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ELISA for Alpha-hemolysin (Hla) in Methicilin-resistant Staphylococcus aureus (MRSA)
酶联免疫吸附试验分析耐甲氧西林葡萄球菌属(MRSA)中的α溶血素(Hla)   

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Abstract

Anti-virulence agents against MRSA inhibit the production of disease-causing virulence factors, such as alpha-hemolysin, but are neither bacteriostatic nor bactericidal. Here we discuss a rapid method to screen for MRSA anti-virulence agents by measuring alpha-hemolysin production through ELISA. This protocol can be used with other alpha-hemolysin producing bacteria or for other excreted toxins to which antibodies exist.

Materials and Reagents

  1. Staphylococcus aureus subsp. aureus USA300
  2. Dry ice
  3. Dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific, catalog number: D128-1 )
  4. Polyclonal anti-Hla antibody (Abcam, catalog number: ab15948 )
  5. Anti-alpha hemolysin (Hla) antibody conjugated to horseradish peroxidase (Abcam, catalog number: ab15949 )
  6. 3,3’,5,5’ tetramethylbenzidine (TMB) (Sigma-Aldrich, catalog number: T4444 )
  7. Stop reagent (Sigma-Aldrich, catalog number: S5689 )
  8. Luria broth (LB) agar plates
  9. Phosphate buffered saline (PBS) (pH 7.2) (Sigma-Aldrich, catalog number: P5119 )
  10. 10 mg/ml Bovine serum albumin (BSA) in PBS (Sigma-Aldrich, catalog number: P3688 ) (see Recipes)
  11. 0.05% Tween 20 in PBS (Sigma-Aldrich, catalog number: P3563 ) (see Recipes)
  12. Trypticase Soy Broth (TSB) (see Recipes)

Equipment

  1. -80 °C freezer
  2. 14 ml polypropylene culture tube (BD, catalog number: 352059 )
  3. Cotton ball or spill resistant caps
  4. Microtiter 96 well EIA/RIA plates (Corning, CostarTM, catalog number: 9017 )
  5. Microplate reader (Molecular Devices, model: SPECTRA Max M2), or any other model capable of measuring a 96 well microtiter plate at OD 650 nm
    Note: If a different model of microplate reader is used, ensure appropriate model of microtiter 96-well plate is used as the model above may not work.
  6. 37 °C shaker
  7. Nitrile gloves
  8. Multichannel pipetman (Eppendorf)
  9. 0.22 µm syringe filter (PVDF) (Thermo Fisher Scientific, FisherbrandTM, catalog number: 09-720-3 )
  10. 3 ml luer lock syringe (BD, catalog number: 309657 )
  11. 1.5 inch needle (BD, catalog number: 305187 )
  12. 2 ml cryotubes
  13. Plastic wrap
  14. Disposable inoculation loops (10 µl, PS, sterile, yellow) (LPS, catalog number: M122002 )

Software

  1. Microsoft Excel or other data processing program

Procedure

  1. Induction of alpha - hemolysin (Hla)
    1. A frozen bacterial stock of MRSA strain USA300 is removed from the -80 °C freezer and placed directly onto dry ice.
      Note: The stock is not to thaw.
    2. Using a sterile disposable inoculator loop, inoculum is scraped off from the bacterial stock and placed into culture tube containing 1.5 ml of TSB.
      Note: Sterile technique is not required, however care should be taken to minimize possible contamination.
    3. A cotton ball is placed in the top of the culture tube and lid is put on loosely.
    4. The culture is incubated overnight, 16-18 h, at 37 °C with shaking.
      Note: The cotton ball is a precautionary measure to prevent the culture from spilling.
    5. The single overnight culture is diluted 1:100 in enough TSB to aliquot 1.960 ml diluted culture for each treatment (compounds to test and DMSO).
      Note: To determine the final volume of TSB needed for the dilution of the culture, the following calculation is used:
      (# of treatments) x 2 ml + 1 ml = ml of TSB.
    6. Prepare solutions 0.05 mg/ml, 0.5 mg/ml or 2.5 mg/ml of the compounds of interested (potential MRSA anti-virulence agents) in 100% DMSO. (One example of a compound of interest is Diflunisal, for more examples see Reference 1.)
    7. 40 µl of 100% DMSO, 0.05 mg/ml, 0.5 mg/ml or 2.5 mg/ml of a compound in 100% DMSO are added to 14 ml plastic culture tubes to yield final concentrations of 2% DMSO, 1 µg/ml, 10 µg/ml, and 50 µg/ml of compound, respectively.
      Notes:
      1. The concentration use varies depending on the nature of the compound of interest.
      2. If compounds are dissolved in a different solvent, 40 µl of that solvent is used instead of DMSO.
    8. 1.960 ml of the 1:100 diluted overnight culture is added to each culture tube containing compound or DMSO.
    9. The remainder of the 1:100 dilution from step A5 is placed on ice.
    10. As with the overnight culture, a cotton ball is placed in the top of each culture tube and the lid is put on loosely.
    11. The tubes from step A8 are incubated for 6 h at 37 °C with shaking.

