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Nov 2018
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Quantification of Serum Ovalbumin-specific Immunoglobulin E Titre via in vivo Passive Cutaneous Anaphylaxis Assay
通过体内被动皮肤过敏试验定量血清卵清蛋白特异性免疫球蛋白E滴度   

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Abstract

Murine models of allergic airway disease are frequently used as a tool to elucidate the cellular and molecular mechanisms of tissue-specific asthmatic disease pathogenesis. Paramount to the success of these models is the induction of experimental antigen sensitization, as indicated by the presence of antigen-specific serum immunoglobulin E. The quantification of antigen-specific serum IgE is routinely performed via enzyme-linked immunosorbent assay. However, the reproducibility of these in vitro assays can vary dramatically in our experience. Furthermore, quantifying IgE via in vitro methodologies does not enable the functional relevance of circulating IgE levels to be considered. As a biologically appropriate alternative method, we describe herein a highly reproducible in vivo passive cutaneous anaphylaxis assay using Sprague Dawley rats for the quantification of ovalbumin-specific IgE in serum samples from ovalbumin-sensitized murine models. Briefly, this in vivo assay involves subcutaneous injections of serum samples on the back of a Sprague Dawley rat, followed 24 h later by intravenous injection of ovalbumin and a blue detection dye. The subsequent result of antigen-IgE mediated inflammation and leakage of blue dye into the initial injection site indicates the presence of ovalbumin-specific IgE within the corresponding serum sample.

Keywords: Asthma (哮喘), Allergy (过敏), Antigen (抗原), Immunoglobulin (免疫球蛋白), IgE (IgE), Ovalbumin (卵清蛋白), Sensitization (致敏), ELISA (ELISA)

Background

Allergic asthma is a complex inflammatory disease, the development of which involves multiple intricate gene-environment interactions. A defining factor of the genetic component for disease inception is the predisposition to developing allergen-specific immunoglobulin (Ig) E to common allergens, otherwise known as atopy (Sly et al., 2008). As such, allergic (atopic) sensitization to innocuous perennial aeroallergens during early childhood is now recognized as a fundamental risk factor in the establishment of potentially life-long allergic asthmatic disease (Illi et al., 2006; Sly et al., 2008), particularly when sensitization occurs to multiple aeroallergens (Stoltz et al., 2013).

Murine models have long provided a means to investigate the cellular and molecular mechanisms that drive onset and progression of allergic airway diseases such as allergic asthma (Kumar et al., 2016). In this regard, mice are typically experimentally sensitized to a model antigen, resulting in CD4+ T-helper (Th) type 2-driven inflammation and the production of antigen-specific IgE (Mincham et al., 2018); the hallmark feature of allergic sensitization. Quantification of circulating serum antigen-specific IgE from antigen sensitized murine models is routinely performed by enzyme-linked immunosorbent assay (ELISA). However, in our experience, these in vitro assays lack the necessary reproducibility for accurate detection of ovalbumin (OVA)-specific serum IgE in both Brown Norway rat and BALB/c mouse models of allergic airway disease. Furthermore, the use of in vitro techniques disregards the functional activity of antigen-specific IgE in a biologically relevant setting.

To combat the disadvantages of in vitro IgE quantification assays, we herein detail a highly reproducible in vivo passive cutaneous anaphylaxis (PCA) assay which enables both the quantification and assessment of biological activity of OVA-specific IgE within serum samples from OVA-sensitized murine models. In this regard, the localized cutaneous anaphylaxis response observed from an OVA-specific IgE positive sample is indicative of mast cell degranulation driven via OVA-induced cross-linking of OVA-specific IgE (Kawakami and Galli, 2002). The resultant release of mast cell granule-derived proinflammatory mediators exemplified by histamine, prostaglandin, leukotriene and tryptase, ultimately stimulates vascular permeability and the subsequent extravasation of the blue detection dye (Bradding and Arthur, 2016). While this protocol has been described for quantifying OVA-specific IgE in murine serum samples, it could be adapted to assess alternative model antigens.

