WO2019129126A1 - Serum marker mmp-7-based biliary atresia diagnosis kit - Google Patents

Serum marker mmp-7-based biliary atresia diagnosis kit Download PDF

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WO2019129126A1
WO2019129126A1 PCT/CN2018/124215 CN2018124215W WO2019129126A1 WO 2019129126 A1 WO2019129126 A1 WO 2019129126A1 CN 2018124215 W CN2018124215 W CN 2018124215W WO 2019129126 A1 WO2019129126 A1 WO 2019129126A1
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mmp
solution
serum
diagnosis
biliary atresia
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PCT/CN2018/124215
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French (fr)
Chinese (zh)
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汤绍涛
阳历
周莹
曹国庆
张茜
许培培
常晓盼
李帅
普佳睿
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华中科技大学同济医学院附属协和医院
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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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)
    • G01N2333/96491—Metalloendopeptidases (3.4.24) with definite EC number
    • G01N2333/96494—Matrix metalloproteases, e. g. 3.4.24.7
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis

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  • the invention relates to the field of biological detection, in particular to a diagnostic kit for biliary atresia based on serum marker MMP-7 protein.
  • Biliary atresia is a neonatal cholestatic disease characterized by progressive inflammation of the extrahepatic bile duct and rapid liver fibrosis. The disease progresses rapidly. Kasai surgery (hepatic jejunostomy) is performed within 3 months. it is good. The incidence of the disease in Western countries is 1/17,000-1/19000, while in Taiwan, Japan is 1/7000-1/13000, and the incidence in mainland China is 1/5000-1/12000 [1-6] Is the most common cause of liver transplantation in children [2,7,8] .
  • GGT is the most commonly used screening index for biliary atresia, but the difference between different children in clinical cases is large, and the accuracy is limited. 3 Other indicators: serum cholic acid, prothrombin concentration, and platelet determination have many influencing factors.
  • abnormal gallbladder constriction of gallbladder, poor contraction
  • hepatic ganglion are characteristic features of the liver in children with biliary atresia by ultrasonography [11,12] , but the hepatic mass is not necessarily in every disease. All of them appear in children, and the results of ultrasound examination vary greatly due to different doctors and machines. Therefore, the sensitivity and specificity of the diagnosis of BA are reported in the literature [10,13-19] .
  • the pathological features of BA are small bile duct hyperplasia, bile thrombosis, capillary bile duct and hepatocyte cholestasis, fibrosis around the portal area or lobule, and hepatic lobular structure can be seen early [1,20,21] .
  • the pathological manifestations of ⁇ 1 anti-trypsin deficiency disease overlap with it.
  • Alagille syndrome, cystic fibrosis, and total parenteral nutrition-related cholestasis can also show similar pathological changes [1] . Therefore, when clinically applied to identify BA and other neonatal cholestasis, the diagnosis and treatment delay is delayed due to low detection accuracy or complicated process.
  • MMP-7 matrix metalloproteinases-7
  • the object of the present invention is to overcome the poor accuracy and complicated operation of the existing diagnostic BA method, and the patient is repeatedly treated in the pediatric medicine and the pediatric surgery, the procedure is cumbersome, the diagnosis is prone to delay, and the delay of treatment directly affects the deficiency of the curative effect.
  • a diagnostic kit for biliary atresia based on the serum marker MMP-7 is provided, which has the characteristics of simple operation, reliable results, high sensitivity and high specificity. To solve the problem of diagnostic technology that cannot be realized by the prior art BA diagnosis method.
  • a diagnostic kit for biliary atresia based on serum marker MMP-7 for early diagnosis of BA, characterized in that the diagnostic kit comprises anti-human MMP-7 antibody Coated ELISA plate, positive control solution, sample diluent (negative control solution), enzyme standard reagent (A, B), enzyme substrate solution, washing solution and stop solution.
  • the positive control solution was serum diluted with MMP-7 in a sample diluent containing a MMP-7 concentration of 40 ng/mL.
  • the sample diluent was 0.01 mol/L pH 7.4 PBS; the negative control solution was the sample dilution.
  • the enzyme labeling reagent A is a biotinylated MMP-7 antibody
  • the enzyme labeling reagent B is an HRP-labeled avidin at a concentration of 0.1-111 g/mL.
  • the enzyme substrate solution is a 3,3',5,5'-tetramethylbenzidine solution (3,3',5,5'-Tetramethylbenzidine, TMB solution), including developer A and developer B.
  • developer A is 500 mL of sodium acetate containing 13.6 g, citric acid 1.6 g and 30% hydrogen peroxide 0.3 mL
  • developer B is 500 mL solution containing TMB 350 mg, dimethyl sulfoxide (DMSO) 20 mL and lemon Acid. H 2 O 5.1 g.
  • the washing solution is 0.01 lmol/L pH 7.4 phosphate-NaCl buffer (PBST), and the PBST contains 0.05% Tween-20, that is, 1 g of the solution contains 8 g of NaCl and 0.2 g of KH 2 P0 4 . 2.9 g Na 2 HP0 4 .12H 2 0, 0.2 g KCl and 0.5 mL Tween-20.
  • PBST pH 7.4 phosphate-NaCl buffer
  • the stop solution was a 2 mol/L H 2 SO 4 solution.
  • the reagents in the kit may be added with a preservative.
  • the antibody used for the anti-human MMP-7 antibody-coated ELISA plate is an anti-human MMP-7 monoclonal antibody.
  • a diagnostic method for the biliary atresia diagnostic kit based on the serum marker MMP-7 which is used for the early diagnosis of BA.
  • the diagnostic kit uses an ELISA technique widely used in clinical practice to quantitatively detect MMP in human serum by indirect method. 7 content, specifically: adding anti-human MMP-7 antibody-coated ELISA plate to the test serum, adding biotinylated MMP-7 antibody, and after washing the unbound biotinylated antibody, adding HRP The labeled avidin forms an enzyme antibody complex. After thorough washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted into a final yellow color under the action of acid. It is positively correlated with the level of MMP-7 in the sample.
  • the present invention has the following advantages:
  • the serum biomarker protein MMP-7 provided has a sensitivity of 100% and a specificity of 95.6%. It has high specificity and high sensitivity.
  • Figure 1 is a standard curve of serum MMP-7 by ELISA
  • FIG. 2A and 2B show the expression of serum MMP-7 in BA group, non-BA group and control group; wherein: Figure 2A shows that serum MMP-7 concentration in BA group is significantly higher than that in non-BA group and CO group in the whole population (Fig. 2A) **P ⁇ 0.001); Figure 2B shows that serum MMP-7 levels in BA group were significantly higher than those in non-BA and CO groups (**P ⁇ 0.001).
