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Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen
人主动脉抗原循环自身抗体的定性和定量检测实验   

编审
Jia Li
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Abstract

Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to detect the presence of autoantibodies as well as to measure the circulating autoantibodies in the sera of patients suffering from large vessel vasculitis (LVV), an autoimmune disease (Chakravarti et al., 2015). Using this assay, we detected the increase in anti-aortic antibodies in LVV patient’s sera. We have further verified the results by independent biochemical techniques and found the specificity to be > 94% (Chakravarti et al., 2015). This method can be uniquely modified to suit any autoimmune, in particular organ specific, disease and thus has wider applications in the detection and quantification of autoantibodies.

Keywords: ELISA(ELISA), Autoantigen(自身抗原), Autoantibody(自身抗体), Vasculitis(血管炎), Serum(血清), Autoimmune disease(自身免疫性疾病)

Background

There are about 80-100 autoimmune diseases wherein immune cells recognize a self-protein as a foreign antigen, and get activated to generate humoral response. Though the trigger for most autoimmune diseases remains a mystery, presence of higher levels of antibodies in the patient’s sera is very common (Wang et al., 2015). Some of the examples include antibodies against neutrophil cytoplasmic antigens in small vessel vasculitis, myelin basic protein in multiple sclerosis, glutamate decarboxylase in type 1 diabetes etc. (Wang et al., 2015). Limited knowledge of autoantigen in most autoimmune diseases presents a challenge for both the detection of disease as well as understanding the pathogenesis. However, ability to detect autoantibodies in the sera provides a diagnostic and prognostic advantage. Discovering a reliable autoantigen in autoimmune diseases are needed to make better clinical decisions. LVV is a spectrum of autoimmune diseases affecting aorta or its primary branch vessels. Like many others, its etiology and cure are not known (Buttgereit et al., 2016). We have developed a qualitative and quantitative assay to detect the presence of autoantibody in the human sera targeting against the human aortic antigen. Our assay provides a tool to find autoantigen in any affected/damaged tissue and in any disease model. We looked for the presence of autoantigen in the human thoracic aortic aneurysms using patients’ sera and tested the specificity of the assay by comparing the sera obtained from related subsets of autoimmune or non-immune diseases. We utilized discarded aortic tissues from aortic reconstruction surgeries and made soluble extracts of aorta to set up the assay (Figure 1). Using this method we performed a high throughput screen for detecting autoantibody in more than 100 sera against aortic antigen (Chakravarti et al., 2015).


Figure 1. Schematic Flow Diagram of ELISA to detect autoantibody in human tissue lysates. Step-by-step outline of the method used to detect and quantify autoantibodies present in the sera against antigen present in tissue.

Materials and Reagents

  1. 5 ml sterile polystyrene round bottom tubes (Corning, Falcon®, catalog number: 352008 )
  2. 1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5500 )
  3. 15 ml centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  4. Greiner 96 well, F-Bottom, clear microplate (Greiner Bio One International, catalog number: 655001 )
  5. 200 μl pipette tip (USA Scientific, catalog number: 1111-1700 )
  6. 1,250 μl pipette tip (USA Scientific, catalog number: 1112-1720 )
  7. Immulon 2 HB: high affinity protein binding plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3455 )
  8. Paper towel
  9. Human Specimen
    Note: Human tissue explant as well as sera should be collected under institutionally approved IRB protocol.
  10. Protease
  11. Phosphatase inhibitor (PhosSTOP) (Roche Diagnostics, catalog number: 04906837001 )
  12. Protein Assay dye (Bio-Rad Laboratories, catalog number: 5000006 )
  13. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  14. Phosphate-buffered saline-Tween (PBS-T) (Fisher Scientific, catalog number: BP293810 )

  15. Antibodies:
    1. Anti-Human IgG (used at 1:5,000) (Thermo Fisher Scientific, Invitrogen, catalog number: 31135 )
    2. HRP-conjugated secondary antibodies (used at 1:3,000) (Bio-Rad Laboratories, catalog numbers: 1706516 ) 