  2. Dilution of cultures for determining colony forming units (CFU)
    1. The 1:100 diluted overnight culture placed on ice in Part A is serial diluted 1:104 and 1:105 into TSB.
    2. 100 µl of the 1:104 and 1:105 dilutions are spread onto LB agar plates and set aside until all of the plates are prepared. This is the zero hour time point.
    3. After 6 h the treated cultures are removed from the 37 °C shaker and first processed following Part C of the protocol before conducting the following dilutions.
    4. The liquid culture remaining in each treated sample is serial diluted to 1:104 and 1:105.
    5. 100 µl of the 1:104 and 1:105 dilutions are spread onto LB agar plates.
    6. All of the plates (including the zero hour time point plate) are incubated at 37 °C overnight.
    7. The numbers of colonies in each of the 1:105 dilution plates are counted.
      Note: If there are less than 30 colonies on a 1:105 plate, the 1:104 dilution plate of that sample is counted. If statistical power is of interest, both plates should be counted.

  3. Bacterial culture sample preparation for ELISA
    1. After 6 h of incubation the bacterial cultures are drawn into a luer lock syringe with a 1.5 inch needle, and the needle is removed.
      Note: The needle is removed using tweezers or hemostat, placed into bleach and disposed of in a hard plastic sharps container.
    2. A 0.22 µm filter is tightly placed onto the syringe and the bacterial cultures are filtered into 2 ml cyrotubes.
      Note: If there is resistance while filtering, applying gentle pressure to the plunger of the syringe will push the filtrate through. Too much pressure can result in filter failure.
    3. The filtrates are immediately frozen using dry ice and stored at -80 °C for future use.
      Note: The filtrates are sterile at this point, but should still be handled with nitrile gloves throughout the protocol.

  4. Alpha-hemolysin (Hla) ELISA
    1. Prepare diluted 1:1,000 Hla antibody. For each 96-well plate used, 10 µl of anti-Hla antibody is added to 10 ml of cold PBS (pH 7.2) (1:1,000 dilution), and mixed gently.
    2. 100 µl of 1:1,000 anti-Hla antibody in PBS is added to each microtiter 96 well, covered with plastic wrap and incubated overnight at 4 °C.
    3. The supernatant is removed and briefly washed (i.e. rinsed) once by adding 230 µl of 0.05% Tween 20 in PBS and then removing the supernatant. Agitation is not required.
    4. 230 µl of 10 mg/ml BSA in PBS block is added to each well and placed at 4 °C for 60 min.
    5. The supernatant is removed and the plate is washed once by adding 230 µl of 0.05% Tween 20 in PBS and then removing the supernatant.
    6. 100 µl of PBS is added to the “blank wells” in the designated row or column.
    7. Test samples (filtered supernatants of bacterial cultures from step D21) are diluted 1:8 or 1:16 with PBS, and 100 µl of the diluted test samples are added to their respective wells.
    8. The plate is covered with plastic wrap and foil then incubated for 1 h at room temperature with gentle rocking.
    9. The supernatant is removed and 230 µl of 0.05% Tween 20 in PBS is added to each well. The supernatant is subsequently removed.
    10. Step B12 is repeated two more times.
    11. 100 µl of the HLA antibody conjugated to horseradish peroxidase, diluted 1:1,000 in 10 mg/ml BSA in PBS, is added to the wells, covered with plastic wrap and foil, and then incubated for 1 h with rocking at room temperature.
    12. The supernatant is removed and the 230 µl of 0.05% Tween 20 in PBS is added to each well. The supernatant is subsequently removed.
    13. Step D33 is repeated 2 more times.
      Note: On the final wash, using a pipette ensure the removal of all 0.05% Tween 20 in PBS.
    14. 230 µl of PBS (pH 7.2) is added to each well and subsequently removed.
    15. Step D35 is repeated.
    16. The plate is placed upside down onto a paper towel until all of the supernatant is drained.
    17. 100 µl of substrate solution 3,3’,5,5’ tetramethylbenzidine (TMB) is added to each well and incubated at room temperature for 10 min.
    18. 100 µl of stop reagent is added to each well.
      Note: Air bubbles may form when stop reagent is added. Prior to measuring absorbance, all air bubbles should be popped. This can be done by blowing air with a pipette, but should be done with care to avoid cross contamination between wells.
    19. For each well in the plate, the OD is read at 650 nm using a microplate reader.