  • 3A, 3B and 3C are age-related cases of serum MMP-7;
  • Figure 3A shows that the serum MMP-7 concentration in the BA group is positively correlated with age (P ⁇ 0.001);
  • Figure 4 is a comparison of the area under the ROC curve (receiver operating characteristic curve, ROC curve) of serum MMP-7 and GGT for diagnosis of BA.
  • the area under the ROC curve of MMP-7 and GGT is 0.996 and 0.678, respectively;
  • Figure 5 shows the expression of MMP-7 gene in liver: the expression of MMP-7 mRNA in BA group was significantly higher than that in BA group and control group;
  • Figure 6 shows that a large number of hyperplasia, disordered small bile ducts in the liver of children with BA express MMP-7 strong positive (immunohistochemical staining, 40 times magnification);
  • 7A, 7B, 7C, 7D, 7E, and 7F show the expression of MMP-7 in the liver of different children (the block in the figure is an enlarged area);
  • Figure 7A magnification 100 times
  • Figure 7B magnification 400 times
  • Figure 7C magnification 100 times
  • Figure 7D magnification 400 times
  • Figure 7E 100-fold magnification
  • Figure 7F 400-fold magnification
  • the abscissa is the absorbance value (OD value), and the ordinate is the MMP-7 concentration. It can be seen from Fig. 1 that the OD value is positively correlated with the MMP-7 concentration.
  • the serum MMP-7 concentration in the BA group was positively correlated with the age of the children, as shown in Fig. 3A; the serum MMP-7 concentration in the non-BA group and the control group was not correlated with age, as shown in Fig. 3B, 3C.
  • the area under the curve of the ROC curve for the diagnosis of BA by serum MMP-7 was 0.996, as shown in Fig. 4.
  • the sensitivity and specificity of the diagnostic BA were 100% and 95.6%, respectively.
  • the whole blood sample was allowed to stand at room temperature for 2 hours or at 4 ° C overnight, and then centrifuged at 1000 g for about 20 minutes, and the supernatant was taken. Immediately test or dispense, and store the specimen at -20 ° C or -80 ° C to avoid repeated freezing and thawing. The thawed sample should be centrifuged again and then tested. NaN 3 was not contained in the sample tested because NaN 3 inhibited the activity of horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • the preparation method of the antibody ELISA plate is:
  • the anti-human MMP-7 monoclonal antibody was diluted to 60 ⁇ g/ml with PBS buffer at pH 8.2, and added to a 96-well microtiter plate at a dose of 100 ⁇ l/well, and left at a temperature of 4 ° C overnight. Then, it was washed with a washing solution containing 0.05% Tween-20, pH 7.4 phosphate buffer, and dried to obtain a 96-well microtiter plate coated with an anti-MMP-7 monoclonal antibody.
  • the specific steps of the diagnostic kit are as follows:
  • Standards and serum samples to be tested 1:100 were diluted to 100 uL with sample buffer and added to the respective antigen assay well plates. Be careful not to have air bubbles. Add the sample to the bottom of the hole of the plum mark plate, try not to touch the hole wall, gently shake the tears, and cover or cover the microplate. If there are more serum samples to be tested, it is recommended to use a multi-tube micro-spirator to load. Standards and samples to be tested are prepared within 15 minutes before use, discarded after use, and freshly prepared standards are used for the next test.
  • Termination 50 uL of stop solution was added to each well in sequence to terminate the reaction.
  • the order of addition of the stop liquid should be as close as possible to the order in which the substrate liquid is added.
  • the stop solution should be added as soon as possible after the substrate reaction time has elapsed.
  • the optical density (OD value) of each well was measured by a microplate reader at a wavelength of 450 nm within 15 minutes after the addition of the stop solution.
  • the detection standard of the test kit is: the concentration of the standard is the ordinate (or logarithmic coordinate), the OD value is the abscissa (or logarithmic coordinate), and the standard curve is drawn using the curve expert 1.30 software (the best equation should be The R 2 value calculated by the regression equation is undetermined, and the R 2 value is closer to 1 is better.
  • the actual concentration of the sample is calculated. Among them: the positive result judgment standard is: MMP-7>55.18 ng/mL.
  • the serum of 300 BA patients (100 children with BA, 100 children with non-BA jaundice, 100 children without liver disease or jaundice) were specifically used for the diagnostic kit of the present invention. Sex and sensitivity testing.
  • the diagnostic kit of the present invention assists the early diagnosis of BA with a specificity of 95.6% and a sensitivity of 100%, which are higher than the prior art BA diagnostic indicators.
  • the invention provides a diagnostic kit for biliary atresia based on serum marker MMP-7, wherein the diagnostic kit comprises an anti-human MMP-7 monoclonal antibody coated ELISA plate, a negative control solution, a positive control solution and an enzyme standard reagent. , enzyme substrate solution, blocking solution, sample diluent, washing solution and stop solution;
  • the negative control solution is a healthy human serum diluted in the sample diluent without MMP-7;
  • the positive control solution is a serum containing MMP-7 diluted in a sample diluent, and the concentration of MMP-7 is 100 U/mL;
  • the enzyme labeling reagent is a horseradish peroxidase (HRP)-labeled secondary antibody 0.1-111 g/mL;
  • the enzyme substrate solution is a 3,3',5,5'-tetramethylbenzidine solution, including a developer A and a developer B, and the developer A is 500 mL of a solution containing 13.6 g of sodium acetate and a lemon. Acid 1.6g and 30% hydrogen peroxide 0.3mL; developer B is 500mL solution containing TMB 350mg, dimethyl sulfoxide 20mL and citric acid. H 2 O 5.1g;
  • the blocking solution is a 0.01 mol/L pH 7.4 phosphate-NaCl buffer solution of 0.5% bovine serum albumin, ie, 1 g of BSA, 8 g of NaCl, 0.2 g of KH 2 P0 4 , and 2.9 g of Na 2 HP0 in 1 liter of solution. 4 .12H 2 O and 0.2g KCl;
  • the sample diluent is 0.01 mol / L pH 7.4 PBS;
  • the washing solution is 0.01 lmol/L pH 7.4 phosphate-NaCl buffer PBST, and PBST contains 0.05% Tween 20, that is, 1 liter of solution contains 8 g of NaCl, 0.2 g of KH 2 P0 4 , and 2.9 g of Na 2 HP0 4 . 12H 2 0, 0.2g KCl and 0.5mL Tween-20;
  • the stop solution was a 2 mol/L H 2 SO 4 solution.