  16. Ultra-TMB (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34029 )
  17. 2 N HCl (Fisher Scientific, catalog number: SA431-500 )
  18. Tris buffer, pH 7.4 (Fischer Scientific, catalog number: BP152-1 )
  19. Sodium chloride (NaCl) (Sigma-Aldrich, product number: S9888 )
  20. Triton X-100 (Bio-Rad Laboratories, catalog number: 1610407 )
  21. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  22. Sodium fluoride (Sigma-Aldrich, catalog number: 919 )
  23. Glycerophosphate (Sigma-Aldrich, catalog number: G9422 )
  24. Sodium pyrophosphate (Sigma-Aldrich, catalog number: P8010 )
  25. Non-fat dry milk powder (Bio-Rad Laboratories, catalog number: 1706404 )
  26. Cell lysis buffer (see Recipes)
  27. Blocking buffer (see Recipes)

Equipment

  1. 2-20 μl pipettes (Alkali Scientific, catalog number: P9280-20U )
  2. 20-200 μl pipettes (Alkali Scientific, catalog number: P9280-200U )
  3. Scissors (Fisher Scientific, catalog number: 08-951-20 )
  4. Table top centrifuge (Labnet International, model: PrismRTM R, catalog number: C2500-R )
  5. Orbital shaker (Benchmark Scientific, model: BT302 )
  6. Refrigerator

  7. Plate reader (BMG LABTECH, model: FLUstar Omega )
  8. Homogenizer (Thomas Scientific, model: SCILOGEX D160 )

Software

  1. MS Excel
  2. GraphPad Prism 7

Procedure

  1. Making tissue lysates
    1. Chop tissue into small pieces (as much possible) and place in a sterile round bottom tube on ice.
    2. Add 5 ml of cold cell lysis buffer (see Recipes) containing protease and phosphatase inhibitors. Homogenize tissue using tissue homogenizer in sterile lysis buffer at cold temperature by keeping the tube covered in ice to prevent protein denaturation.
    3. Perform 6-8 cycles, 20-30 sec each, of homogenization. After each cycle, wait for 2 min and evaluate suspension manually for the presence of large pieces of tissues (Figure 2).
      Note: If the intact tissues pieces are observed, continue homogenization to improve solubilization. Make sure that tissue lysate stays cold during homogenization.
    4. Centrifuge homogenized lysate at 12,000 x g for 20 min under refrigeration at 4 °C. Transfer supernatants to sterile Eppendorf tubes and keep them cold. If needed, lysate can be stored at -80 °C for long-term.


      Figure 2. Tissue homogenization. A. Cartoon showing round tube containing red colored pieces of tissue in lysis buffer and a sharp metal homogenizer; B. Representative picture of soluble tissue lysate.

  2. Measuring protein concentration in tissue lysate
    1. Pipette a Greiner 96-well plate with 200 µl of pre-diluted (1:5) Bio-Rad Protein Assay dye into each well corresponding to a sample.
      Note: BSA standards and tissue lysate usually measured in the same plate, however separated here for clarity.
    2. First, prepare a protein standard curve using BSA concentration in the range of 0-1 mg/ml at the increment of 0.1 mg/ml. To each well containing assay dye, add 1 µl of BSA solution mixing solution with pipette tip, and incubate for 5 min. Each concentration of BSA is assayed in triplicate. Absorbance of wells is measured using plate reader at 595 nm. Wells containing no protein solution (no BSA) are used as blank for subtraction from the actual samples. Average absorbance (blank subtracted average of triplicates) and respective concentrations of BSA samples are plotted in an excel file to obtain the linear equation (Figure 3).
      Note: Standard equation of linear functions is: Y = MX + C, where Y is absorbance, X is BSA concentration, M is slope and C is intercept on y-axis.