Materials and Reagents

  1. 1.5 ml screw cap micro tube (SARSTEDT, catalog number: 72.703.600)
  2. 5 ml screw cap tube (SARSTEDT, catalog number: 60.9921.524)
  3. 27 G x ½” 1 ml Insulin syringe (Terumo, catalog number: SS-10M2713A)
  4. 26 G x ½” needle (Terumo, catalog number: NN-2613R)
  5. 23 G x 1¼” needle (Terumo, catalog number: NN-2332R)
  6. 3 ml syringe (Terumo, catalog number: SS-03L)
  7. 5 ml syringe (Terumo, catalog number: SS-05L)
  8. 80 μm syringe filter (Sartorius, Minisart®, catalog number: 16592)
  9. 45 μm syringe filter (Sartorius, Minisart®, catalog number: 16555)
  10. 96-well microplate (Thermo Fisher Scientific, NuncTM MicroWellTM, catalog number: 260860)
  11. Cotton wool roll (BNS Medical, catalog number: 71841-14)
  12. Marker pen
  13. Aluminum foil
  14. Male Sprague Dawley rats > 10 weeks old (Animal Resource Centre, Murdoch, Australia)
  15. Serum samples derived from ovalbumin-sensitized murine models
  16. Evans blue (Sigma-Aldrich, catalog number: E2129-10G), store at room temperature
  17. Ovalbumin (OVA) lyophilized powder (Sigma-Aldrich, catalog number: A5503), store at 4 °C
  18. Sterile Water for Irrigation (Baxter, catalog number: 2F7114), store at room temperature
  19. Sterile Water for Injection (Pfizer, catalog number: 25020010), store below 25 °C
  20. Chloral hydrate crystalized (Sigma-Aldrich, catalog number: 23100-250G)
  21. Sterile Phosphate buffered saline (PBS) (made in house)
  22. Topical ophthalmic ointment (Novartis, Viscotears®, catalog number: 1AD031591)
  23. Pentobarbitone sodium (Virbac, Lethabarb®, catalog number: LETH-1)
  24. Isoflurane (Henry Schein, IsoThesia®, catalog number: 988-3244), store below 25 °C in the dark
  25. 5.71% chloral hydrate solution (C2H3Cl3O2) (see Recipes)
  26. 1% Evans Blue dye stock solution (see Recipes)
  27. 10 mg/ml ovalbumin stock solution (see Recipes)
  28. 1:1 Ovalbumin-Evans blue dye solution for one rat (see Recipes)

Equipment

  1. 100 ml Schott bottle (Sigma-Aldrich, Duran®, catalog number: Z305170)
  2. 500 ml Bottle Top Filter, 0.2 μm pore size (Thermo Fisher Scientific, Nalgene® Rapid-FlowTM, catalog number: 595-4520)
  3. Animal hair clippers (Wahl, KM CordlessTM, catalog number: WA-9596-212)
  4. Warming pad (Kent Scientific Corporation, catalog number: DCT-25)
  5. Small animal anesthesia system (Kent Scientific Corporation, SumnoSuite®, catalog number: SS-01)
  6. 4 °C refrigerator
  7. -20 °C freezer

Software

  1. GraphPad Prism (GraphPad software, version 7.0a)

Procedure

  1. Serial 1 in 2 dilutions of serum samples
    1. Using a 96-well microplate, perform serial 1 in 2 dilutions of serum in PBS to a final sample volume of 55 μl.
      Notes:
      1. Serum samples are obtained from OVA-sensitized and aerosol challenged murine models.
      2. Final serum dilutions include: neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128 samples.
      3. Duplicate 55 μl PBS only samples are required as negative controls.
    2. A total of 8 individual experimental samples can be analyzed per rat, in addition to duplicate negative control samples.

  2. Male Sprague Dawley rat anesthetization
    1. Place male Sprague Dawley rat in an anesthetic induction chamber and anesthetize via isoflurane for approximately 3 min.
    2. Once sufficiently anesthetized, intraperitoneally inject the male Sprague Dawley rat with 4 ml 5.71% chloral hydrate solution using a 23 G x 1¼” needle and 5 ml syringe.
      Notes:
      1. This volume of 5.71% chloral hydrate solution will anesthetize an adult male Sprague Dawley rat (> 10 weeks old) for approximately 1 h.
      2. The male Sprague Dawley rat has reached sufficient deep plane anesthesia when the pedal withdrawal reflex is absent or barely noticeable.
    3. Place a small amount of topical ophthalmic ointment on the eyes of the anesthetized rat to stop them drying out during the procedure.

