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Isolation of Circulating Immune Complexes from TB Patient Serum for Serodiagnosis
从结核病人血清中分离免疫蛋白复合物用于诊断   

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Abstract

Estimation of circulating immune complex in tuberculosis patients has shown better insight to the infection. Isolating circulating immune complex helps quantifying both antigen and antibody in the serum. It’s a simple procedure to improve the sensitivity and specificity in serodiagnosis of tuberculosis. This protocol may be modified to detect antigen/antibody in other infectious diseases.

Keywords: Circulating Immune complex(循环免疫复合物), Serodiagnosis(血清学诊断), Tuberculosis(肺结核), TB diagnosis(结核病的诊断), Antigen and antibody detection(抗原和抗体检测)

Materials and Reagents

  1. Boric acid (Sigma-Aldrich, catalog number: B6768 )
  2. Disodium tetraborate (Borax) (Sigma-Aldrich, catalog number: 71996 )
  3. NaCI (Sigma-Aldrich, catalog number: S3014 )
  4. Polyethylene glycol (PEG 8000) (Sigma-Aldrich, catalog number: 89510 )
  5. Phosphate buffer saline (Life Technologies, Gibco®, catalog number: 20012-027 )
  6. NaHCO3 (Sigma-Aldrich, catalog number: S6014 )
  7. Na2CO3 (Sigma-Aldrich, catalog number: 330361 )
  8. BSA (Sigma-Aldrich, catalog number: A7030 )
  9. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  10. Antihuman affinipure IgG/lgA/lgM HRP [The Jackson Laboratory, USA, catalog number: 709-036-149 (IgG); 109-036-011 (IgA); 309-036-043 (IgM)]
  11. Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
  12. Citric acid (Sigma-Aldrich, catalog number: 251275 )
  13. O-Phenylenediamine (OPD) (Sigma-Aldrich, catalog number: P5412 )
  14. H2SO4 (Sigma-Aldrich, catalog number: 320501 )
  15. 0.1 M borate buffer (pH 8.4) (see Recipes)
  16. 7% polyethylene glycol (PEG) (see Recipes)
  17. Phosphate buffer saline (PBS) (see Recipes)
  18. 0.06 M Carbonate buffer (pH 9.6) (see Recipes)
  19. Blocking solution (see Recipes)
  20. Primary antibody (see Recipes)
  21. Secondary antibody (see Recipes)
  22. Substrate buffer (see Recipes)
  23. Substrate (see Recipes)
  24. Stop solution (see Recipes)

Equipment

  1. Refrigerated bench-top centrifuges
  2. 96 wells flat bottom ELISA plates (Nunc®, catalog number: 44-2404-21 )
  3. ELISA washer (Organon Teknika)
  4. Spectromax ELISA reader (Molecular Devices)

Procedure

  1. Circulating immune complex preparation
    1. Two hundred microliters of the test sera were centrifuged at 10,000 rpm for 5 min to remove any suspended particles.
    2. Next, 150 μl of the upper layer of the sera was collected and mixed with equal volume of 7% PEG. This gives a final concentration of 3.5% PEG in the sera.
    3. The sera were then vortexed and left overnight at 4 °C for precipiation of the immune complex.
    4. The next day, the precipitated immune complex was pelleted by centrifugation at 13,000 rpm for 10 min. The supernatant was discarded and the pellet was washed two times with 3.5% PEG.
    5. The circulating immune complex was finally dissolved in 0.01 M PBS and made up to the original volume of 150 μl.