      Figure 3. Sample standard curve using BSA with the fitted line and equation. This BSA curve and equation will be used to calculate protein concentration from tissue lysate.

    3. Next, add 1 µl of supernatant from the tissue lysate to each well containing 200 µl of protein assay reagent. In case of high concentration of proteins, dilute tissue lysates in 1:10 or 1:20 with cell lysis buffer before the protein estimation. Measure each sample in triplicate.
    4. Measure absorbances at 595 nm using plate reader.
    5. After the blank subtraction, average absorbance values for each sample. Plot final absorbance of each sample on the BSA standard curve to calculate the protein concentrations of the tissue lysate (see step B2). In case tissue lysates are diluted, dilution factor is included for the final calculation.
      Note: When calculating the concentration of the sample undergoing ELISA procedure, input the absorbance measured into equation as Y value, and use slope (M) and y-intercept (C) from BSA line equation as constants to calculate protein concentration (X).

  3. ELISA
    1. Coat Immulon 2 HB plates with 100 µl of soluble tissue lysate by adding in each well. We recommend testing 3 different protein dilutions (ranging from 1:10 to 1:1,000) in PBS buffer for this analysis in triplicate. Use BSA solution of the same protein concentration as tissue lysates. Incubate at 4 °C overnight. In case of any known antigen, use purified proteins of concentrations (5-100 µg/ml) as positive control in the experiment.
    2. Remove the protein solution by decanting the plate. Wash the wells 3 times with PBS-T (200 µl) by shaking for 5 min at room temperature each time. Tap it upside down on the paper towel to get rid of any retained liquid.
    3. Block the wells by adding 200 µl of blocking buffer (see Recipes) in each well and incubate for 2 h at room temperature (RT) while shaking at 100 rpm.
    4. Remove the blocking buffer by decanting.
    5. Add 200 µl of diluted sera (typical dilutions range from 1-10-1:1,000) in the blocking buffer. We recommend using 3 dilutions of each sera to figure out the best dilutions for subsequent experiments. Also include no sera control for tissue lysate coated wells.
      Note: Frozen sera should always be thawed on ice before making dilutions.
    6. Incubate the plate on shaker at 100 rpm for 2 h at RT or overnight at 4 °C.
    7. Remove sera and wash wells 3 times with PBS-T. Tap it upside down on a paper towel to get rid of any retained liquid.
    8. Add 200 µl of mouse anti-human IgG antibody, the primary antibody, diluted with blocking buffer in the ratio of 1:5,000. Shake the plate at RT for 2 h at 100 rpm.
    9. Remove the primary antibody and wash the plate 3 times with PBS-T as in step C7.
    10. Add 200 µl of anti-mouse IgG conjugated with HRP (diluted in blocking buffer at 1:3,000) and shake the plate for 1 h at RT.
    11. Remove the secondary antibody by decanting the plate and wash the plate as in step C7.
      Note: While performing washing step, prepare the plate reader for plate reading. Set up the program for the wells to be read at 450 nm.
    12. Add 100 µl of ultra-TMB and observe the blue color development. Depending upon the antigen (protein concentration of tissue lysates) and autoantibodies (sera dilutions), some wells may change color to blue before others. Change of color in 5-30 min is desirable. Very quick change of color would require lowering the amount of tissue lysates or increasing the serum dilution. If color change is very slow, takes more than 60 min, increasing the tissue lysates or decreasing the sera dilution, may be needed.
    13. As desirable change in color is achieved, add 100 µl of 2 N HCl to each well that will change the color to yellow (Figure 4).
    14. Read plate at 450 nm and note absorbance in each well.


      Figure 4. Image showing final readout of ELISA. Addition of HCL to the TMB containing well changes the color of solution from blue to yellow. Arrow shows the desirable intensity of yellow color that should be within the linear range of absorbance of the plate reader.