  3. Subcutaneous injections of serum dilutions
    1. Place the anesthetized male Sprague Dawley rat on a warming pad and closely shave the entire back of the rat from the base of the neck to the hips.
    2. Using a marker pen, mark an 8 x 8 dot grid covering the entire back of the rat. Space the dots approximately 1 cm apart (Figure 1).
      Notes:
      1. Do not use a blue marker as this will be hard to distinguish from the Evans blue dye. 
      2. An adult male Sprague Dawley rat will fit approximately 8 individual serum samples.
      3. Include 2 additional dots below the 8 x 8 sample grid for duplicate negative control samples.
    3. Starting with the highest dilution (1:128), subcutaneously inject 50 μl of the sample under the first marked dot using a 27 G x ½” 1 ml Insulin syringe. Continue down the first row of dots injecting each of the serial dilutions.
      Note: Start injections from the right-hand side of the rat if you inject with your right hand. This will avoid disturbing the injection sites as you progress across the back (Figure 1).
    4. Subcutaneously inject the 7 remaining individual samples and the duplicate PBS negative control samples, as per Step C3.
      Note: Always use a new sterile syringe for each individual sample.
    5. Once all samples have been injected, cover the back of the rat in cotton wool roll and place back in the housing cage.
    6. Monitor the anesthetized male Sprague Dawley rat periodically until full consciousness is regained.


      Figure 1. Representative diagram of steps performed in Procedure C

  4. Evans blue dye intravenous injection
    1. Twenty-four hours after subcutaneously injecting the serum samples, re-anesthetize the rat as per Procedure B.
    2. Place anesthetized rat on its back and intravenously inject 2 ml of the 1:1 OVA-Evans blue dye solution (see Recipes) into the penile vein using a 26 G x ½” needle and 3 ml syringe.
    3. Place rat on its stomach and allow samples to incubate for 15-30 min.
    4. Following injection, subcutaneous serum samples that are positive for OVA-specific IgE will develop a blue color (Figure 2). Record the highest dilution for each individual serum sample that returns a positive indication.
    5. Once all data has been recorded, humanely euthanize the male Sprague Dawley rat by intravenously injecting 600 μl of Lethabarb® Pentobarbitone sodium into the heart using a 27 G x ½” 1 ml syringe.


      Figure 2. Extravasation of Evans blue detection dye indicating the presence of OVA-specific IgE in the serum sample. Subcutaneously injected serial dilutions of serum samples that contain OVA-specific IgE will become blue following the intravenous injection of an OVA-Evans blue dye solution. Record the highest dilution at which a blue injection site is present for each sample (i.e., 1:4 dilution for Sample 2).

Data analysis

Plot data on a log2 scale to account for the serial dilutions (i.e., neat = 1, 1:2 = 2, 1:4 = 3, 1:8 = 4, 1:16 = 5, 1:32 = 6, 1:64 = 7, 1:128 = 8) using a graphing program such as GraphPad prism. Depending on the number of experimental groups, statistical significance can be determined by Student’s t-test or one-way ANOVA. As detailed in the original research article studying BALB/c mice (Mincham et al., 2018; Figure 1A), by additional research conducted within our laboratory studying Brown Norway rats (Leffler et al., 2018) and in Figure 3 below comparing gender-specific responses following early life OVA sensitization and aerosol challenge (Protocol ID: 1802387), OVA-specific serum IgE titres are significantly greater in OVA sensitized and aerosol challenged animals compared to naïve control animals.


Figure 3. Enhanced serum OVA-specific IgE titers in early life OVA sensitized and aerosol challenge BALB/c mice. Comparison of male and female serum OVA-specific IgE titers as measured by passive cutaneous anaphylaxis assay. Data are presented from individual animals comparing PBS control versus OVA sensitized and aerosol challenged male (shaded) and female (white) BALB/c mice and displayed as box and whisker plots showing minimum to maximum of n ≥ 6 independent experiments. Statistical significance was determined using Student’s t-test and presented as **P < 0.01, ****P < 0.0001.