  5. Calculations
    1. The colony forming units (CFU) per ml for each treated sample is calculated by: [(# of colonies of treated sample) x (culture dilution factor)] / (# ml plated) = CFU/ml. This step can be done using Microsoft Excel or similar program.
      Notes:
      1. 0.1 ml of diluted culture is generally plated.
      2. The comparison CFU/ml of treated with the CFU/ml of the time zero plate is used to determine if the compounds are bactericidal, bacteriostatic, or have no effect on growth. If the CFU/ml of treated samples is statistically the same as CFU/ml of time zero, then the compound inhibits bacterial growth. If the CFU/ml of treated samples is statistically less than that of time zero, then the compound kills the bacteria.
    2. The amount of Hla production is calculated from the OD650 by: [(average OD sample) – (average OD blank)] x (sample dilution factor) / (# ml sample added) = units/ml.
      Notes:
      1. 0.1 ml of sample is generally added per well.
      2. This is easily done using Excel or similar program.
    3. The amount of Hla is first determined with respect to CFU by: (# units/ml) / (# CFU/ml) = units/CFU.
    4. The percent Hla produced is defined as: [(units/CFU treated) / (units/CFU DMSO)] x 100 = % Hla production.
      Notes:
      1. If % Hla production is below 100%, then the compound inhibits Hla production with respect to DMSO.
      2. A master Excel template (attached here) is provided with example data for a 1:8 dilution of the bacterial filtrate. The template will calculate steps E41-44 automatically once OD values and colony counts are added. Take note that the template is made for the volumes and dilutions specified in this protocol. If other volumes or dilutions are used, adjustments will need to be made in the template.


    Figure 1. Example graph of percent Hla production relative to a DMSO treated MRSA bacterial culture (from example Excel template)

Recipes

  1. 10 mg/ml BSA in PBS
    The solution is made as per manufacturer’s directions
  2. 0.05% Tween 20 in PBS
    The solution is made as per manufacturer’s directions
  3. Trypticase Soy Broth (TSB)
    17.0 g Bacto Tryptone
    3.0 g Bacto Soytone
    2.5 g Dextrose
    5.0 g sodium chloride
    2.5 g dipotassium hydrogen phosphate (pH to 7.3)

Acknowledgments

We thank Barbara Truitt and Dr. Michelle Pesho for their contributions to the development of this protocol. This protocol has been adapted from John R Crowther, (2000). Funding for this work was provided by a Grant-in-Aid from the American Heart Association, Great Rivers Affiliate, and by a grant from the Steris Foundation.

References

  1. Crowther, J. R. (2000). The ELISA guidebook. Methods Mol Biol 149: III-IV, 1-413.
  2. Khodaverdian, V., Pesho, M., Truitt, B., Bollinger, L., Patel, P., Nithianantham, S., Yu, G., Delaney, E., Jankowsky, E. and Shoham, M. (2013). Discovery of antivirulence agents against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 57(8): 3645-3652.

