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Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress
采用环核苷酸(cAMP和cGMP)试验和捕获酶联免疫吸附法量化分析恶性疟原虫红内期外出   

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Abstract

Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the merozoite surface. This shedding, which takes place even in the absence of invasion, is therefore a surrogate marker for the degree of egress taking place in a culture, and can be measured using a specific capture ELISA to quantify AMA1 levels in culture supernatants (Collins et al., 2013). The assay uses a monoclonal antibody specific for AMA1 (called 4G2dc1) (Kocken et al., 1998; Collins et al., 2009) to capture and immobilize the protein from culture supernatants, then uses a specific rabbit polyclonal antiserum to detect the immobilized antigen. A phosphatase-conjugated goat anti-rabbit antibody is finally used to quantify the binding of the second antibody. Egress is absolutely dependent upon the activity of a parasite cGMP-dependent protein kinase, PKG, and so is influenced by levels of intracellular cGMP (Collins et al., 2013). This is regulated by the interplay between guanylate cyclases and phosphodiesterases. The latter enzymes may also degrade cAMP, so it may also be informative to measure intracellular cAMP levels.

Materials and Reagents

  1. Plasmodium falciparum schizonts
  2. DetectX Direct cGMP or cAMP immunoassay kit (Arbor Assays, catalog number: K020-H1 or K019-H1 )
  3. Protein-free RPMI 1640 (Life Technologies, InvitrogenTM)
  4. Albumax
  5. Zaprinast (Sigma-Aldrich, catalog number: Z0878 )
    Note: Make up as a 50 mM stock solution in DMSO and stored at -20 °C.
  6. PKG inhibitor compound 1 {4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine} (Merck KGaA)
    Note: Make up as a 10 mM stock solution in DMSO and stored at -20 °C.
  7. Anti-AMA1 monoclonal antibody (clone 4G2dc1) (Kocken et al., 1998)
  8. Complete RPMI 1640 culture medium (Blackman, 1994)
  9. Tween 20
  10. Rabbit polyclonal anti-AMA1 serum (Collins et al., 2009)
  11. Phosphatase-conjugated goat anti-rabbit IgG (whole molecule) (Sigma-Aldrich, catalog number: A3687 )
  12. Phosphatase substrate (Sigma-Aldrich, catalog number: S0942 )
  13. Phosphate-buffered saline (PBS)
  14. Sodium azide [10% (w/v) stock solution in water]
  15. Dry ice-ethanol mix for freezing
  16. Gelatine-tween stock solution (blocking buffer) (see Recipes)
  17. 0.1 M Sodium carbonate/bicarbonate (pH 9.6) (see Recipes)
  18. Diethanolamine buffer (pH 9.8) (see Recipes)

Equipment

  1. Centrifuge
  2. 96-well Immulon plates (Nunc®)
  3. ELISA reader
  4. Incubator

Procedure

  1. Assay of cGMP levels in the malaria parasite
    1. For measurement of intracellular cyclic nucleotide levels, Percoll-enriched schizonts were suspended at a 5% haematocrit in gassed, protein-free RPMI 1640 at 37 °C. Standard procedures were used to culture the parasites and enrich schizonts (Blackman, 1994; Yeoh et al., 2007). The suspension was divided into aliquots each containing 2 x 108 schizonts (2 x 108 schizonts corresponds to ~20 μl packed cells).
    2. Duplicate zero time samples (each containing 2 x 108 schizonts) were pelleted by centrifugation (1 min, 13,000 x g) and snap-frozen in a dry ice-ethanol mix.
    3. Further duplicate samples were then taken at various time intervals following addition of 75 µM zaprinast, zaprinast plus the 2.5 µM PKG inhibitor compound 1, or vehicle only (DMSO), and similarly processed. Zaprinast is an inhibitor of parasite phosphodiesterase (which degrades cGMP and cAMP), and so results in an increase in cGMP levels, inducing egress.
    4. All samples were stored at -70 °C until required for assay.
    5. cGMP and cAMP levels in schizont extracts were measured using a DetectX Direct cGMP or cAMP immunoassay kit , precisely as directed by the manufacturer.