Recipes

  1. 5.71% chloral hydrate solution (C2H3Cl3O2)
    1. Dissolve 5.71 g chloral hydrate in 100 ml sterile water for injection at room temperature
    2. Cover (100 ml Schott bottle) with aluminum foil to protect from light and store at 4 °C
  2. 1% Evans Blue dye stock solution
    1. Dissolve 1 g Evans blue powder in 100 ml PBS
    2. Store at 4 °C
  3. 10 mg/ml ovalbumin stock solution
    1. Dissolve 1 g OVA lyophilized powder in 100 ml sterile water for irrigation by placing lyophilized OVA powder on the water surface and allow to slowly dissolve at room temperature without stirring the solution
    2. Once dissolved, filter the 10 mg/ml ovalbumin solution into a sterile 100 ml Schott bottle using a 500 ml bottle top filter
    3. Aliquot the sterile 10 mg/ml OVA stock solution into 1 ml aliquots (1.5 ml screw cap micro tubes)
    4. Store at -20 °C
  4. 1:1 Ovalbumin-Evans blue dye solution for one rat
    1. Add 400 μl of 10 mg/ml OVA stock solution to 600 μl PBS to yield a final concentration of 4 mg/ml OVA solution in a total volume of 1 ml
    2. Add 1 ml 1% Evans blue dye to 1 ml 4 mg/ml OVA solution
      Note: Filter the necessary volume of 1% Evans blue dye stock solution required for the experiment through 80 μm and 45 μm syringe filters prior to use to remove precipitation.
    3. Store at 4 °C until required
      Note: Make up the 1:1 OVA-Evans blue dye solution immediately before anesthetizing the male Sprague Dawley rat.

Acknowledgments

The authors would like to acknowledge the animal technicians at the Telethon Kids Institute Bioresources facility. The original research article (Mincham et al., 2018) utilizing this protocol was funded by the National Health and Medical Research Council of Australia.

Competing interests

The authors declare no financial or non-financial competing interests related to this work.

Ethics

All animal experiments relating to this protocol were formally approved by the Telethon Kids Institute Animal Ethics Committee, which operates under guidelines developed by the National Health and Medical Research Council of Australia for the care and use of animals in scientific research.

References

  1. Bradding, P. and Arthur, G. (2016). Mast cells in asthma--state of the art. Clin Exp Allergy 46(2): 194-263.
  2. Illi, S., von Mutius, E., Lau, S., Niggemann, B., Gruber, C., Wahn, U. and Multicentre Allergy Study, g. (2006). Perennial allergen sensitisation early in life and chronic asthma in children: a birth cohort study. Lancet 368(9537): 763-770.
  3. Kawakami, T. and Galli, S. J. (2002). Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2(10): 773-786.
  4. Kumar, R. K., Herbert, C. and Foster, P. S. (2016). Mouse models of acute exacerbations of allergic asthma. Respirology 21(5): 842-849.
  5. Leffler, J., Mincham, K. T., Mok, D., Blank, F., Holt, P. G., Stumbles, P. A. and Strickland, D. H. (2018). Functional differences in airway dendritic cells determine susceptibility to IgE-sensitization. Immunol Cell Biol 96(3): 316-329.
  6. Mincham, K. T., Scott, N. M., Lauzon-Joset, J. F., Leffler, J., Larcombe, A. N., Stumbles, P. A., Robertson, S. A., Pasquali, C., Holt, P. G. and Strickland, D. H. (2018). Transplacental immune modulation with a bacterial-derived agent protects against allergic airway inflammation. J Clin Invest 128(11): 4856-4869.
  7. Sly, P. D., Boner, A. L., Bjorksten, B., Bush, A., Custovic, A., Eigenmann, P. A., Gern, J. E., Gerritsen, J., Hamelmann, E., Helms, P. J., Lemanske, R. F., Martinez, F., Pedersen, S., Renz, H., Sampson, H., von Mutius, E., Wahn, U. and Holt, P. G. (2008). Early identification of atopy in the prediction of persistent asthma in children. Lancet 372(9643): 1100-1106.
  8. Stoltz, D. J., Jackson, D. J., Evans, M. D., Gangnon, R. E., Tisler, C. J., Gern, J. E. and Lemanske, R. F., Jr. (2013). Specific patterns of allergic sensitization in early childhood and asthma & rhinitis risk. Clin Exp Allergy 43(2): 233-241.

