检测实例2
以PAD1作为肿瘤血液标志物，对患者的血液进行检测，检测方法同实施例1。同时，以现有技术中的CEA、CA199、F/PSA、CA125、PSA、CA242、AFP、HCG、NSE作为肿瘤血液标志物，对肿瘤患者的血液进行检测(检测方法为现有技术中的常规方法；这些现有技术中的肿瘤血液标志物的血样检测数据为某医院的患者门诊数据，该数据可以作为诊断患者是否患有肿瘤的依据之一；本发明又拿来部分患者的血样检测PAD1)。对比各肿瘤血液标志物的阳性率，以及PAD1与各肿瘤标志物的交叉比较。
结果：患者的例数(共906例)以及检测阳性的例数见表3。通过表3可以看出，PAD1可以作为肿瘤血液标志物进行应用，其特异性良好。
表3 PAD1与各肿瘤标志物的交叉比较
Figure PCTCN2015077352-appb-000001
Figure PCTCN2015077352-appb-000002
实施例3 PAD1作为血液标志物的检测实例3
以PAD1作为血液标志物，对乙肝、普通炎症(白细胞、中性粒细胞增多，淋巴细胞比率降低的患者)、肾病(包括肾病综合征、肾衰竭、肾炎)、尿毒症(包括尿毒症和尿路感染)患者的血液进行检测，患者的例数以及所患疾病的种类详见表4。
检测方法同实施例1。
结果：阳性例数及阳性率见表4，通过表4可以看出，PAD1作为血液标志物，也可以用于检测乙肝、普通炎症(具有白细胞、中性粒细胞增多，淋巴细胞比率降低的症状)、肾病(包括肾病综合征、肾衰竭、肾炎)、尿毒症(包括尿毒症和尿路感染)等疾病，检测结果可以作为临床诊断依据之一。
表4
疾病类别 总例数 阳性例数 阳性率
乙肝 394 34 8.6％
普通炎症 225 8 3.6％
肾病 133 8 6.0％
尿毒症 110 20 18.2％

Claims (9)

  1. 肽基精氨酸脱亚胺酶1在制备诊断肿瘤的临床诊断试剂或检测物中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述肿瘤选自子宫癌、肝癌、胃癌、食管癌、卵巢癌、肺癌。
  3. 肽基精氨酸脱亚胺酶1在制备诊断炎症的临床诊断试剂或检测物中的应用，所述炎症选自乙肝、普通炎症、肾病、尿毒症。
  4. 根据权利要求1或2或3所述的应用，其特征在于：以常规方法制备抗肽基精氨酸脱亚胺酶1抗体，建立检测肽基精氨酸脱亚胺酶1的定性或定量方法及配套试剂盒。
  5. 一种诊断肿瘤的血液诊断试剂，其特征在于：包括肽基精氨酸脱亚胺酶1抗体、HRP-IgG抗体，以及血液诊断试剂中的常规试剂。
  6. 根据权利要求5所述的诊断肿瘤的血液诊断试剂，其特征在于：所述肿瘤选自子宫癌、肝癌、胃癌、食管癌、卵巢癌、肺癌。
  7. 根据权利要求5或6所述的诊断肿瘤的血液诊断试剂，其特征在于：所述血液诊断试剂中的常规试剂包括碳酸盐缓冲液、PBST洗液、封闭液、显色液和终止液。
  8. 一种诊断炎症的血液诊断试剂，其特征在于：包括肽基精氨酸脱亚胺酶1抗体、HRP-IgG抗体，以及血液诊断试剂中的常规试剂；所述炎症选自乙肝、普通炎症、肾病、尿毒症。
  9. 根据权利要求8所述的诊断炎症的血液诊断试剂，其特征在于：所述血液诊断试剂中的常规试剂包括碳酸盐缓冲液、PBST洗液、封闭液、显色液和终止液。
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