简介

针对MRSA的抗毒剂抑制引起疾病的毒力因子如α-溶血素的产生,但既不是抑菌性的也不是杀细菌性的。 在这里我们讨论一种快速方法来筛选MRSA抗毒力试剂通过测量α-溶血素的生产通过ELISA。 该方案可以与其它产生α-溶血素的细菌或存在抗体的其它排泄毒素一起使用。

材料和试剂

  1. 金黄色葡萄球菌亚种 aureus USA300
  2. 干冰
  3. 二甲基亚砜(DMSO)(Thermo Fisher Scientific,目录号:D128-1)
  4. 多克隆抗Hla抗体(Abcam,目录号:ab15948)
  5. 与辣根过氧化物酶偶联的抗α溶血素(Hla)抗体(Abcam,目录号:ab15949)
  6. 3,3',5,5'-四甲基联苯胺(TMB)(Sigma-Aldrich,目录号:T4444)
  7. 停止试剂(Sigma-Aldrich,目录号:S5689)
  8. Luria肉汤(LB)琼脂平板
  9. 磷酸盐缓冲盐水(PBS)(pH7.2)(Sigma-Aldrich,目录号:P5119)
  10. 10mg/ml PBS中的牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:P3688)(参见Recipes)
  11. 0.05%Tween 20的PBS溶液(Sigma-Aldrich,目录号:P3563)(参见Recipes)
  12. 胰蛋白酶大豆肉汤(TSB)(参见配方)

设备

  1. -80°C冰箱
  2. 14ml聚丙烯培养管(BD,目录号:352059)
  3. 棉球或防溅帽
  4. 微量滴定96孔EIA/RIA板(Corning,Costar TM ,目录号:9017)
  5. 微量板读数器(Molecular Devices,型号:SPECTRA Max M2)或能够测量OD 650nm的96孔微量滴定板的任何其它模型
    注意:如果使用不同型号的酶标仪,请确保使用适当的微量滴定板96孔板样品,因为上述样品可能无法正常工作。
  6. 37℃摇床
  7. 丁腈手套
  8. 多通道移液器(Eppendorf)
  9. 0.22μm注射器过滤器(PVDF)(Thermo Fisher Scientific,Fisherbrand TM ,目录号:09-720-3)
  10. 3ml鲁尔锁注射器(BD,目录号:309657)
  11. 1.5英寸针(BD,目录号:305187)
  12. 2 ml cryotubes
  13. 塑料包装
  14. 一次性接种环(10μl,PS,无菌,黄色)(LPS,目录号:M122002)

软件

  1. Microsoft Excel或其他数据处理程序

程序

  1. 诱导α溶血素(Hla)
    1. 从-80℃冰箱中取出MRSA菌株USA300的冷冻菌群,并直接置于干冰上。
      注意:股票不解冻。
    2. 使用无菌一次性接种器环,刮下接种物 从细菌原液中并置于含有1.5ml的培养管中 的TSB。
      注意:不需要使用无菌技术,但应注意尽量减少可能的污染。
    3. 将棉球放在培养管的顶部,并将盖子松松地放置
    4. 将培养物在37℃振荡培养过夜,16-18小时 注意:棉球是防止文化溢出的预防措施。
    5. 将单个过夜培养物在足够的TSB中以1:100稀释至等分   每个处理使用1.960ml稀释的培养物(待测化合物) DMSO) 注意:为了确定培养物稀释所需的TSB的最终体积,使用以下计算:
      (治疗次数)x 2 ml + 1 ml = ml TSB。
    6. 制备溶液0.05mg/ml,0.5mg/ml或2.5mg/ml的化合物 的感兴趣(潜在的MRSA抗毒力剂)在100%DMSO中。 (一 感兴趣的化合物的实例是二氟尼柳,更多实例参见 参考1.)
    7. 40μl的100%DMSO,0.05mg/ml,0.5mg/ml或2.5 将mg/ml的化合物在100%DMSO中的溶液加入到14ml塑料培养物中 以产生最终浓度为2%DMSO,1μg/ml,10μg/ml,和 50μg/ml的化合物。
      注意:
      1. 浓度使用取决于目标化合物的性质。
    8. 将1.960ml 1:100稀释的过夜培养物加入含有化合物或DMSO的每个培养管中
    9. 将来自步骤A5的1:100稀释液的剩余物置于冰上
    10. 与过夜培养一样,将棉球放置在每个培养管的顶部,并且松散地放置盖子。
    11. 将来自步骤A8的管在37℃下振荡孵育6小时。