简介

过敏性气道疾病的小鼠模型经常被用作阐明组织特异性哮喘病发病机理的细胞和分子机制的工具。这些模型成功的关键是诱导实验性抗原致敏,如抗原特异性血清免疫球蛋白E的存在所示。抗原特异性血清IgE的定量通常通过酶联免疫吸附测定进行。然而,这些体外检测的可重复性在我们的经验中可能有很大差异。此外,通过体外方法定量IgE不能考虑循环IgE水平的功能相关性。作为生物学上合适的替代方法,我们在本文中描述了使用Sprague Dawley大鼠的高度可再现的体内被动皮肤过敏试验,用于定量来自卵清蛋白致敏鼠模型的血清样品中的卵清蛋白特异性IgE。简而言之,该体内测定包括在Sprague Dawley大鼠背部皮下注射血清样品,24小时后通过静脉内注射卵清蛋白和蓝色检测染料。随后抗原-IgE介导的炎症和蓝色染料渗入初始注射部位的结果表明在相应的血清样品中存在卵清蛋白特异性IgE。
【背景】过敏性哮喘是一种复杂的炎性疾病,其发展涉及多种复杂的基因 - 环境相互作用。疾病初始的遗传成分的决定因素是将过敏原特异性免疫球蛋白(Ig)E发展为常见过敏原的倾向,也称为特应性(Sly et al。,2008)。因此,儿童早期对无害多年生空气过敏原的过敏(特应性)致敏现在被认为是建立潜在终身过敏性哮喘病的一个基本风险因素(Illi et al。,2006; Sly et al。,2008),特别是当多种气源性过敏原发生致敏时(Stoltz et al。,2013)。

小鼠模型长期以来提供了一种手段来研究驱动过敏性气道疾病(如过敏性哮喘)发病和进展的细胞和分子机制(Kumar et al。,2016)。在这方面,小鼠通常在实验上对模型抗原敏感,导致CD4 + T辅助(Th)2型驱动的炎症和抗原特异性IgE的产生(Mincham et al。,2018);过敏致敏的标志性特征。通过酶联免疫吸附测定(ELISA)常规地对来自抗原致敏鼠模型的循环血清抗原特异性IgE进行定量。然而,根据我们的经验,这些体外检测缺乏必要的重现性,无法准确检测Brown Norway大鼠和过敏性气道疾病BALB / c小鼠模型中的卵清蛋白(OVA)特异性血清IgE。此外,体外技术的使用忽视了生物学相关环境中抗原特异性IgE的功能活性。

为了克服体外 IgE定量分析的缺点,我们在此详细介绍了一种高度可重复的体内被动皮肤过敏反应(PCA)分析,该分析能够对生物活性进行定量和评估。来自OVA致敏的鼠模型的血清样品中的OVA特异性IgE。在这方面,从OVA特异性IgE阳性样品观察到的局部皮肤过敏反应表明通过OVA诱导的OVA特异性IgE交联驱动的肥大细胞脱粒(Kawakami和Galli,2002)。由组胺,前列腺素,白三烯和类胰蛋白酶所例证的肥大细胞颗粒衍生的促炎介质的释放最终刺激血管通透性和随后的蓝色检测染料的外渗(Bradding和Arthur,2016)。虽然已经描述了该方案用于量化小鼠血清样品中的OVA特异性IgE,但是它可以适用于评估替代模型抗原。