Data analysis

  1. Since all of our samples are tested in triplicate, we averaged the absorbance obtained for each of our patient samples.
  2. All the samples were clustered in the set of diseases (e.g., sera of patients with large vessel vasculitis or healthy).
  3. Absorbance of each sera under each set was plotted in a chart (Figure 5).


    Figure 5. Increased anti-aortic antigen antibody levels in the sera from LVV patient. Sera from patients of LVV (large vessel vasculitis), SLE (systemic lupus erythematosus), ANA (antinuclear antibody) positive, HSP (Henoch-Schonlein purpura), GPA (granulomatosis with polyangitis), matched controls (thoracic aortic aneurysmal control), and healthy individuals were tested for the presence of antibodies against aortic proteins. Adopted from Chakravarti et al., 2015.

  4. Mean absorbance of each set of sera was plotted using column graphs and average option in the GraphPad Prism 7.
  5. Student’s t-test can be used to compare statistically significant differences between two sets of sera. We preferred two-tailed test to provide stringency to our analysis. Alternatively one can use Mann-Whitney test (parametric option).
  6. In case of a known antigen, use purified antigen coated plate and known amount of commercial antibodies to prepare a standard curve to facilitate quantification of autoantibodies in the sera. In case where details of antigen are not known, this assay provides a semi-quantitative assay to compare different sets, however exact quantitation can’t be made.

Notes

  1. This method, when tested with other biochemical analysis, shows significant sensitivity (> 94%). However, it is always recommended to include a positive control and negative control in his assay. Positive control can be a known antigen and antibody (we used commercial antibody to 14-3-3 proteins that we suspected to be present in aorta), on the other hand, we used no sera added as our negative control and blank for absorbance subtraction. Having internal controls allows us to correlate amongst different sets of experiments.
  2. When comparing human samples, it is common to compare disease vs. normal (or healthy). In our case, we compared autoantibodies in the sera set from patients with LVV with other rheumatic diseases (e.g., GPA, RA, HSP, SLE etc.), patients having thoracic aortic aneurysms due to non-inflammatory causes (e.g., bicuspid valve, matrix disorder etc.), and healthy individuals. This provided additional layers for comparison and strengthened our analysis.
  3. To verify if the LVV patient has increased autoantibody against aortic protein(s), we performed western analysis and immunohistological analysis, as detailed elsewhere (Chakravarti et al., 2015).

Recipes

  1. Cell lysis buffer

    50 mM Tris buffer, pH 7.4
    150 mM NaCl
    0.1% Triton X-100
    1 mM sodium orthovanadate
    10 mM sodium fluoride
    10 mM glycerophosphate
    5 mM sodium pyrophosphate
  2. Blocking buffer
    5% Non-fat dry milk powder
    PBS-T

Acknowledgments

We are thankful to American Heart Association Scientist Development Grant (15SDG2308025) and the University of Toledo start-up funds to RC. The protocol and result are adopted from our previously published work (Chakravarti et al., 2015) which was carried out at Cleveland Clinic.

References

  1. Buttgereit, F., Dejaco, C., Matteson, E. L. and Dasgupta, B. (2016). Polymyalgia rheumatica and giant cell arteritis: a stematic review. JAMA 315(22): 2442-2458.
  2. Chakravarti, R., Gupta, K., Swain, M., Willard, B., Scholtz, J., Svensson, L. G., Roselli, E. E., Pettersson, G., Johnston, D. R., Soltesz, E. G., Yamashita, M., Stuehr, D., Daly, T. M. and Hoffman, G. S. (2015). 14-3-3 in thoracic aortic aneurysms: identification of a novel autoantigen in large vessel vasculitis. Arthritis Rheumatol 67(7): 1913-1921.
  3. Wang, L., Wang, F. S. and Gershwin, M. E. (2015). Human autoimmune diseases: a comprehensive update. J Intern Med 278(4): 369-395.