  2. Enzyme linked immunosorbent assay
    1. Polystyrene ELISA plates (96 wells, Nunc Maxisorp, flat bottom) were coated with 5 μg/ml of CFA, 1 μg/ml for 38 and 30 kDa and 0.2 μg/ml for 16 kDa of purified mycobacterial antigen in Carbonate buffer (pH 9.6). The plates were incubated with antigen overnight at 4 °C.
      Note: Mycobacterial antigen is purified in the lab.
    2. The plates were washed four times with PBS buffer containing 0.1 % Tween 20 by the automatic ELISA washer.
    3. The non-specific sites in the wells were blocked with blocking solution for 1 h at 37 °C. After four washes with PBST, the plates were incubated for one hour at 37 °C with the precipitated circulating immune complex in 1/100 dilution. The plates were washed four times after 1 h incubation at 37 °C .The plates were then incubated with antihuman IgG peroxidase conjugate.
    4. At the end of 1 h of incubation at 37 °C and washing, the colour was developed by the addition of 100 μl of the substrate to each well.
    5. After arresting the reaction with 50 μl of 8 N H2SO4, the optical density was read in the Spectromax ELISA reader at 490 nm wavelength.
      Note: IgA and IgM antibody determination were also carried out among the same patients and healthy subjects. For this, Antihuman IgA and Antihuman IgM were used in the dilution of 1:500 and 1:1, 000 respectively.
    6. Antibody estimation for IgG, A and M isotypes were carried out for CIC precipitated patients and normal healthy serum. The ELISA positivity was calculated based on the OD values of the mean plus two standard deviation of the normal healthy serum. This value was used as the cut off value to decide the Elisa positivity of the unknown serum.

Notes

The samples to be assayed in a plate were randomly allocated to different wells within the plate and were also coded to conceal the identity of the specimens. In each of the experimental replicates of a positive reference serum (serum which has a strong positive response with high concentration of antibody from a smear microscopy confirmed tuberculous patient) was included. The mean value of the positive reference serum in independent experiments was taken as a constant reference value to assess plate-to-plate and day-to-day variations. Each plate had blank wells which represents the wells without antibody binding.

Recipes

  1. 0.1 M borate buffer (pH 8.4)
    0.1 M boric acid 6.184 g
    0.5 mM disodium tetraborate 9.5 g
    75 mM NaCI 4.4 g
    Made up to 1 L with distilled water
    The pH should be 8.4 without adjusting
  2. 7% PEG
    PEG 7 g
    Borate buffer 100 ml
  3. 3.5% PEG
    7% PEG 50 ml
    Borate buffer 50 ml
  4. Phosphate buffer saline (PBS)
    1. 0.25 M PBS (25x stock solution)
    2. 0.01 M PBS (1x buffer)
    Reagents a and b are prepared as mentioned in immunoblot section
  5. 0.06 M Carbonate buffer (pH 9.6)
    Solution A: [NaHCO3 (1 M)] 8.4 g/100 ml
    Solution B: [Na2CO3 (1 M)] 10.6 g/100 ml
    45.3 ml of A + 11 ml of B and made up to 1 L
  6. Blocking solution
    1 % BSA in PBS+0.1% Tween 20
  7. Primary antibody
    Circulating immune complex (CIC) 1:100 dilution
  8. Secondary antibody
    Antihuman affinipure IgG/lgA/lgM HRP
  9. Substrate buffer (phosphate citrate buffer)
    1. Solution A
      0.2 M Na2HPO4 8.517 g
      Milli Q water (to make up to) 300 ml
    2. Solution B
      0.1 M Citric acid 5.254 g
      Milli Q water (to make up to) 250 ml
    3. 257 ml of solution A and 243 ml of solution B were mixed with 500 ml of Milli Q water to give 1 L of phosphate citrate buffer (pH 5.0)
  10. Substrate
    OPD 10 mg (1 tablet)
    Dissolved in 25 ml of 0.05 M phosphate-citrate buffer (pH 5.0)
    30% H2O2 10 μl added to the above.
  11. Stop solution (8 N H2SO4)
    36 N H2SO4 222.2 ml
    Milli Q water (made upto) 1,000 ml

Acknowledgments

This protocol was adopted from previous work published by Raja et al. (1995). 

References

  1. Raja, A., Ranganathan, U. D. and Ramalingam, B. (2006). Clinical value of specific detection of immune complex-bound antibodies in pulmonary tuberculosis. Diagn Microbiol Infect Dis 56(3): 281-287.
  2. Raja, A., Narayanan, P. R., Mathew, R. and Prabhakar, R. (1995). Characterization of mycobacterial antigens and antibodies in circulating immune complexes from pulmonary tuberculosis. J Lab Clin Med 125(5): 581-587.
  3. Uma Devi, K. R., Ramalingam, B., Brennan, P. J., Narayanan, P. R. and Raja, A. (2001). Specific and early detection of IgG, IgA and IgM antibodies to Mycobacterium tuberculosis 38 kDa antigen in pulmonary tuberculosis. Tuberculosis (Edinb) 81(3): 249-253.