  2. Capture ELISA for quantitative analysis of P. falciparum blood-stage egress
    1. Coat 96-well Immulon plates with 100 μl/well of a stock solution of purified monoclonal antibody 4G2dc1 made up at 2.5 μg/ml in carbonate/bicarbonate buffer (pH 9.6).
    2. Seal the plates and incubate overnight in a humidified atmosphere at 4 °C. This and all subsequent incubations are done whilst stationary.
    3. Wash the plates once with PBS 0.5% (v/v) Tween 20 (PBS/T), then block by adding 250 μl/well of a 1:4 dilution of gelatine-Tween stock solution, and incubating for 2 h at room temperature.
    4. Prepare serial 2-fold dilutions of test culture supernatant samples in a separate 96-well plate. Dilute the samples into RPMI 1640 culture medium containing 0.5% Albumax. For negative control wells, use RPMI Albumax medium only. Add samples to the washed ELISA plates, then seal and incubate the plates for 1 h at room temperature.
    5. Wash plates 3x in PBS/T.
    6. Make up a solution of rabbit polyclonal anti-AMA1 serum diluted 1:1,000 in PBS/T. Add 100 μl/well, then seal and incubate the plates a further 1 h at room temperature.
    7. Wash plates 3x with PBS.
      Note: No Tween at this stage, as it interferes with the alkaline phosphatase activity in the next step of the assay.
    8. Add 100 μl/well phosphatase-conjugated goat anti-rabbit IgG (whole molecule), diluted 1:30,000 in PBS. Seal the plates, then incubate 1 h at room temperature.
    9. Wash plates 5x with PBS (no tween) then add 50 μl/well substrate (1 tablet of phosphatase substrate, in 5 ml diethanolamine buffer). Incubate in the dark at 37 °C for at least 30 min. Determine OD405 using an ELISA reader.
    10. Use the data to produce titration curves of antigen levels in the various culture supernatants.

Recipes

  1. Gelatine-tween stock solution (blocking buffer)
    5 g Gelatine
    2.5 ml Tween-20
    150 μl 10% (w/v) sodium azide
    500 ml PBS
    Warm at 37 °C to dissolve the gelatine
    Use at 1: 4 dilution in PBS
    Stored stock at 4 °C
    May require warming before coaxing out of the bottle
  2. 0.1 M Sodium carbonate/bicarbonate (pH 9.6)
    Make up the two component buffers first
    These are
    7.155 g Na2CO3.10H2O made up to 250 ml water
    2.1 g NaHCO3 made up to 250 ml water
    Mix the above buffers to obtain a buffer with a pH of 9.6
    As an approximate measure, this requires ~37 ml 0.1 M Na2CO3.10H2O plus 73 ml 0.1 M NaHCO3
  3. Diethanolamine buffer (pH 9.8)
    48.5 ml Diethanolamine
    982 μl 1 M MgCl2
    2.5 ml 10% (w/v) sodium azide
    Add 400 ml water then adjust pH to 9.8 with 1 M HCl before making up to a total of 500 ml with water
    Stored in the dark at 4 °C

Acknowledgments

This method is adapted from an original method described in Collins et al. (2013). The authors are grateful to Alan Thomas (Biomedical Primate Research Centre, Rijswijk, The Netherlands) for the gift of monoclonal antibody 4G2dc1. This work was supported by the UK Medical Research Council (U117532063), and received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement N° 242095 (EviMalAR).

References

  1. Blackman, M. J. (1994). Purification of Plasmodium falciparum merozoites for analysis of the processing of merozoite surface protein-1. Methods Cell Biol 45: 213-220. 
  2. Collins, C. R., Withers-Martinez, C., Hackett, F. and Blackman, M. J. (2009). An inhibitory antibody blocks interactions between components of the malarial invasion machinery. PLoS Pathog 5(1): e1000273.
  3. Collins, C. R., Hackett, F., Strath, M., Penzo, M., Withers-Martinez, C., Baker, D. A. and Blackman, M. J. (2013). Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog 9(5): e1003344.
  4. Kocken, C. H., van der Wel, A. M., Dubbeld, M. A., Narum, D. L., van de Rijke, F. M., van Gemert, G. J., van der Linde, X., Bannister, L. H., Janse, C., Waters, A. P. and Thomas, A. W. (1998). Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization. J Biol Chem 273(24): 15119-15124. 
  5. Yeoh, S., O'Donnell, R. A., Koussis, K., Dluzewski, A. R., Ansell, K. H., Osborne, S. A., Hackett, F., Withers-Martinez, C., Mitchell, G. H., Bannister, L. H., Bryans, J. S., Kettleborough, C. A. and Blackman, M. J. (2007). Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131(6): 1072-1083.