简介

人血清中自身抗体量的增加是自身免疫性疾病的标志(Wang等人,2015)。 在已知抗原的情况下,使用基于实验室的方法检测自身抗体。 然而,在大多数自身免疫性疾病中,自身抗原的知识仍然是模糊的。 我们已经开发了基于ELISA的定量测定法来检测自身抗体的存在以及测量患有大血管血管炎(LVV),自身免疫疾病(Chakravarti等人)的患者的血清中的循环自身抗体, ,2015)。 使用该测定,我们检测到LVV患者血清中抗主动脉抗体的增加。 我们通过独立生物化学技术进一步验证了结果,发现特异度 94%(Chakravarti等人,2015)。 该方法可以被独特地修改以适应任何自身免疫,特别是器官特异性疾病,因此在自身抗体的检测和定量中具有更广泛的应用。
【背景】约有80-100种自身免疫性疾病,其中免疫细胞将自身蛋白识别为外源抗原,并被激活以产生体液应答。尽管大多数自​​身免疫性疾病的触发因素仍然是一个谜,患者血清中存在较高水平的抗体是非常普遍的(Wang等人,2015)。一些实例包括抗小血管血管炎中的嗜中性粒细胞胞浆抗原,多发性硬化中的髓磷脂碱性蛋白,1型糖尿病中的谷氨酸脱羧酶等。(Wang等人, 2015年)。自身抗原在大多数自身免疫性疾病中的有限知识对于疾病的检测以及了解发病机理都是一个挑战。然而,在血清中检测自身抗体的能力提供了诊断和预后的优点。发现自身免疫性疾病中可靠的自身抗原需要做出更好的临床决策。 LVV是影响主动脉或其主要分支血管的自身免疫性疾病的一系列。像许多其他人一样,其病因和治疗方法尚不清楚(Buttgereit 等人,2016)。我们已经开发了一种定性和定量的检测方法来检测针对人类主动脉抗原的人血清中自身抗体的存在。我们的检测提供了一种在任何受影响/受损组织和任何疾病模型中发现自身抗原的工具。我们使用患者血清寻找人类胸主动脉瘤中自身抗原的存在,并通过比较从自身免疫性疾病或非免疫性疾病相关子集获得的血清来测试该检测的特异性。我们利用主动脉重建手术中的主动脉组织丢弃,并制作主动脉可溶性提取物进行检测(图1)。使用这种方法,我们进行了高通量筛选,用于检测超过100种血清中主动脉抗原的自身抗体(Chakravarti等人,2015)。


图1.用于检测人组织裂解物中自身抗体的ELISA的示意图流程图。用于检测和定量存在于组织中存在的抗原的血清中的自身抗体的方法的逐步概述。

关键字:ELISA, 自身抗原, 自身抗体, 血管炎, 血清, 自身免疫性疾病

材料和试剂

  1. 5 ml无菌聚苯乙烯圆底管(Corning,Falcon ®,目录号:352008)
  2. 1.5ml Eppendorf管(USA Scientific,目录号:1615-5500)
  3. 15ml离心管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339650)
  4. Greiner 96孔,F-Bottom,透明微孔板(Greiner Bio One International,目录号:655001)
  5. 200μl移液器吸头(USA Scientific,目录号:1111-1700)
  6. 1,250μl移液器吸头(USA Scientific,目录号:1112-1720)
  7. Immulon 2 HB:高亲和力蛋白质结合板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3455)
  8. 纸巾
  9. 人体样本
    注意:人体组织外植体以及血清应在制度上批准的IRB方案下收集。
  10. 蛋白酶
  11. 磷酸酶抑制剂(PhosSTOP)(Roche Diagnostics,目录号:04906837001)
  12. 蛋白质分析染料(Bio-Rad Laboratories,目录号:5000006)
  13. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  14. 磷酸盐缓冲盐水 - 吐温(PBS-T)(Fisher Scientific,目录号:BP293810)
  15. 抗体:
    1. 抗人IgG(以1:5000使用)(Thermo Fisher Scientific,Invitrogen,目录号:31135)
    2. HRP偶联的二抗(1:3,000使用)(Bio-Rad Laboratories,目录号:1706516)
  16. Ultra-TMB(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:34029)
  17. 2 N HCl(Fisher Scientific,目录号:SA431-500)
  18. Tris缓冲液,pH 7.4(Fischer Scientific,目录号:BP152-1)
  19. 氯化钠(NaCl)(Sigma-Aldrich,商品编号:S9888)
  20. Triton X-100(Bio-Rad Laboratories,目录号:1610407)
  21. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
  22. 氟化钠(Sigma-Aldrich,目录号:919)
  23. 甘油磷酸酯(Sigma-Aldrich,目录号:G9422)
  24. 焦磷酸钠(Sigma-Aldrich,目录号:P8010)
  25. 无脂干奶粉(Bio-Rad Laboratories,目录号:1706404)
  26. 细胞裂解缓冲液(参见食谱)
  27. 阻塞缓冲区(见配方)