简介

结核病患者中循环免疫复合物的估计已经更好地了解感染。 分离循环免疫复合物有助于定量血清中的抗原和抗体。 这是一个简单的过程,以提高结核病血清诊断的灵敏度和特异性。 可以修改该方案以检测其他感染性疾病中的抗原/抗体。

关键字:循环免疫复合物, 血清学诊断, 肺结核, 结核病的诊断, 抗原和抗体检测

材料和试剂

  1. 硼酸(Sigma-Aldrich,目录号:B6768)
  2. 二硼酸四钠(Borax)(Sigma-Aldrich,目录号:71996)
  3. NaCl(Sigma-Aldrich,目录号:S3014)
  4. 聚乙二醇(PEG 8000)(Sigma-Aldrich,目录号:89510)
  5. 磷酸盐缓冲盐水(Life Technologies,Gibco ,目录号:20012-027)
  6. NaHCO 3(Sigma-Aldrich,目录号:S6014)
  7. Na 2 CO 3(Sigma-Aldrich,目录号:330361)
  8. BSA(Sigma-Aldrich,目录号:A7030)
  9. 吐温20(Sigma-Aldrich,目录号:P9416)
  10. 抗人Affinipure IgG/IgA/IgM HRP [The Jackson Laboratory,USA,目录号:709-036-149(IgG); 109-036-011(IgA); 309-036-043(IgM)]
  11. Na 2 HPO 4(Sigma-Aldrich,目录号:S3264)
  12. 柠檬酸(Sigma-Aldrich,目录号:251275)
  13. O-苯二胺(OPD)(Sigma-Aldrich,目录号:P5412)
  14. H 2 SO 4(Sigma-Aldrich,目录号:320501)
  15. 0.1M硼酸盐缓冲液(pH 8.4)(参见配方)
  16. 7%聚乙二醇(PEG)(参见配方)
  17. 磷酸盐缓冲盐水(PBS)(见配方)
  18. 0.06 M碳酸盐缓冲液(pH 9.6)(参见配方)
  19. 阻止解决方案(参见配方)
  20. 一抗(见配方)
  21. 二抗(见配方)
  22. 底物缓冲液(参见配方)
  23. 衬底(参见配方)
  24. 停止解决方案(参见配方)

设备

  1. 制冷台式离心机
  2. 96孔平底ELISA板(Nunc ,目录号:44-2404-21)
  3. ELISA洗涤器(Organon Teknika)
  4. Spectromax ELISA读数器(Molecular Devices)

程序

  1. 循环免疫复合物制备
    1. 将200微升测试血清在10,000rpm离心5分钟以除去任何悬浮颗粒
    2. 接下来,收集150μl血清的上层,并与等体积的7%PEG混合。 这使得血清中PEG的最终浓度为3.5%。
    3. 然后将血清涡旋并在4℃下放置过夜以沉淀免疫复合物
    4. 第二天,通过在13,000rpm离心10分钟使沉淀的免疫复合物沉淀。 弃去上清液,将沉淀用3.5%PEG洗涤两次。
    5. 将循环的免疫复合物最终溶解于0.01M PBS中并补足至150μl的原始体积
  2. 酶联免疫吸附测定
    1. 用5μg/ml CFA,1μg/ml的38和30kDa和0.2μg/ml的16kDa纯化的分枝杆菌抗原在碳酸盐缓冲液中包被聚苯乙烯ELISA平板(96孔,Nunc Maxisorp, (pH9.6)。将板在4℃下与抗原温育过夜 注意:分枝杆菌抗原在实验室中纯化。
    2. 通过自动ELISA洗涤器将板用含有0.1%Tween 20的PBS缓冲液洗涤四次。
    3. 孔中的非特异性位点用封闭溶液在37℃封闭1小时。用PBST洗涤四次后,将平板在37℃下与1/100稀释的沉淀的循环免疫复合物温育1小时。在37℃下温育1小时后将板洗涤4次。然后将板与抗人IgG过氧化物酶缀合物一起温育。
    4. 在37℃孵育1小时并洗涤结束时,通过向每个孔中加入100μl底物显色。
    5. 在用50μl8N H 2 SO 4终止反应后,在Spectromax ELISA读数器中在490nm波长下读取光密度。
      注意:在相同的患者和健康受试者中也进行IgA和IgM抗体测定。为此,抗人IgA和抗人IgM分别以1:500和1:1000的稀释度使用。
    6. 对于CIC沉淀的患者和正常健康血清进行IgG,A和M同种型的抗体估计。基于正常健康血清的平均值加两个标准偏差的OD值计算ELISA阳性。该值被用作截断值以决定未知血清的Elisa阳性