  • the reagents in the kit may be added with a preservative.
  • a diagnostic method for the biliary atresia diagnostic kit based on the serum marker MMP-7 which is used for the early diagnosis of BA.
  • the diagnostic kit uses an ELISA technique widely used in clinical practice to quantitatively detect MMP in human serum by indirect method. 7 content, specifically: adding anti-human MMP-7 antibody-coated ELISA plate to the test serum, adding biotinylated MMP-7 antibody, and after washing the unbound biotinylated antibody, adding HRP The labeled avidin forms an enzyme antibody complex. After thorough washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted into a final yellow color under the action of acid. It is positively correlated with the level of MMP-7 in the sample.

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Abstract

The invention relates to a serum marker MMP-7-based biliary atresia diagnosis kit, the diagnosis kit comprises an anti-human MMP-7 monoclonal antibody coated ELISA plate, a negative control solution, a positive control solution, an enzyme labeling reagent, an enzyme substrate solution, a blocking solution, a sample diluent, a washing solution and a stopping solution. Compared with the prior art, the present invention provides a novel sensitive, safe, reliable and easily-operated commercial kit for quantitatively detecting the level of MMP-7 in human serum, which is helpful for early diagnosis of BA; the BA diagnosis sensitivity of the serum biomarker protein MMP-7 is 100%, and the BA diagnosis specificity is 95.6%, the kit has the characteristics of high specificity and high sensitivity; the kit improves the early diagnosis rate of BA, reduces misdiagnosis, and improves the own liver survival rate of the BA.

Description

基于血清标志物MMP-7的胆道闭锁诊断试剂盒Diagnostic kit for biliary atresia based on serum marker MMP-7 技术领域Technical field
本发明涉及生物检测领域，具体涉及一种基于血清标志物MMP-7蛋白的胆道闭锁诊断试剂盒。The invention relates to the field of biological detection, in particular to a diagnostic kit for biliary atresia based on serum marker MMP-7 protein.
背景技术Background technique
胆道闭锁(Biliary atresia，BA)是以肝外胆管进行性炎症、快速肝纤维化为特征的新生儿胆汁淤积性疾病，疾病进展迅速，3月以内行Kasai手术(肝门空肠吻合术)效果较好。该疾病在西方国家的发生率为1/17000-1/19000，而在台湾地区、日本为1/7000-1/13000，大陆地区的发生率为1/5000-1/12000 [1-6]，是小儿肝移植最常见病因 [2,7,8]。 Biliary atresia (BA) is a neonatal cholestatic disease characterized by progressive inflammation of the extrahepatic bile duct and rapid liver fibrosis. The disease progresses rapidly. Kasai surgery (hepatic jejunostomy) is performed within 3 months. it is good. The incidence of the disease in Western countries is 1/17,000-1/19000, while in Taiwan, Japan is 1/7000-1/13000, and the incidence in mainland China is 1/5000-1/12000 [1-6] Is the most common cause of liver transplantation in children [2,7,8] .
现有的BA筛查和诊断血清学指标包括：①血清总胆红素，直接胆红素；其作为最初筛查指标。新生儿血清总胆红素>2.0mg/dL(42–51μmol/L)，或直接胆红素>1.0mg/dL(17μmol/L)需进行BA排查，但对于诊断BA特异性差；②GGT：大样本调查研究表明，不同年龄组中，BA组GGT含量均明显高于其他胆汁淤积疾病组 [9]。Liu等人 [10]报道GGT>300U/L时，其诊断BA的精确度为60％-85％诊断。GGT是胆道闭锁最常用的筛查指标，但临床病例中不同患儿之间差异较大，准确性受到局限；③其他指标：血清胆酸、凝血酶原浓度、血小板测定影响因素较多。影像学检查中，异常胆囊(胆囊缩小，收缩差)和肝门部纤维块是超声检查胆道闭锁患儿肝脏的特征性表现 [11,12]，但肝门部纤维块不一定在每个患儿中均出现，并且超声检查因不同医生、机器等观察结果差异较大。因此文献中报道诊断BA的敏感性与特异性各不相同 [10,13-19]。近期，有Meta分析报道，胆囊闭锁和肝门部纤维块的特异性均可达到99％，然而相对应的敏感性为28％和80％ [10]。其它检查虽然可以给胆道闭锁的诊断提供一些线索，但受到不同程度的限制。如十二指肠肠液检测操作困难；放射性核素检查可不同程度加重患儿梗阻性黄疸 [2,20]；3个月龄以内的婴儿胆管直径小、胆管内液体少 [21]，磁共振胰胆管成像检查诊断胆道闭锁的高假阳性率不可避免。此外，BA的病理特征为小胆管增生、胆栓形成、毛细胆管和肝细胞胆汁淤积、汇管区或小叶周围纤维化，早期尚可见肝小叶结构 [1,20,21]。而α1抗胰 蛋白酶缺乏疾病的病理表现与之重叠，偶尔Alagille综合症、囊性纤维化、全胃肠外营养相关的胆汁淤积也可表现为相似的病理改变 [1]。因此，临床应用于鉴别BA与其他新生儿胆汁淤积症时，因检测准确性低或过程复杂导致BA诊断和治疗的延迟。 Current BA screening and diagnostic serological indicators include: 1 serum total bilirubin, direct bilirubin; as an initial screening indicator. Neonatal serum total bilirubin>2.0mg/dL (42–51μmol/L), or direct bilirubin>1.0mg/dL (17μmol/L) requires BA investigation, but poorly specific for diagnosis of BA; 2GGT: large Sample survey studies showed that the GGT levels in the BA group were significantly higher in the different age groups than in the other cholestasis groups [9] . Liu et al [10] reported that the accuracy of the diagnosis of BA was 60%-85% when GGT>300U/L. GGT is the most commonly used screening index for biliary atresia, but the difference between different children in clinical cases is large, and the accuracy is limited. 3 Other indicators: serum cholic acid, prothrombin concentration, and platelet determination have many influencing factors. In imaging examination, abnormal gallbladder (constriction of gallbladder, poor contraction) and hepatic ganglion are characteristic features of the liver in children with biliary atresia by ultrasonography [11,12] , but the hepatic mass is not necessarily in every disease. All of them appear in children, and the results of ultrasound examination vary greatly due to different doctors and machines. Therefore, the sensitivity and specificity of the diagnosis of BA are reported in the literature [10,13-19] . Recently, a meta-analysis reported that the specificity of gallbladder atresia and hepatic mass can reach 99%, but the corresponding sensitivity is 28% and 80% [10] . Other tests may provide some clues for the diagnosis of biliary atresia, but are subject to varying degrees of restriction. Such as duodenal intestinal fluid detection operation is difficult; radionuclide examination can aggravate obstructive jaundice in children [2,20] ; infants within 3 months of age have small bile duct diameter and less fluid in the bile duct [21] , magnetic resonance The high false positive rate of cholangiopancreatography in the diagnosis of biliary atresia is inevitable. In addition, the pathological features of BA are small bile duct hyperplasia, bile thrombosis, capillary bile duct and hepatocyte cholestasis, fibrosis around the portal area or lobule, and hepatic lobular structure can be seen early [1,20,21] . The pathological manifestations of α1 anti-trypsin deficiency disease overlap with it. Occasionally, Alagille syndrome, cystic fibrosis, and total parenteral nutrition-related cholestasis can also show similar pathological changes [1] . Therefore, when clinically applied to identify BA and other neonatal cholestasis, the diagnosis and treatment delay is delayed due to low detection accuracy or complicated process.