简介

在体外破裂镰状疟原虫(恶性疟原虫)裂殖体(一种称为外出的事件)时,裂殖子释放到培养基中。裂殖子侵入新鲜的红细胞,该过程涉及从裂殖子表面脱落称为顶膜抗原-1(AMA1)的微米蛋白蛋白质。因此,即使在不存在侵袭的情况下发生的脱落也是培养物中出现的出口程度的替代标记,并且可以使用特异性捕获ELISA来测量,以定量培养物上清液中的AMA1水平(Collins, et al。,2013)。该测定使用特异于AMA1的单克隆抗体(称为4G2dc1)(Kocken等人,1998; Collins等人,2009),以捕获和固定来自培养物的蛋白质然后使用特异性兔多克隆抗血清来检测固定的抗原。最终使用磷酸酶缀合的山羊抗兔抗体来定量第二抗体的结合。出口绝对依赖于寄生虫cGMP依赖性蛋白激酶PKG的活性,因此受细胞内cGMP水平的影响(Collins等人,2013)。这由鸟苷酸环化酶和磷酸二酯酶之间的相互作用调节。后者酶也可以降解cAMP,因此它也可以是信息性的测量细胞内cAMP水平

材料和试剂

  1. 恶性疟原虫裂殖体
  2. DetectX Direct cGMP或cAMP免疫测定试剂盒(Arbor Assays,目录号:K020-H1或K019-H1)
  3. 无蛋白的RPMI 1640(Life Technologies,Invitrogen TM
  4. Albumax
  5. Zaprinast(Sigma-Aldrich,目录号:Z0878)
    注意:用DMSO制成50mM储备溶液,-20℃保存。
  6. PKG抑制剂化合物1 {4- [2-(4-氟苯基)-5-(1-甲基哌啶-4-基)-1H-吡咯-3-基]吡啶}(Merck KGaA)
    注意:制成10mM的DMSO储备溶液,储存于-20℃。
  7. 抗AMA1单克隆抗体(克隆4G2dc1)(Kocken等人,1998)
  8. 完全RPMI 1640培养基(Blackman,1994)
  9. 吐温20
  10. 兔多克隆抗AMA1血清(Collins等人,2009)
  11. 磷酸酶结合的山羊抗兔IgG(全分子)(Sigma-Aldrich,目录号:A3687)
  12. 磷酸酶底物(Sigma-Aldrich,目录号:S0942)
  13. 磷酸盐缓冲盐水(PBS)
  14. 叠氮化钠[10%(w/v)储备水溶液]
  15. 干冰 - 乙醇混合物冻结
  16. 明胶 - 吐温储备液(封闭缓冲液)(见配方)
  17. 0.1 M碳酸钠/碳酸氢钠(pH 9.6)(见配方)
  18. 二乙醇胺缓冲液(pH 9.8)(见配方)

设备

  1. 离心机
  2. 96孔Immulon平板(Nunc )
  3. ELISA读数器
  4. 孵化器

程序

  1. 疟原虫中cGMP水平的测定
    1. 对于细胞内环核苷酸水平的测量,将富含Percoll的裂殖体以5%血细胞比容在37℃下在充气,无蛋白的RPMI 1640中悬浮。使用标准程序培养寄生虫并富集裂殖体(Blackman,1994; Yeoh等人,2007)。将悬浮液分成各含有2×10 8个裂殖体(2×10 8个裂殖体对应于〜20μl填充细胞)的等分试样。
    2. 通过离心(1分钟,13,000×g)使重复的零时间样品(每个含有2×10 8个裂殖体)沉淀并在干冰 - 乙醇混合物中快速冷冻。
    3. 然后在加入75μM托普司特,托普司特加2.5μMPKG抑制剂化合物1或仅媒介物(DMSO)后,在不同的时间间隔取另外的重复样品,类似地 处理。 扎普司特是寄生虫磷酸二酯酶(其降解cGMP和cAMP)的抑制剂,因此导致cGMP水平增加,诱导出体。
    4. 将所有样品储存在-70℃直至需要测定。
    5. 使用DetectX Direct cGMP或cAMP免疫测定试剂盒,准确地按照制造商的指导测量裂殖体提取物中的cGMP和cAMP水平。