笔记

将板中待测定的样品随机分配到板内的不同孔中,并且还编码以隐藏样品的身份。 在每个实验重复的阳性参考血清(具有来自涂片显微镜确认的结核病患者的高浓度抗体的强阳性反应的血清)。 将独立实验中阳性参考血清的平均值作为恒定参考值,以评估板与板和日间变化。 每个板具有代表没有抗体结合的孔的空白孔。

食谱

  1. 0.1M硼酸盐缓冲液(pH 8.4) 0.1M硼酸6.184g
    0.5mM四硼酸二钠9.5g
    75mM NaCl 4.4g
    用蒸馏水
    补足至1升 pH值应该为8.4,而不调整
  2. 7%PEG
    PEG 7g
    硼酸缓冲液100 ml
  3. 3.5%PEG
    7%PEG50ml
    硼酸缓冲液50 ml
  4. 磷酸盐缓冲盐水(PBS)
    1. 0.25 M PBS(25x储备液)
    2. 0.01M PBS(1x缓冲液)
    如免疫印迹部分
    中所述制备试剂a和b
  5. 0.06M碳酸盐缓冲液(pH9.6) 溶液A:[NaHCO 3(1M)] 8.4g/100ml
    溶液B:[Na 2 CO 3(1M)] 10.6g/100ml
    45.3ml A + 11ml B,并加至1L
  6. 封锁解决方案
    1%BSA的PBS + 0.1%吐温20
  7. 一抗
    循环免疫复合物(CIC)1:100稀释
  8. 二抗
    抗人亲和IgG/lgA/lgM HRP
  9. 底物缓冲液(磷酸盐柠檬酸盐缓冲液)
    1. 解决方案A
      0.2M Na 2 HPO 4 8.517g
      Milli Q水(达到)300 ml
    2. 解决方案B
      0.1M柠檬酸5.254g
      Milli Q水(达到)250 ml
    3. 将257ml溶液A和243ml溶液B与500ml Milli Q水混合,得到1L磷酸盐柠檬酸盐缓冲液(pH5.0)。
  10. 基板
    OPD 10 mg(1片)
    溶于25ml的0.05M磷酸盐 - 柠檬酸盐缓冲液(pH5.0)中 30%H 2 O 2 O 210μl加到上述中。
  11. 停止溶液(8N H 2 SO 4)
    36 N H 2 SO 4 222/2ml
    Milli Q水(制成)1000 ml

致谢

该协议从Raja等人在1995年出版的前期工作中获得。

参考文献

  1. Raja,A.,Ranganathan,U. D.和Ramalingam,B。(2006)。 肺结核中免疫复合物结合抗体的特异性检测的临床价值。 Diagn Microbiol Infect Dis 56(3):281-287。
  2. Raja,A.,Narayanan,P.R.,Mathew,R。和Prabhakar,R。(1995)。 分枝杆菌抗原和抗体在肺结核的循环免疫复合物中的表征。 J Lab Clin Med 125(5):581-587
  3. Uma Devi,K.R.,Ramalingam,B.,Brennan,P.J.,Narayanan,P.R.and Raja,A。(2001)。 特异性和早期检测肺结核中结核分枝杆菌38 kDa抗原的IgG,IgA和IgM抗体。 Tuberculosis(Edinb) 81(3):249-253。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ranganathan, U. D., Bethunaickan, R. and Raja, A. (2012). Isolation of Circulating Immune Complexes from TB Patient Serum for Serodiagnosis. Bio-protocol 2(11): e188. DOI: 10.21769/BioProtoc.188.
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