设备

  1. 2-20μl移液器(Alkali Scientific,目录号:P9280-20U)
  2. 20-200μl移液器(Alkali Scientific,目录号:P9280-200U)
  3. 剪刀(Fisher Scientific,目录号:08-951-20)
  4. 台式离心机(Labnet International,型号:PrismR TM,目录号:C2500-R)
  5. 轨道摇床(Benchmark Scientific,型号:BT302)
  6. 冰箱
  7. 读卡器(BMG LABTECH,型号:FLUstar Omega)
  8. 均质器(Thomas Scientific,型号:SCILOGEX D160)

软件

  1. MS Excel
  2. GraphPad Prism 7

程序

  1. 制作组织裂解物
    1. 将组织切碎成小块(尽可能地),并放置在冰上的无菌圆底管中。
    2. 加入5ml含有蛋白酶和磷酸酶抑制剂的冷细胞裂解缓冲液(见食谱)。使用组织匀浆器在无菌裂解缓冲液中在冷的温度下均匀化组织,保持管覆盖在冰中以防止蛋白质变性。
    3. 进行6-8次循环,每次20-30秒,均化。每个循环后,等待2分钟,并手动评估悬浮液是否存在大块组织(图2) 注意:如果观察到完整的组织块,继续匀浆以改善溶解。确保组织裂解物在匀浆期间保持冷。
    4. 在4℃冷冻下,以12,000×g离心匀浆裂解物20分钟。将上清液转移到无菌Eppendorf管中并保持冷。如果需要,裂解液可以在-80°C长期储存。


      图2.组织匀浆 A.在裂解缓冲液和锋利的金属均化器中显示含有红色彩色片的圆管的卡通图; B.可溶性组织裂解液的代表性图片。

  2. 测量组织裂解物中的蛋白质浓度
    1. 移取一个Greiner 96孔板,将200μl预先稀释的(1:5)Bio-Rad蛋白质分析染料移至与样品相对应的每个孔中。
      注意:BSA标准品和组织裂解液通常在同一块板中测量,但为了清楚起见,此处分离。
    2. 首先,以0.1mg / ml的增量,使用0-1mg / ml范围内的BSA浓度制备蛋白质标准曲线。向含有测定染料的每个孔中加入1μlBSA溶液与移液管吸头混合溶液,孵育5 min。测定每种浓度的BSA一式三份。使用595nm的平板读数器测量孔的吸光度。使用不含蛋白质溶液(不含BSA)的孔作为实际样品减去的空白。平均吸光度(一式三份的空白相减平均值)和BSA样品的各自浓度绘制在excel文件中以获得线性方程(图3)。
      注意:线性函数的标准方程为:Y = MX + C,其中Y为吸光度,X为BSA浓度,M为斜率,C为y轴上的截距。 >