疾病早期，BA的临床表现、生物化学指标、影像学和组织学特征与其它胆汁淤积疾病存在重叠性，鉴别诊断过程曲折、复杂，容易导致诊断延迟。因此探索简单、特异性诊断方法从未停止。以往研究显示基质金属蛋白酶7(Matrix metalloproteinases-7，MMP-7)在慢性肝脏疾病中与肝纤维化形成有关 [22]。最近发现MMP-7可激活BA胆管上皮先天性免疫反应并参与胆管的炎性损伤 [23-25]。本项目通过病例对照研究MMP-7在BA患儿血清和肝内胆管组织中的表达情况，证明血清MMP-7水平在胆道闭锁诊断中具有应用价值。 In the early stage of the disease, the clinical manifestations, biochemical indicators, imaging and histological features of BA overlap with other cholestasis diseases. The differential diagnosis process is tortuous and complicated, which may lead to delayed diagnosis. Therefore, exploring simple, specific diagnostic methods has never stopped. Previous studies have shown that matrix metalloproteinases-7 (MMP-7) is associated with liver fibrosis in chronic liver disease [22] . Recently, MMP-7 was found to activate the innate immune response of BA bile duct epithelium and participate in inflammatory injury of the bile duct [23-25] . In this study, the expression of MMP-7 in serum and intrahepatic bile duct tissues of children with BA was studied by case-control study. It is proved that serum MMP-7 level has application value in the diagnosis of biliary atresia.
参考文献references
[1]Sokol RJ,Mack C,Narkewicz MR,等。胆道闭锁发病机制和预后的最新观念。儿科胃肠病学与营养学,2003,37(1):4-21.[1] Sokol RJ, Mack C, Narkewicz MR, et al. The latest concept of pathogenesis and prognosis of biliary atresia. Pediatric Gastroenterology and Nutrition, 2003, 37(1): 4-21.
[2]Hartley JL,Davenport M,Kelly DA。胆道闭锁。柳叶刀,2009,374(9702):1704-1713.[2] Hartley JL, Davenport M, Kelly DA. Biliary atresia. Lancet, 2009, 374 (9702): 1704-1713.
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发明内容Summary of the invention
本发明的目的在于克服现有诊断BA方法准确性差，操作复杂，病人在儿内科和儿外科反复就诊，程序繁琐，容易延误诊断，导致治疗的延迟，直接影响疗效的不足。而提供一种基于血清标志物MMP-7的胆道闭锁诊断试剂盒，它具有操作简单，结果可靠，高灵敏性、高特异性的特点。以解决现有技术中的BA诊断方法不能实现的诊断技术问题。The object of the present invention is to overcome the poor accuracy and complicated operation of the existing diagnostic BA method, and the patient is repeatedly treated in the pediatric medicine and the pediatric surgery, the procedure is cumbersome, the diagnosis is prone to delay, and the delay of treatment directly affects the deficiency of the curative effect. A diagnostic kit for biliary atresia based on the serum marker MMP-7 is provided, which has the characteristics of simple operation, reliable results, high sensitivity and high specificity. To solve the problem of diagnostic technology that cannot be realized by the prior art BA diagnosis method.
本发明的目的是通过如下技术方案来实现的：一种基于血清标志物MMP-7的胆道闭锁诊断试剂盒，用于BA的早期诊断，其特征是该诊断试剂盒包括抗人MMP-7抗体包被的酶标板、阳性对照液、样品稀释液(阴性对照液)、酶标试剂(A、B)、酶底物溶液、洗涤液和终止液。The object of the present invention is achieved by the following technical solution: a diagnostic kit for biliary atresia based on serum marker MMP-7, for early diagnosis of BA, characterized in that the diagnostic kit comprises anti-human MMP-7 antibody Coated ELISA plate, positive control solution, sample diluent (negative control solution), enzyme standard reagent (A, B), enzyme substrate solution, washing solution and stop solution.
所述阳性对照液为含有MMP-7的稀释于样品稀释液中血清，含MMP-7浓度为40ng/mL。The positive control solution was serum diluted with MMP-7 in a sample diluent containing a MMP-7 concentration of 40 ng/mL.
所述样品稀释液为0.01mol/L pH 7.4 PBS；阴性对照液即为样品稀释液。The sample diluent was 0.01 mol/L pH 7.4 PBS; the negative control solution was the sample dilution.
所述酶标试剂A为生物素化的MMP-7抗体，酶标试剂B为HRP标记的亲和素，浓度为0.1-111g/mL。The enzyme labeling reagent A is a biotinylated MMP-7 antibody, and the enzyme labeling reagent B is an HRP-labeled avidin at a concentration of 0.1-111 g/mL.
所述酶底物溶液为3,3',5,5'-四甲基联苯胺溶液(3,3',5,5'-Tetramethylbenzidine,TMB溶液)，包括显色剂A和显色剂B，显色剂A为500mL溶液中含有醋酸纳13.6g、柠檬酸1.6g和30％双氧水0.3mL；显色剂B为500mL溶液中含有TMB350mg、二甲亚砜(Dimethyl Sulphoxide,DMSO)20mL和柠檬酸.H 2O 5.1g。 The enzyme substrate solution is a 3,3',5,5'-tetramethylbenzidine solution (3,3',5,5'-Tetramethylbenzidine, TMB solution), including developer A and developer B. , developer A is 500 mL of sodium acetate containing 13.6 g, citric acid 1.6 g and 30% hydrogen peroxide 0.3 mL; developer B is 500 mL solution containing TMB 350 mg, dimethyl sulfoxide (DMSO) 20 mL and lemon Acid. H 2 O 5.1 g.