关键字:哮喘, 过敏, 抗原, 免疫球蛋白, IgE, 卵清蛋白, 致敏, ELISA

材料和试剂

  1. 1.5毫升螺旋盖微管(SARSTEDT,目录号:72.703.600)
  2. 5毫升螺旋盖管(SARSTEDT,目录号:60.9921.524)
  3. 27 Gx½“1 ml胰岛素注射器(Terumo,目录号:SS * 10M2713A)
  4. 26 Gx½“针(Terumo,目录号:NN + 2613R)
  5. 23 Gx1¼“针(Terumo,目录号:NN * 2332R)
  6. 3毫升注射器(Terumo,目录号:SS + 03L)
  7. 5毫升注射器(Terumo,目录号:SS + 05L)
  8. 80μm注射器过滤器(Sartorius,Minisart ®,目录号:16592)
  9. 45μm注射器过滤器(Sartorius,Minisart ®,目录号:16555)
  10. 96孔微孔板(Thermo Fisher Scientific,Nunc TM MicroWell TM ,目录号:260860)
  11. 棉毛卷(BNS Medical,目录号:71841-14)
  12. 记号笔
  13. 铝箔
  14. 10周大(动物资源中心,澳大利亚默多克)
  15. 血清样品来源于卵清蛋白致敏的鼠模型
  16. 伊文思蓝(Sigma-Aldrich,目录号:E2129-10G),在室温下储存
  17. 卵清蛋白(OVA)冻干粉(Sigma-Aldrich,目录号:A5503),储存于4°C
  18. 用于灌溉的无菌水(Baxter,目录号:2F7114),在室温下储存
  19. 无菌注射用水(辉瑞,目录号:25020010),储存温度低于25°C
  20. 水合氯醛(Sigma-Aldrich,目录号:23100-250G)
  21. 无菌磷酸盐缓冲盐水(PBS)(内部制造)
  22. 局部眼用软膏(诺华,Viscotears ®,目录号:1AD031591)
  23. 戊巴比妥钠(Virbac,Lethabarb ®,目录号:LETH-1)
  24. 异氟醚(Henry Schein,IsoThesia ®,目录号:988-3244),在黑暗中储存在25°C以下
  25. 5.71%水合氯醛溶液(C 2 H 3 Cl 3 O 2 )(见食谱)
  26. 1%伊文思蓝染料储备液(见食谱)
  27. 10 mg / ml卵白蛋白原液(见食谱)
  28. 1:1一只大鼠的卵清蛋白 - 伊文思蓝染料溶液(见食谱)

设备

  1. 100毫升Schott瓶(Sigma-Aldrich,Duran ®,目录号:Z305170)
  2. 500毫升瓶顶过滤器,0.2微米孔径(Thermo Fisher Scientific,Nalgene ® Rapid-Flow TM ,目录号:595-4520)
  3. 动物理发推子(Wahl,KM Cordless TM ,目录号:WA-9596-212)
  4. 保温垫(Kent Scientific Corporation,目录号:DCT-25)
  5. 小动物麻醉系统(Kent Scientific Corporation,SumnoSuite ®,目录号:SS-01)
  6. 4°C冰箱
  7. -20°C冰柜

软件

  1. GraphPad Prism(GraphPad软件,版本7.0a)

程序

  1. 连续1对2稀释的血清样品
    1. 使用96孔微量培养板,在PBS中进行连续1对2稀释的血清,最终样品体积为55μl。
      注意:
      1. 血清样品获自OVA致敏和气溶胶攻击的鼠模型。
      2. 最终血清稀释液包括:纯净,1:2,1:4,1:8,1:16,1:32,1:64和1:128样品。
      3. 重复55μl仅PBS样品作为阴性对照。
    2. 除了重复的阴性对照样品外,每只大鼠可以分析总共8个单独的实验样品。
  2. 雄性Sprague Dawley大鼠麻醉
    1. 将雄性Sprague Dawley大鼠置于麻醉诱导室中并通过异氟烷麻醉约3分钟。
    2. 一旦充分麻醉,使用23 Gx1¼“针头和5 ml注射器腹腔注射雄性Sprague Dawley大鼠4 ml 5.71%水合氯醛溶液。
      注意:
      1. 该5.71%水合氯醛溶液将麻醉成年雄性Sprague Dawley大鼠(> 10周龄)约1小时。
      2. 雄性Sprague Dawley大鼠已经达到足够的深度平面麻醉,当踩踏撤退反射不存在或几乎不可察觉时。
    3. 在麻醉的大鼠的眼睛上放置少量局部眼用软膏,以防止它们在手术过程中变干。
  3. 皮下注射血清稀释液
    1. 将麻醉的雄性Sprague Dawley大鼠置于保温垫上,并将大鼠的整个背部从颈部基部到臀部紧密剃毛。
    2. 使用记号笔,标记覆盖整个大鼠背部的8 x 8点网格。将点间隔大约1厘米(图1)。
      注意:
      1. 不要使用蓝色标记,因为这很难与埃文斯蓝色染料区分开来。&nbsp;
      2. 成年雄性Sprague Dawley大鼠将适合大约8个单独的血清样本。
      3. 在8 x 8样本网格下方包含2个额外的点,用于复制阴性对照样本。
    3. 从最高稀释度(1:128)开始,使用27 G x 1/2“1 ml胰岛素注射器在第一个标记点下皮下注射50μl样品。继续向下注入每个连续稀释液的第一排点。
      注意:如果用右手注射,从大鼠右侧开始注射。当您向后穿过时,这将避免干扰注射部位(图1)。
    4. 按照步骤C3,皮下注射剩余的7个单独样品和重复的PBS阴性对照样品。
      注意:每个样品都要使用新的无菌注射器。
    5. 一旦注射完所有样品,用棉花卷覆盖大鼠背部并放回到外壳笼中。
    6. 定期监测麻醉的雄性Sprague Dawley大鼠,直至恢复全意识。