      图3.使用拟合线和方程的BSA的样品标准曲线。 此BSA曲线和方程将用于计算来自组织裂解液的蛋白质浓度
    3. 接下来,将来自组织裂解物的1μl上清液加入到含有200μl蛋白质测定试剂的每个孔中。在蛋白质浓度高的情况下,在蛋白质估计之前用细胞裂解缓冲液以1:10或1:20稀释组织裂解物。测量每个样品一式三份
    4. 使用读卡器测量595nm处的吸光度。
    5. 空白减法后,每个样品的平均吸光度值。绘制BSA标准曲线上每个样品的最终吸光度,以计算组织裂解物的蛋白质浓度(参见步骤B2)。在组织裂解物稀释的情况下,包括稀释因子进行最终计算 注意:在计算样品进行ELISA测定的浓度时,将测定的吸光度输入方程式为Y值,并用BSA线方程的斜率(M)和y截距(C)作为常数计算蛋白质浓度X)。

  3. ELISA
    1. 通过在每个孔中加入100μl可溶性组织裂解物的大衣Immulon 2 HB板。我们建议在PBS缓冲液中测试3种不同的蛋白质稀释液(范围为1:10至1:1,000),以进行一式三份的分析。使用与组织裂解物相同的蛋白质浓度的BSA溶液。在4℃下孵育过夜。在任何已知抗原的情况下,在实验中使用浓度(5-100μg/ ml)的纯化蛋白作为阳性对照
    2. 通过滗析板去除蛋白质溶液。每次在室温下振荡5分钟,用PBS-T(200μl)洗涤孔3次。将其倒在纸巾上,以除去任何残留液体。
    3. 通过在每个孔中加入200μl封闭缓冲液(见配方)并在室温(RT)下孵育2小时,同时以100rpm摇动来阻断孔。
    4. 通过倾析移除阻塞缓冲区。
    5. 在封闭缓冲液中加入200μl稀释血清(典型稀释度范围为1-10-1:1,000)。我们建议使用每种血清的3种稀释液来确定后续实验的最佳稀释度。也不包括组织裂解物包被的孔的血清控制 注意:冷冻血清在进行稀释前应始终在冰上解冻。
    6. 将振荡器上的板以100rpm在室温下孵育2小时或在4℃下过夜。
    7. 用PBS-T去除血清和洗涤孔3次。将其倒置在纸巾上,以除去任何残留液体。
    8. 加入200μl小鼠抗人IgG抗体,一抗,用封闭缓冲液以1:5000的比例稀释。在室温下摇动板2小时,以100转/分
    9. 取出第一抗体,并用PBS-T洗板3次,如步骤C7。
    10. 加入200μl与HRP缀合的抗小鼠IgG(以1:3,000封闭缓冲液稀释),并在室温下振荡板1小时。
      2508/5000
    11. 通过滗析板并按照步骤C7清洗板清除二抗。
      注意:在进行洗涤步骤时,准备读板器进行读板。设置要在450 nm读取的孔的程序。
    12. 加入100μl超TMB,观察蓝色发育。根据抗原(组织裂解物的蛋白质浓度)和自身抗体(血清稀释度),一些孔可能在其他孔之前将颜色变为蓝色。 5-30分钟的颜色变化是可取的。颜色的非常快速变化将需要降低组织裂解物的量或增加血清稀释度。如果颜色变化非常慢,需要60分钟以上,可能需要增加组织裂解液或降低血清稀释度。
    13. 当达到理想的颜色变化时,向每个孔中加入100μl的2N HCl,将颜色变为黄色(图4)。
    14. 在450nm读板,并记录每个孔中的吸光度。


      图4.显示ELISA最终读数的图像。将HCL添加到含有TMB的孔中,将溶液的颜色从蓝色变为黄色。箭头显示所需的黄色强度,其应在读板器的吸光度的线性范围内

数据分析

  1. 由于我们所有的样品都一式三份地进行了测试,所以我们对每个患者样本的吸光度进行了平均
  2. 所有样品聚集在一组疾病(例如,,具有大血管血管炎或健康的患者的血清)中。
  3. 每组血清的吸光度绘制在图表中(图5)