所述洗涤液为0.0lmol/L pH 7.4磷酸盐-NaCl缓冲液(PBST)，PBST内含0.05％吐温20(Tween-20)，即1升溶液中含8g NaCl、0.2g KH 2P0 4、2.9g Na 2HP0 4.12H 20、0.2g KCl和0.5mL Tween-20。 The washing solution is 0.01 lmol/L pH 7.4 phosphate-NaCl buffer (PBST), and the PBST contains 0.05% Tween-20, that is, 1 g of the solution contains 8 g of NaCl and 0.2 g of KH 2 P0 4 . 2.9 g Na 2 HP0 4 .12H 2 0, 0.2 g KCl and 0.5 mL Tween-20.
所述终止液为2mol/L H 2S0 4溶液。 The stop solution was a 2 mol/L H 2 SO 4 solution.
所述试剂盒中的试剂均可加入防腐剂。The reagents in the kit may be added with a preservative.
所述抗人MMP-7抗体包被的酶标板所用抗体为抗人MMP-7单克隆抗体。The antibody used for the anti-human MMP-7 antibody-coated ELISA plate is an anti-human MMP-7 monoclonal antibody.
基于血清标志物MMP-7的胆道闭锁诊断试剂盒的诊断方法，用于BA早期的诊断，所述诊断试剂盒采用的是现在临床广泛使用的ELISA技术，应用间接法定量检测人血清中MMP-7含量，具体是：向抗人MMP-7抗体包被的酶标板中加入待测血清，加入生物素化的MMP-7抗体，在将未结合的生物素化抗体洗净后，加入HRP标记的亲和素，形成酶抗体复合物，经过彻底洗涤后加底物TMB显色，TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色，颜色的深浅和样品中的MMP-7的水平呈正相关。A diagnostic method for the biliary atresia diagnostic kit based on the serum marker MMP-7, which is used for the early diagnosis of BA. The diagnostic kit uses an ELISA technique widely used in clinical practice to quantitatively detect MMP in human serum by indirect method. 7 content, specifically: adding anti-human MMP-7 antibody-coated ELISA plate to the test serum, adding biotinylated MMP-7 antibody, and after washing the unbound biotinylated antibody, adding HRP The labeled avidin forms an enzyme antibody complex. After thorough washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted into a final yellow color under the action of acid. It is positively correlated with the level of MMP-7 in the sample.
与现有技术相比，本发明有以下优点:Compared with the prior art, the present invention has the following advantages:
1、提供一种新的灵敏、安全、可靠、易操作的商品化试剂盒，定量测定人血清中MMP-7的水平，有助于早期诊断BA；1. Provide a new sensitive, safe, reliable and easy-to-operate commercial kit to quantitatively determine the level of MMP-7 in human serum, which is helpful for early diagnosis of BA;
2、提供的血清生物标志物蛋白质MMP-7诊断BA的敏感性为100％，特异性为95.6％，具有高特异性和高敏感性的特点。2. The serum biomarker protein MMP-7 provided has a sensitivity of 100% and a specificity of 95.6%. It has high specificity and high sensitivity.
3、提高BA的早期诊断率，减少误诊，提高BA自身肝脏生存率。3. Improve the early diagnosis rate of BA, reduce misdiagnosis, and improve the survival rate of BA's own liver.
附图说明DRAWINGS
图1为ELISA测定血清MMP-7的标准曲线；Figure 1 is a standard curve of serum MMP-7 by ELISA;
图2A和图2B为血清MMP-7在BA组、非BA组和对照组中的表达情况；其中：图2A表示整体人群中BA组血清MMP-7浓度明显高于非BA组和CO组(**P<0.001)；图2B表示年龄小于60d患儿中，BA组血清MMP-7浓度明显高于非BA组及CO组(**P<0.001)2A and 2B show the expression of serum MMP-7 in BA group, non-BA group and control group; wherein: Figure 2A shows that serum MMP-7 concentration in BA group is significantly higher than that in non-BA group and CO group in the whole population (Fig. 2A) **P<0.001); Figure 2B shows that serum MMP-7 levels in BA group were significantly higher than those in non-BA and CO groups (**P<0.001).
图3A、图3B和图3C为血清MMP-7与年龄相关性情况；3A, 3B and 3C are age-related cases of serum MMP-7;
其中：图3A表示BA组血清MMP-7浓度与年龄呈正相关趋势(P<0.001)；图3B表示非BA组血清MMP-7浓度与年龄不相关(P＝0.11)；图3C表示对照组血清MMP-7浓度与年龄无相关趋势(P＝0.29)；Among them: Figure 3A shows that the serum MMP-7 concentration in the BA group is positively correlated with age (P < 0.001); Figure 3B shows that the non-BA group serum MMP-7 concentration is not related to age (P = 0.11); Figure 3C shows the control serum There was no correlation between MMP-7 concentration and age (P=0.29);
图4为血清MMP-7与GGT诊断BA的ROC曲线(受试者工作特征曲线receiver operating characteristic curve，简称ROC曲线)下面积比较，MMP-7与GGT的ROC曲 线下面积分别为0.996和0.678；Figure 4 is a comparison of the area under the ROC curve (receiver operating characteristic curve, ROC curve) of serum MMP-7 and GGT for diagnosis of BA. The area under the ROC curve of MMP-7 and GGT is 0.996 and 0.678, respectively;
图5为肝脏MMP-7基因表达情况：BA组MMP-7mRNA表达明显高于分BA组和对照组；Figure 5 shows the expression of MMP-7 gene in liver: the expression of MMP-7 mRNA in BA group was significantly higher than that in BA group and control group;
图6表示BA患儿肝脏中可见大量增生、排列紊乱的小胆管表达MMP-7强阳性(免疫组织化学染色，放大40倍)；Figure 6 shows that a large number of hyperplasia, disordered small bile ducts in the liver of children with BA express MMP-7 strong positive (immunohistochemical staining, 40 times magnification);
图7A、图7B、图7C、图7D、图7E、图7F为MMP-7在不同患儿肝脏中的表达情况比较(图中的方框部分为放大的区域)；7A, 7B, 7C, 7D, 7E, and 7F show the expression of MMP-7 in the liver of different children (the block in the figure is an enlarged area);
其中：图7A(放大100倍),图7B(放大400倍)表示BA患儿肝脏汇管区可见胆管上皮细胞强阳性染色；图7C(放大100倍),图7D(放大400倍)非BA(婴儿肝炎)患儿肝脏汇管区，仅可见部分胆管上皮细胞MMP-7染色；图7E(放大100倍),图7F(放大400倍)正常肝脏汇管区中未见MMP-7表达。Among them: Figure 7A (magnification 100 times), Figure 7B (magnification 400 times) showed strong positive staining of biliary epithelial cells in the liver portal area of children with BA; Figure 7C (magnification 100 times), Figure 7D (magnification 400 times) non-BA ( In infants with hepatitis), only MMP-7 staining was observed in some of the bile duct epithelial cells; Figure 7E (100-fold magnification), Figure 7F (400-fold magnification) No MMP-7 expression was observed in the normal liver portal area.