      图1.在程序C中执行的步骤的代表性图

  4. 伊文思蓝染料静脉注射
    1. 皮下注射血清样品后24小时,按照方法B重新麻醉大鼠。
    2. 将麻醉的大鼠放在其背部,并使用26 G x 1/2“针头和3 ml注射器静脉注射2 ml 1:1 OVA-Evans蓝色染料溶液(参见食谱)到阴茎静脉。
    3. 将大鼠放在胃上,让样品孵育15-30分钟。
    4. 注射后,OVA特异性IgE阳性的皮下血清样品将呈蓝色(图2)。记录每个血清样本的最高稀释度,返回阳性指示。
    5. 记录完所有数据后,使用27 Gx½“1 ml注射器静脉注射600μlLethabarb®戊巴比妥钠,雄性安乐死雄性Sprague Dawley大鼠。


      图2.伊文思蓝检测染料的外渗,表明血清样本中存在OVA特异性IgE。静脉注射含有OVA特异性IgE的血清样品的连续稀释液将变为蓝色。 OVA-Evans蓝色染料溶液。记录每个样品中蓝色注射部位的最高稀释度(即,样品2的1:4稀释度)。

数据分析

在log 2 刻度上绘制数据以考虑序列稀释(即,neat = 1,1:2 = 2,1:4 = 3,1:8 = 4,1:16 = 5,1:32 = 6,1:64 = 7,1:128 = 8)使用GraphPad prism等图形程序。根据实验组的数量,可以通过学生的 t - 测试或单向ANOVA确定统计学显着性。正如在研究BALB / c小鼠的原始研究文章(Mincham et al。,2018;图1A)中所详述,通过在我们的实验室内进行的研究Brown Norway大鼠的另外研究(Leffler et al。 ,2018)和下图3比较早期OVA致敏和气溶胶攻击后的性别特异性反应(方案ID:1802387),OVA致敏和气溶胶攻击动物的OVA特异性血清IgE滴度显着高于天真的控制动物。


图3.早期生活中增强的血清OVA特异性IgE滴度OVA致敏和气溶胶攻击BALB / c小鼠。通过被动皮肤过敏试验测量的男性和女性血清OVA特异性IgE滴度的比较。数据来自个体动物,比较PBS对照与OVA致敏和气溶胶攻击的雄性(阴影)和雌性(白色)BALB / c小鼠,并显示为显示最小至最大n≥6个独立实验的框和须图。使用Student's t -test确定统计显着性,并表示为** P &lt; 0.01,**** P &lt; 0.0001。