  2. 培养物的稀释以确定菌落形成单位(CFU)
    1. 将置于冰上的1:100稀释的过夜培养物在A部分中以1:10 4和1:10 5的比例连续稀释到TSB中。
    2. 将100μl的1:10 4和1:10稀释物铺在LB琼脂上 并搁置,直到所有的板都准备好。 这是 零小时时间点。
    3. 6小时后,处理的培养物 从37℃摇床中取出,并按照C部分进行第一次处理 在进行以下稀释之前的协议
    4. 将每个处理的样品中剩余的液体培养物连续稀释至1:10 和1:10 5
    5. 将100μl的1:10和1:10稀释物铺在LB琼脂平板上。
    6. 所有板(包括零时时间点板)在37℃下孵育过夜
    7. 对每个1:10稀释板中的菌落数进行计数 注意:   如果在1:10平板上有少于30个菌落,则计数该样品的1:10稀释板。 如果统计功效是 利息,两盘都应计算。

  3. 用于ELISA的细菌培养物样品制备
    1. 在孵育6小时后,将细菌培养物吸入鲁尔 用1.5英寸针头锁定注射器,然后取出针 注意:用镊子或止血钳取出针头,放入 漂白并在硬的塑料锐器容器中处理。
    2. 将0.22μm过滤器紧紧放置在注射器上,将细菌培养物过滤到2ml电泳管中 注意:   如果在过滤时存在阻力,则对其施加轻微的压力   注射器的柱塞将推动滤液通过。 太多了 压力可能导致过滤器故障。
    3. 将滤液立即用干冰冷冻并储存在-80℃备用 注意:此时的滤液是无菌的,但在整个方案中仍应使用丁腈手套处理。

  4. α-溶血素(Hla)ELISA
    1. 准备稀释1:1000 Hla抗体。 对于每个使用的96孔板, μl的抗Hla抗体加入到10ml冷PBS(pH7.2)中, (1:1,000稀释),并轻轻混合
    2. 100μl的1:1000 将抗-H1a抗体的PBS加入每个微量滴定板96孔中,盖上 用塑料包裹并在4℃下孵育过夜
    3. 的 除去上清液并通过加入短暂洗涤(即,冲洗)一次 230μl的0.05%Tween 20的PBS溶液,然后除去上清液。 不需要搅拌。
    4. 向每个孔中加入230μl在PBS溶液中的10mg/ml BSA,并置于4℃下60分钟
    5. 除去上清液,通过加入230将板洗涤一次 μl的0.05%Tween 20的PBS溶液,然后除去上清液
    6. 100μl的1:1000 将抗-H1a抗体的PBS加入每个微量滴定板96孔中,盖上 用塑料包裹并在4℃下孵育过夜
    7. 的 除去上清液并通过加入短暂洗涤(即,冲洗)一次 230μl的0.05%Tween 20的PBS溶液,然后除去上清液。 不需要搅拌。
    8. 向每个孔中加入230μl在PBS溶液中的10mg/ml BSA,并置于4℃下60分钟
    9. 除去上清液,通过加入230将板洗涤一次 μl的0.05%Tween 20的PBS溶液,然后除去上清液
    10. ... 100 µl of the HLA antibody conjugated to horseradish peroxidase, diluted 1:1,000 in 10 mg/ml BSA in PBS, is added to the wells, covered with plastic wrap and foil, and then incubated for 1 h with rocking at room temperature.
    11. The supernatant is removed and the 230 µl of 0.05% Tween 20 in PBS is added to each well. The supernatant is subsequently removed.
    12. Step D33 is repeated 2 more times.
      Note: On the final wash, using a pipette ensure the removal of all 0.05% Tween 20 in PBS.
    13. 230 µl of PBS (pH 7.2) is added to each well and subsequently removed.
    14. Step D35 is repeated.
    15. The plate is placed upside down onto a paper towel until all of the supernatant is drained.
    16. 100 µl of substrate solution 3,3’,5,5’ tetramethylbenzidine (TMB) is added to each well and incubated at room temperature for 10 min.
    17. 100 µl of stop reagent is added to each well.
      Note: Air bubbles may form when stop reagent is added. Prior to measuring absorbance, all air bubbles should be popped. This can be done by blowing air with a pipette, but should be done with care to avoid cross contamination between wells.
    18. For each well in the plate, the OD is read at 650 nm using a microplate reader.