具体实施方式Detailed ways
以下结合附图对本发明做进一步详细的说明，但它们并不构成对本发明的限定，仅作举例，同时通过说明本发明的优点将变得更加清林和容易理解。The invention will be further described in detail below with reference to the accompanying drawings, which are not to be construed as limiting.
参阅附图可知：1、血清MMP-7浓度检测及比较：Referring to the attached drawings, we can see that: 1. Detection and comparison of serum MMP-7 concentration:
按照试剂盒说明书，做标准曲线如图1。According to the kit instructions, the standard curve is shown in Figure 1.
图中横坐标为吸光度值(OD值)，纵坐标为MMP-7浓度，从图1可以看出OD值与MMP-7浓度呈正相关系。In the figure, the abscissa is the absorbance value (OD value), and the ordinate is the MMP-7 concentration. It can be seen from Fig. 1 that the OD value is positively correlated with the MMP-7 concentration.
整体人群及年龄小于60天患儿血清MMP-7水平分别如图2A和图2B所示，BA组均明显高于非BA组和对照组(P<0.001)。The overall population and serum MMP-7 levels in children younger than 60 days were shown in Figure 2A and Figure 2B, respectively, and the BA group was significantly higher than the non-BA group and the control group (P < 0.001).
BA组血清MMP-7浓度与患儿年龄呈正相关趋势，如图3A；非BA组和对照组血清MMP-7浓度与年龄无相关性，如图3B，3C。The serum MMP-7 concentration in the BA group was positively correlated with the age of the children, as shown in Fig. 3A; the serum MMP-7 concentration in the non-BA group and the control group was not correlated with age, as shown in Fig. 3B, 3C.
血清MMP-7诊断BA的ROC曲线计算曲线下面积为0.996，如图4。当血清MMP-7浓度>55.18ng/mL时,其诊断BA的灵敏度与特异度分别为100％和95.6％。The area under the curve of the ROC curve for the diagnosis of BA by serum MMP-7 was 0.996, as shown in Fig. 4. When the serum MMP-7 concentration was >55.18 ng/mL, the sensitivity and specificity of the diagnostic BA were 100% and 95.6%, respectively.
2、MMP-7在不同患儿肝脏组织中基因和蛋白的表达鉴定及比较：2. Identification and comparison of gene and protein expression in liver tissues of different children with MMP-7:
RT-PCR检测显示，BA组肝脏MMP-7基因mRNA表达为非BA组的8倍，明显高于非BA组(P<0.001)和对照组(P<0.001)。非BA组与对照组无明显差异(P＝0.15)， 如图5。RT-PCR analysis showed that the expression of MMP-7 mRNA in liver of BA group was 8 times higher than that in non-BA group, which was significantly higher than that in non-BA group (P<0.001) and control group (P<0.001). There was no significant difference between the non-BA group and the control group (P=0.15), as shown in Fig. 5.
3、BA组在低倍镜下可见肝小叶周围存在大量增生、排列紊乱的小胆管，这些增生的小胆管表达MMP-7强阳性表达，如图6。高倍镜下可见胆管、疸栓强阳性染色，如图7A,7B；在非BA组中，仅可见部分胆管上皮细胞MMP-7染色，如图7C,7D；对照组中未见MMP-7表达，如图7E,7F。3. In the BA group, there were a large number of small bile ducts with hyperplasia and disordered arrangement around the hepatic lobules. These hyperplastic bile ducts expressed strong positive expression of MMP-7, as shown in Fig. 6. High-powered microscopy showed strong staining of bile duct and sputum plug, as shown in Figures 7A and 7B. In the non-BA group, only partial biliary epithelial cells were stained with MMP-7, as shown in Figure 7C, 7D; no MMP-7 expression was observed in the control group. , as shown in Figures 7E, 7F.
4、血清样品的准备：4. Preparation of serum samples:
全血标本于室温放置2小时或4℃过夜后于1000g离心20分钟左右，取上清即可。立即检测或进行分装，并将标本放于-20℃或-80℃保存，避免反复冻融。解冻后的样品应再次离心，然后检测。所检测样品中不能含有NaN 3，因为NaN 3抑制辣根过氧化物酶(HRP)的活性。 The whole blood sample was allowed to stand at room temperature for 2 hours or at 4 ° C overnight, and then centrifuged at 1000 g for about 20 minutes, and the supernatant was taken. Immediately test or dispense, and store the specimen at -20 ° C or -80 ° C to avoid repeated freezing and thawing. The thawed sample should be centrifuged again and then tested. NaN 3 was not contained in the sample tested because NaN 3 inhibited the activity of horseradish peroxidase (HRP).
5、抗体酶标板的制备方法为：5. The preparation method of the antibody ELISA plate is:
用pH为8.2的PBS缓冲液将抗人MMP-7单克隆抗体稀释至60μg/m1，按100μ1/孔的加入量加于96孔酶标板中，在温度为4℃的条件下放置过夜，然后用洗涤液洗涤，所述洗涤液为含有0.05％Tween-20，pH为7.4的磷酸盐缓冲液，甩干，得包被有抗MMP-7单克隆抗体的96孔酶标板。The anti-human MMP-7 monoclonal antibody was diluted to 60 μg/ml with PBS buffer at pH 8.2, and added to a 96-well microtiter plate at a dose of 100 μl/well, and left at a temperature of 4 ° C overnight. Then, it was washed with a washing solution containing 0.05% Tween-20, pH 7.4 phosphate buffer, and dried to obtain a 96-well microtiter plate coated with an anti-MMP-7 monoclonal antibody.