    图5.来自LVV患者血清中抗主动脉抗原抗体水平的提高 LVV患者血清(大血管血管炎),SLE(系统性红斑狼疮),ANA(抗核抗体)阳性,HSP (Henoch-Schonlein紫癜),GPA(多发性肉芽肿),匹配对照(胸主动脉瘤控制)和健康个体测试了存在抗主动脉蛋白的抗体。
    从Chakravarti 采用
  4. 使用GraphPad Prism 7中的列图和平均选项绘制每组血清的平均吸光度。
  5. Student's t -test可用于比较两组血清之间的统计学显着性差异。我们首选双尾测试来提供严格的分析。或者,可以使用Mann-Whitney测试(参数选项)。
  6. 在已知抗原的情况下,使用纯化的抗原包被的平板和已知量的商业抗体来制备标准曲线以便于定量血清中的自身抗体。如果抗原的细节未知,则该测定法提供半定量测定以比较不同的组,但是不能进行精确的定量。

笔记

  1. 当用其他生物化学分析测试时,该方法显示出显着的灵敏度(> 94%)。然而,总是建议在其测定中包括阳性对照和阴性对照。阳性对照可以是已知的抗原和抗体(我们使用我们怀疑存在于主动脉的14-3-3蛋白质的商业抗体),另一方面,我们不使用添加的血清作为阴性对照,空白用于吸光度减去。内部控制使我们能够在不同的实验组之间进行关联。
  2. 当比较人类样本时,通常将疾病与正常(或健康)进行比较。在我们的例子中,我们比较了患有LVV的患者与其他风湿性疾病(例如,GPA,RA,HSP,SLE等等)的血清中的自身抗体,胸部患者主动脉瘤由于非炎性原因(例如,二尖瓣,基质障碍等)和健康个体。这提供了更多的层次来进行比较和加强我们的分析。
  3. 为了验证LVV患者是否增加了抗主动脉蛋白的自身抗体,我们进行了西方分析和免疫组织学分析,其他地方(Chakravarti等人,2015年)。

食谱

  1. 细胞裂解缓冲液
    50mM Tris缓冲液,pH 7.4
    150 mM NaCl
    0.1%Triton X-100
    1mM原钒酸钠
    10 mM氟化钠
    10mM甘油磷酸盐
    5mM焦磷酸钠
  2. 阻塞缓冲区
    5%不含脂肪的干奶粉
    PBS-T

致谢

我们感谢美国心脏协会科学家发展资助(15SDG2308025)和托莱多大学启动资金给RC。我们以前在克利夫兰诊所进行的工作(Chakravarti等人,2015)采用了方案和结果。

参考

  1. Buttgereit,F.,Dejaco,C.,Matteson,EL和Dasgupta,B.(2016)。  风湿性多肌痛和巨细胞性动脉炎:一个病例综述。 JAMA 315(22):2442-2458。
  2. Chakravarti,R.,Gupta,K.,Swain,M.,Willard,B.,Scholtz,J.,Svensson,LG,Roselli,EE,Pettersson,G.,Johnston,DR,Soltesz,EG,Yamashita, ,Stuehr,D.,Daly,TM和Hoffman,GS(2015)。 14-3-3:鉴定大血管血管炎中的新型自身抗原。关节炎风湿关节炎67(7):1913-1921。 >
  3. Wang,L.,Wang,FS和Gershwin,ME(2015)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26212387”target = “_blank”>人类自身免疫性疾病:全面更新。 J Intern Med 278(4):369-395。
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引用:Veerman, B. and Chakravarti, R. (2017). Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen. Bio-protocol 7(13): e2367. DOI: 10.21769/BioProtoc.2367.
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