  5. Calculations
    1. The colony forming units (CFU) per ml for each treated sample is calculated by: [(# of colonies of treated sample) x (culture dilution factor)]/(# ml plated) = CFU/ml. This step can be done using Microsoft Excel or similar program.
      Notes:
      1. 0.1 ml of diluted culture is generally plated.
      2. The comparison CFU/ml of treated with the CFU/ml of the time zero plate is used to determine if the compounds are bactericidal, 抑菌,或对生长没有影响。 如果CFU/ml治疗 样品在统计学上与时间零的CFU/ml相同,则 化合物抑制细菌生长。 如果处理的样品的CFU/ml 统计学上小于时间零的,那么化合物杀死 细菌。
    2. Hla产量的量由下式计算 OD650通过:[(平均OD样品) - (平均OD空白)]×(样品稀释度 因子)/(加入#ml样品)=单位/ml 注意:
      1. 通常每孔加入0.1ml样品。
      2. 使用Excel或类似程序即可轻松完成此操作。
    3. 首先通过以下方式相对于CFU确定Hla的量:(#单位/ml)/(#CFU/ml)=单位/CFU。
    4. 产生的百分比Hla定义为:[(单位/CFU处理)/(单位/CFU DMSO)]×100 =%Hla产生。 注意:
      1. 如果%Hla产量低于100%,那么该化合物抑制Hla相对于DMSO的产生。
      2. 主Excel模板(附加此处)提供了示例数据 对于细菌滤液的1:8稀释。 模板将 一旦OD值和菌落计数自动计算步骤E41-44 。 请注意,模板是为卷和 稀释度。 如果其他体积或稀释度 则需要在模板中进行调整。


    图1.相对于DMSO处理的MRSA细菌培养物的百分比Hla产生的示例图(来自示例Excel模板

食谱

  1. 10mg BSA/PBS中 解决方案是按照制造商的指示
  2. 0.05%Tween 20的PBS溶液
    解决方案是按照制造商的指示
  3. 胰蛋白酶大豆肉汤(TSB)
    17.0g细菌胰蛋白胨 3.0克Bacto Soytone
    2.5克右旋糖 5.0克氯化钠
    2.5g磷酸氢二钾(pH至7.3)

致谢

我们感谢Barbara Truitt和Michelle Pesho博士对本议定书的发展所作的贡献。 该协议已经改编自John R Crowther,(2000)。 这项工作的资金由来自美国心脏协会,大河伙伴关系公司的授权和Steris基金会的资助提供。

参考文献

  1. Crowther,J.R。(2000)。 ELISA指南。 Methods Mol Biol 149:III -IV,1-413。
  2. Khodaverdian,V.,Pesho,M.,Truitt,B.,Bollinger,L.,Patel,P.,Nithianantham,S.,Yu,G.,Delaney,E.,Jankowsky,E。和Shoham, 2013)。 发现抗甲氧西林耐药金黄色葡萄球菌的抗病毒剂。 Antimicrob Agents Chemother 57(8):3645-3652。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Khodaverdian, V. Y. and Shoham, M. (2014). ELISA for Alpha-hemolysin (Hla) in Methicilin-resistant Staphylococcus aureus (MRSA). Bio-protocol 4(9): e1118. DOI: 10.21769/BioProtoc.1118.
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