6、ELISA法测定血清中MMP-7的浓度，以辅助早期诊断BA:6. ELISA method to determine the concentration of MMP-7 in serum to assist early diagnosis of BA:
诊断试剂盒的具体操作步骤如下:The specific steps of the diagnostic kit are as follows:
6.1标准品与样品的稀释与加样：将标准品与待测血清样品1:100用样品缓冲液稀释至100uL，加入到各自的抗原测定孔板中。注意不要有气泡，加样将样品加于梅标板孔底部，尽量不触及孔壁，轻轻晃动泪匀，酶标板上加盖或覆膜。若待测血清样品较多，建议使用多管微量加液器加样。标准品及待检测样品临用前15分钟内配制，用完丢弃，下次检测使用新鲜配制的标准品。6.1 Dilution and loading of standards and samples: Standards and serum samples to be tested 1:100 were diluted to 100 uL with sample buffer and added to the respective antigen assay well plates. Be careful not to have air bubbles. Add the sample to the bottom of the hole of the plum mark plate, try not to touch the hole wall, gently shake the tears, and cover or cover the microplate. If there are more serum samples to be tested, it is recommended to use a multi-tube micro-spirator to load. Standards and samples to be tested are prepared within 15 minutes before use, discarded after use, and freshly prepared standards are used for the next test.
6.2温育：酶标板置于37℃反应60分钟，甩净孔中液体，不用洗涤。6.2 Incubation: The plate was placed at 37 ° C for 60 minutes to clean the liquid in the well without washing.
6.3加酶：每孔加酶标试剂A 100uL，37℃，60分钟。甩净孔中液体，洗板3次，拍干。每孔加酶标试剂B 100uL，37℃，30分钟。甩净孔中液体，洗板5次，拍干。6.3 Add enzyme: add 100 μL of enzyme-labeled reagent A per well at 37 ° C for 60 minutes. Clean the liquid in the hole, wash the plate 3 times, and pat dry. Enzyme-labeled reagent B 100 uL per well, 37 ° C, 30 minutes. Clean the liquid in the hole, wash the plate 5 times, and pat dry.
6.4显色：各孔TMB底物溶液90uL，轻轻震荡混匀，37℃避光显色15分钟。6.4 Color development: 90 uL of TMB substrate solution in each well, gently shake and mix, and develop color at 37 ° C for 15 minutes.
6.5终止：依序每孔加终止液50uL，终止反应。终止液的加入顺序应尽量与底物液的加入顺序相同。底物反应时间到后应尽快加入终止液。6.5 Termination: 50 uL of stop solution was added to each well in sequence to terminate the reaction. The order of addition of the stop liquid should be as close as possible to the order in which the substrate liquid is added. The stop solution should be added as soon as possible after the substrate reaction time has elapsed.
6.6结果判定：6.6 Result judgment:
在加入终止液后15min内，用酶标仪在450nm波长测量各孔的光密度(OD值)。检测试剂盒的检测标准为：以标准品的浓度为纵坐标(或对数坐标)，OD值为横坐标(或对数坐标)，使用curve expert 1.30软件绘出标准曲线(最佳方程式应依回归方程计算的R 2值未定，以R2值越趋近于1为好)，计算样品的实际浓度。其中：阳性结果判断标准为：MMP-7>55.18ng/mL。 The optical density (OD value) of each well was measured by a microplate reader at a wavelength of 450 nm within 15 minutes after the addition of the stop solution. The detection standard of the test kit is: the concentration of the standard is the ordinate (or logarithmic coordinate), the OD value is the abscissa (or logarithmic coordinate), and the standard curve is drawn using the curve expert 1.30 software (the best equation should be The R 2 value calculated by the regression equation is undetermined, and the R 2 value is closer to 1 is better. The actual concentration of the sample is calculated. Among them: the positive result judgment standard is: MMP-7>55.18 ng/mL.
对108例BA患儿血清、87例非BA患儿血清、52例对照患儿血清的ROC分析确立了以上参考值。The above reference values were established for ROC analysis of 108 BA children's serum, 87 non-BA children's serum, and 52 control children's serum.
7、特异性和敏感性检测：采用300份BA相关患者的血清(BA患儿100例、非BA黄疸患儿100例、无肝病或黄疸患儿100例)对本发明的诊断试剂盒进行了特异性和敏感性检测。本发明的诊断试剂盒辅助早期诊断BA的特异性为95.6％，敏感性为100％，均高于现有技术中BA诊断的指标。7. Specificity and sensitivity test: The serum of 300 BA patients (100 children with BA, 100 children with non-BA jaundice, 100 children without liver disease or jaundice) were specifically used for the diagnostic kit of the present invention. Sex and sensitivity testing. The diagnostic kit of the present invention assists the early diagnosis of BA with a specificity of 95.6% and a sensitivity of 100%, which are higher than the prior art BA diagnostic indicators.
本发明一种基于血清标志物MMP-7的胆道闭锁诊断试剂盒，所述诊断试剂盒包括抗人MMP-7单克隆抗体包被的酶标板、阴性对照液、阳性对照液、酶标试剂、酶底物溶液、封闭液、样品稀释液、洗涤液和终止液；The invention provides a diagnostic kit for biliary atresia based on serum marker MMP-7, wherein the diagnostic kit comprises an anti-human MMP-7 monoclonal antibody coated ELISA plate, a negative control solution, a positive control solution and an enzyme standard reagent. , enzyme substrate solution, blocking solution, sample diluent, washing solution and stop solution;
所述阴性对照液为不含MMP-7的稀释于样品稀释液中的健康人血清；The negative control solution is a healthy human serum diluted in the sample diluent without MMP-7;
所述阳性对照液为含有MMP-7的稀释于样品稀释液中血清，含MMP-7浓度为100U/mL；The positive control solution is a serum containing MMP-7 diluted in a sample diluent, and the concentration of MMP-7 is 100 U/mL;
所述酶标试剂为含辣根过氧化物酶(HRP)标记的二抗0.1-111g/mL；The enzyme labeling reagent is a horseradish peroxidase (HRP)-labeled secondary antibody 0.1-111 g/mL;
所述酶底物溶液为3,3',5,5'-四甲基联苯胺溶液，包括显色剂A和显色剂B，显色剂A为500mL溶液中含有醋酸纳13.6g、柠檬酸1.6g和30％双氧水0.3mL；显色剂B为500mL溶液中含有TMB 350mg、二甲亚砜20mL和柠檬酸.H 2O 5.1g； The enzyme substrate solution is a 3,3',5,5'-tetramethylbenzidine solution, including a developer A and a developer B, and the developer A is 500 mL of a solution containing 13.6 g of sodium acetate and a lemon. Acid 1.6g and 30% hydrogen peroxide 0.3mL; developer B is 500mL solution containing TMB 350mg, dimethyl sulfoxide 20mL and citric acid. H 2 O 5.1g;
所述封闭液为0.5％牛血清白蛋白的0.01mol/L pH 7.4磷酸盐-NaCl缓冲液溶液，即1升溶液中含5g BSA、8g NaCl、0.2g KH 2P0 4、2.9g Na 2HP0 4.12H 2O和0.2g KCl； The blocking solution is a 0.01 mol/L pH 7.4 phosphate-NaCl buffer solution of 0.5% bovine serum albumin, ie, 1 g of BSA, 8 g of NaCl, 0.2 g of KH 2 P0 4 , and 2.9 g of Na 2 HP0 in 1 liter of solution. 4 .12H 2 O and 0.2g KCl;
所述样品稀释液为0.01mol/L pH 7.4PBS；The sample diluent is 0.01 mol / L pH 7.4 PBS;
所述洗涤液为0.0lmol/L pH 7.4磷酸盐-NaCl缓冲液PBST，PBST内含0.05％吐温20，即1升溶液中含8g NaCl、0.2g KH 2P0 4、2.9gNa 2HP0 4.12H 20、0.2g KCl和0.5mL Tween-20； The washing solution is 0.01 lmol/L pH 7.4 phosphate-NaCl buffer PBST, and PBST contains 0.05% Tween 20, that is, 1 liter of solution contains 8 g of NaCl, 0.2 g of KH 2 P0 4 , and 2.9 g of Na 2 HP0 4 . 12H 2 0, 0.2g KCl and 0.5mL Tween-20;
所述终止液为2mol/L H 2S0 4溶液。 The stop solution was a 2 mol/L H 2 SO 4 solution.