食谱

  1. 5.71%水合氯醛溶液(C 2 H 3 Cl 3 O 2
    1. 将5.71g水合氯醛溶于100ml无菌水中,在室温下注射
    2. 用铝箔盖上(100毫升Schott瓶)以防光照并在4°C下储存
  2. 1%伊文思蓝染料储备液
    1. 将1克伊文思蓝粉末溶于100毫升PBS中
    2. 储存在4°C
  3. 10mg / ml卵清蛋白原液
    1. 将1g OVA冻干粉末溶于100ml无菌水中,通过将冻干的OVA粉末置于水表面上并在室温下缓慢溶解而不搅拌溶液
    2. 溶解后,使用500 ml瓶顶过滤器将10 mg / ml卵清蛋白溶液过滤到无菌的100 ml Schott瓶中
    3. 将无菌10 mg / ml OVA储备液分装到1 ml等分试样(1.5 ml螺帽微管)中
    4. 储存在-20°C
  4. 1:1一只大鼠的卵清蛋白 - 伊文思蓝染料溶液
    1. 向600μlPBS中加入400μl10mg / ml OVA储备液,使最终浓度为4 mg / ml OVA溶液,总体积为1 ml
    2. 将1ml 1%伊文思蓝染料加入1ml 4mg / ml OVA溶液中 注意:在使用之前,通过80μm和45μm注射器过滤器过滤必要体积的1%伊文思蓝染料储备溶液,以去除沉淀。
    3. 储存在4°C直至需要
      注意:在麻醉雄性Sprague Dawley大鼠之前立即补充1:1 OVA-Evans蓝色染料溶液。

致谢

作者要感谢Telethon Kids Institute Bioresources工厂的动物技术人员。利用该协议的原始研究文章(Mincham et al。,2018)由澳大利亚国家健康与医学研究委员会资助。

利益争夺

作者声明与此工作无关的财务或非财务竞争利益。

伦理

与该协议相关的所有动物实验均由Telethon Kids Institute动物伦理委员会正式批准,该委员会根据澳大利亚国家健康与医学研究委员会制定的关于科学研究中动物护理和使用的指南进行操作。

参考

  1. Bradding,P。和Arthur,G。(2016)。 哮喘肥大细胞 - 最先进的技术。 Clin Exp Allergy 46(2):194-263。
  2. Illi,S.,von Mutius,E.,Lau,S.,Niggemann,B.,Gruber,C.,Wahn,U。和Multicentre Allergy Study,g。 (2006年)。 儿童早期多年生过敏原致敏和儿童慢性哮喘:一项出生队列研究。 Lancet 368(9537):763-770。
  3. Kawakami,T。和Galli,S.J。(2002)。 通过IgE调节肥大细胞和嗜碱性粒细胞功能和存活率。 Nat Rev Immunol 2(10):773-786。
  4. Kumar,R。K.,Herbert,C。和Foster,P。S.(2016)。 过敏性哮喘急性发作的小鼠模型。 Respirology 21(5):842-849。
  5. Leffler,J.,Mincham,K.T.,Mok,D.,Blank,F.,Holt,P.G.,Stumbles,P.A。和Strickland,D.H。(2018)。 气道树突状细胞的功能差异决定了对IgE致敏的易感性。 Immunol Cell Biol 96(3):316-329。
  6. Mincham,K.T.,Scott,N.M.,Lauzon-Joset,J.F.,Leffler,J.,Larcombe,A.N.,Stumbles,P.A.,Robertson,S.A.,Pasquali,C.,Holt,P.G。和Strickland,D.H。(2018)。 用细菌衍生的药物进行经胎盘免疫调节可预防过敏性气道炎症。 J Clin Invest 128(11):4856-4869。
  7. Sly,PD,Boner,AL,Bjorksten,B.,Bush,A.,Custovic,A.,Eigenmann,PA,Gern,JE,Gerritsen,J.,Hamelmann,E.,Helms,PJ,Lemanske,RF,Martinez ,F.,Pedersen,S.,Renz,H.,Sampson,H.,von Mutius,E.,Wahn,U。和Holt,PG(2008)。 早期识别儿童持续性哮喘预测中的特应性。 柳叶刀 372(9643):1100-1106。
  8. Stoltz,D.J.,Jackson,D.J.,Evans,M.D.,Gangnon,R.E。,Tisler,C.J。,Gern,J.E。和Lemanske,R.F。,Jr。(2013)。 儿童早期和哮喘过敏性过敏的特定模式&amp;鼻炎风险。 Clin Exp Allergy 43(2):233-241。
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引用:Mincham, K. T., Leffler, J., Scott, N. M., Lauzon-Joset, J., Stumbles, P. A., Holt, P. G. and Strickland, D. H. (2019). Quantification of Serum Ovalbumin-specific Immunoglobulin E Titre via in vivo Passive Cutaneous Anaphylaxis Assay. Bio-protocol 9(5): e3184. DOI: 10.21769/BioProtoc.3184.
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