  2. 捕获ELISA用于定量分析P。 恶性疟原虫血液阶段出口
    1. 用100μl/孔的在碳酸盐/碳酸氢盐缓冲液(pH9.6)中以2.5μg/ml制备的纯化单克隆抗体4G2dc1的储备液涂覆96孔Immulon平板。
    2. 密封板并在4℃的潮湿气氛中孵育过夜。 这和所有后续的孵化在静止时进行
    3. 用PBS 0.5%(v/v)Tween 20(PBS/T)洗涤板一次,然后通过加入250μl/孔的1:4稀释的明胶 - 吐温储备溶液阻断,并在室温下孵育2小时。
    4. 在单独的96孔板中制备测试培养上清液样品的系列2倍稀释液。将样品稀释到含有0.5%Albumax的RPMI 1640培养基中。对于阴性对照孔,仅使用RPMI Albumax培养基。向洗涤的ELISA板中加入样品,然后密封并在室温下孵育板1小时
    5. 在PBS/T中洗涤板3x。
    6. 组成在PBS/T中1:1000稀释的兔多克隆抗AMA1血清的溶液。加入100μl/孔,然后密封,并将板在室温下再孵育1小时
    7. 用PBS洗涤板3x。
      注意:此阶段不使用Tween,因为它会干扰测定下一步中的碱性磷酸酶活性。
    8. 加入在PBS中1:30,000稀释的100μl/孔磷酸酶缀合的山羊抗兔IgG(完整分子)。 密封板,然后在室温下孵育1小时
    9. 用PBS(无吐温)洗涤板5x,然后加入50μl/孔底物(1片磷酸酶底物,在5ml二乙醇胺缓冲液中)。 在黑暗中37℃孵育至少30分钟。 使用ELISA读数器测定OD 405。
    10. 使用数据产生各种培养上清液中抗原水平的滴定曲线

食谱

  1. 明胶 - 吐温储备溶液(封闭缓冲液)
    5克明胶
    2.5ml Tween-20 150μl10%(w/v)叠氮化钠
    500 ml PBS
    在37℃温热以溶解明胶
    以1:4稀释在PBS中使用
    储存在4°C
    可能需要加热,然后晾出瓶子
  2. 0.1M碳酸钠/碳酸氢钠(pH9.6) 首先组成两个组件缓冲区
    这些是
    7.155g Na 2 CO 3 sub 3+ sup 10H 2 O由250ml水组成
    2.1g NaHCO 3水溶液加至250ml水
    混合上述缓冲液,得到pH为9.6的缓冲液 作为一个近似的测量,这需要约37ml 0.1M Na 2 CO 3 SO 3。加10 3 H 2 O加73 ml 0.1M NaHCO 3/v/v
  3. 二乙醇胺缓冲液(pH 9.8)
    48.5ml二乙醇胺
    982μl1 M MgCl 2
    2.5ml 10%(w/v)叠氮化钠 加入400ml水,然后用1M HCl调节pH至9.8,然后用水补足至总共500ml 储存在4℃的黑暗中

致谢

该方法根据Collins等人(2013)中描述的原始方法改编。 作者感谢Alan Thomas(生物医学灵长类研究中心,荷兰,Rijswijk)的单克隆抗体4G2dc1的礼物。 这项工作得到英国医学研究委员会(U117532063)的支持,并在授予协议N°242095(EviMalAR)下获得欧洲共同体第七框架计划(FP7/2007-2013)的资助。

参考文献

  1. Blackman,M.J。(1994)。 纯化恶性疟原虫子突孢子用于分析裂殖子表面蛋白的加工-1。 Methods Cell Biol 45:213-220。
  2. Collins,C.R.,Withers-Martinez,C.,Hackett,F。和Blackman,M.J。(2009)。 抑制性抗体阻断疟疾侵袭机制组件之间的相互作用。 Pathog 5(1):e1000273。
  3. Collins,C.R.,Hackett,F.,Strath,M.,Penzo,M.,Withers-Martinez,C.,Baker,D.A。和Blackman,M.J。(2013)。 疟疾寄生虫cGMP依赖性蛋白激酶调节血液阶段裂殖子分泌细胞器释放和排出。 PLoS Pathog 9(5):e1003344。
  4. Kocken,CH,van der Wel,AM,Dubbeld,MA,Narum,DL,van de Rijke,FM,van Gemert,GJ,van der Linde,X.,Bannister,LH,Janse,C.,Waters, ,AW(1998)。 在恶性疟原虫衍生的转基因中表达的精确时间>伯氏疟原虫是随后亚细胞定位的关键决定因素。 273(24):15119-15124。
  5. Yeverh,S.,O'Donnell,RA,Koussis,K.,Dluzewski,AR,Ansell,KH,Osborne,SA,Hackett,F.,Withers-Martinez,C.,Mitchell,GH,Bannister,LH,Bryans, JS,Kettleborough,CA和Blackman,MJ(2007)。 丝氨酸蛋白酶的亚细胞放电介导从宿主红细胞释放入侵性疟疾寄生虫。 em> Cell 131(6):1072-1083。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hackett, F., Collins, C. R., Strath, M. and Blackman, M. J. (2014). Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress. Bio-protocol 4(5): e1055. DOI: 10.21769/BioProtoc.1055.
  2. Collins, C. R., Hackett, F., Strath, M., Penzo, M., Withers-Martinez, C., Baker, D. A. and Blackman, M. J. (2013). Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog 9(5): e1003344.
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