所述试剂盒中的试剂均可加入防腐剂。The reagents in the kit may be added with a preservative.
基于血清标志物MMP-7的胆道闭锁诊断试剂盒的诊断方法，用于BA早期的诊断，所述诊断试剂盒采用的是现在临床广泛使用的ELISA技术，应用间接法定量检测人血清中MMP-7含量，具体是:向抗人MMP-7抗体包被的酶标板中加入待测血清，加入生物素化的MMP-7抗体，在将未结合的生物素化抗体洗净后，加入HRP标记的亲和素，形成酶抗体复合物，经过彻底洗涤后加底物TMB显色，TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色，颜色的深浅和样品中的MMP-7的水平呈正相关。A diagnostic method for the biliary atresia diagnostic kit based on the serum marker MMP-7, which is used for the early diagnosis of BA. The diagnostic kit uses an ELISA technique widely used in clinical practice to quantitatively detect MMP in human serum by indirect method. 7 content, specifically: adding anti-human MMP-7 antibody-coated ELISA plate to the test serum, adding biotinylated MMP-7 antibody, and after washing the unbound biotinylated antibody, adding HRP The labeled avidin forms an enzyme antibody complex. After thorough washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted into a final yellow color under the action of acid. It is positively correlated with the level of MMP-7 in the sample.
其它未详细说明的均为现有技术。Others not described in detail are prior art.

Claims (3)

  1. 一种基于血清标志物MMP-7蛋白的胆道闭锁诊断试剂盒，该诊断试剂盒包括抗人MMP-7抗体包被的酶标板，阳性对照液，样品稀释液，酶标试剂A、酶标试剂B，酶底物溶液，洗涤液和终止液；A diagnostic kit for biliary atresia based on serum marker MMP-7 protein, the diagnostic kit includes an anti-human MMP-7 antibody-coated ELISA plate, a positive control solution, a sample diluent, an enzyme standard reagent A, and an enzyme label Reagent B, enzyme substrate solution, washing solution and stop solution;
    所述阳性对照液为含有MMP-7的稀释于样品稀释液中血清，含MMP-7浓度为40ng/mL。The positive control solution was serum diluted with MMP-7 in a sample diluent containing a MMP-7 concentration of 40 ng/mL.
    所述样品稀释液为0.01mol/L pH 7.4 PBS；阴性对照液即为样品稀释液。The sample diluent was 0.01 mol/L pH 7.4 PBS; the negative control solution was the sample dilution.
    所述酶标试剂A为生物素化的MMP-7抗体，酶标试剂B为HRP标记的亲和素，浓度为0.1-111g/mL。The enzyme labeling reagent A is a biotinylated MMP-7 antibody, and the enzyme labeling reagent B is an HRP-labeled avidin at a concentration of 0.1-111 g/mL.
    所述酶底物溶液为3,3',5,5'-四甲基联苯胺溶液，包括显色剂A和显色剂B，显色剂A为500mL溶液中含有醋酸纳13.6g、柠檬酸1.6g和30％双氧水0.3mL；显色剂B为500mL溶液中含有TMB350mg、二甲亚砜20mL和柠檬酸.H 2O 5.1g。 The enzyme substrate solution is a 3,3',5,5'-tetramethylbenzidine solution, including a developer A and a developer B, and the developer A is 500 mL of a solution containing 13.6 g of sodium acetate and a lemon. Acid 1.6g and 30% hydrogen peroxide 0.3mL; developer B is 500mL solution containing TMB 350mg, dimethyl sulfoxide 20mL and citric acid. H 2 O 5.1g.
    所述洗涤液为0.0lmol/L pH 7.4磷酸盐-NaCl缓冲液PBST，PBST内含0.05％吐温20，即1升溶液中含8g NaCl、0.2g KH 2P0 4、2.9g Na 2HP0 4.12H 2O、0.2g KCl和0.5mL Tween-20。 The washing liquid is 0.01 lmol/L pH 7.4 phosphate-NaCl buffer PBST, and PBST contains 0.05% Tween 20, that is, 1 liter of solution contains 8 g of NaCl, 0.2 g of KH 2 P0 4 , and 2.9 g of Na 2 HP0 4 . .12H 2 O, 0.2 g KCl and 0.5 mL Tween-20.
    所述终止液为2mol/L H 2S0 4溶液。 The stop solution was a 2 mol/L H 2 SO 4 solution.
  2. 根据权利要求1所述的一种基于血清标志物MMP-7的胆道闭锁诊断试剂盒，其特征在于:所述试剂盒中的试剂均可加入防腐剂。The biliary atresia diagnostic kit based on serum marker MMP-7 according to claim 1, wherein the reagent in the kit can be added with a preservative.
  3. 根据权利要求1或2所述的一种基于血清标志物MMP-7的胆道闭锁诊断试剂盒，其特征在于:所述抗人MMP-7抗体包被的酶标板所用抗体为抗人MMP-7单克隆抗体。The biliary atresia diagnostic kit based on serum marker MMP-7 according to claim 1 or 2, wherein the antibody against the human MMP-7 antibody-coated ELISA plate is anti-human MMP- 7 monoclonal antibodies.
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