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Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24Gag
荧光标记抗原法(FLAQ)对HIV-1 p24Gag快速定量   

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Abstract

The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.

Keywords: Viral Production(病毒生产), HIV(艾滋病咨询门诊), Supernatant(上清), Elisa(ELISA), P24Gag(p24gag)

Materials and Reagents

  1. Sphero Protein A Polystyrene Particles (6-8 µm) (Spherotech, catalog number: PAP-60-5 )
  2. Human anti-p24Gag HIV-1 IIIB polyclonal antibodies (ImmunoDX, catalog number 2503 )
  3. Normal Human IgG (Sigma-Aldrich, catalog number: I2511 )
  4. p24Gag recombinant protein (Abcam, catalog number: ab43037 )
  5. Anti-p24Gag KC57 clone conjugated to fluorescein isothiocyanate (FITC) or to phycoerythrin also called RD1 (Beckman Coulter, catalog numbers: 6604665 and 6604667 )
  6. 1x phosphate-buffered saline (PBS) without calcium and magnesium (Corning, catalog number: 21-031-CV )
  7. Bovine serum albumin (BSA) (Axenia Biologix, catalog number: S200 )
  8. Triton X-100 (Thermo Fisher Scientific, catalog number: BP151-500 )
  9. 16% paraformaldehyde (PFA)  solution (Electron Microscopy Sciences, catalog number: 15710 )
  10. Fluorescence-activated cell sorting (FACS) staining buffer (see Recipes)

Equipment

  1. Eppendorf centrifuge
  2. Rotating mixer
  3. 96-well V-bottom plate (optional) (Thermo Fisher Scientific, catalog number: 12-565-216 )
  4. Thermo Fisher lids for 96-well microplates (Thermo Fisher Scientific, catalog number: 14-245-53A )
  5. Plate sealers (VWR International, catalog number: 62402-921 )
  6. Eppendorf tubes
  7. Multichannel pipetman (optional)
  8. Pipetman
  9. Tips (1 ml, 200 µl, and 10 µl)
  10. Reagent reservoirs (optional)
  11. Tissue culture centrifuge for Eppendorf tubes or for 96 well plates (optional)
  12. 37 °C incubator
  13. Flow cytometer
    Note: The FLAQ requires a flow cytometer equipped with a blue laser excitation (488 nm) and one measurement parameter. The photomultiplicator tube (PMT) with a 525/50 nm or a 585/42 band pass filter is used for the detection of FITC or RD1 (phycoerythrin) signals, respectively.

Software

  1. Forecyt (Intellicyt)
  2. FlowJoX software (Tree Star) or other FACS analysis software

Procedure

  1. Preparation of the FLAQ beads coated with p24Gag HIV-1 IIIB polyclonal antibodies
    1. Pipet 1 ml of Sphero Protein A Polystyrene Particles (referred hereafter as beads) into an Eppendorf tube.
    2. Centrifuge for 2 min at 12,000 rpm to pellet the beads.
    3. Remove supernatant.
    4. Add 1 ml of 1x PBS to beads pellet and centrifuge at 12,000 rpm for 2 min.
    5. Resuspend the beads pellet with 80 µl of the commercial vial of human anti-p24Gag HIV-1 IIIB polyclonal antibodies sold at the concentration 1 mg/ml. Pipet three times up and down and incubate for 15 min at room temperature on a rotating mixer.
    6. Centrifuge for 2 min at 12,000 rpm to pellet the beads.
    7. Remove and keep aside the antibody-containing supernatant.
    8. Add 1 ml of 1x PBS to the beads pellet.
    9. Centrifuge for 2 min at 12,000 rpm.
    10. Add another 1 ml of 1x PBS.
    11. Centrifuge for 2 min at 12,000 rpm.
    12. Add to the beads pellet 100 µl of a 1x PBS solution containing 4.8 mg/ml of Human normal IgG blocking reagent. Mix well by pipetting up and down 3 times.
    13. Incubate for 15 min at room temp on the rotating mixer.
    14. Centrifuge for 2 min at 12,000 rpm to pellet the beads.
    15. Discard the supernatant.
    16. Wash the beads twice with 1 ml of 10 mg/ml of BSA in PBS, spinning 2 min at 12,000 rpm each time.
    17. Resuspend the beads in 1 ml of 10 mg/ml of BSA in PBS and store these “FLAQ beads” at 4 °C.
    18. Repeat steps A1-17 using the supernatant containing p24Gag HIV-1 IIIB polyclonal antibodies from step 7 (Note 1).

  2. Measuring p24Gag content in viral preparations
    1. Resuspend the Abcam p24Gag protein in FACS buffer at 8 µg/ml. Aliquot and freeze.
    2. Prepare 200 µl of the Abcam p24Gag protein standard by serial dilution ½ in FACS buffer with 0.5% Triton-X100. Typically we perform 8 serial dilutions ranging from 10 ng/ml to 78.12 pg/ml.
    3. Harvest viral supernatants and prepare dilutions in FACS buffer supplemented with 0.5% Triton-X100 (Note 2). 160 µl per sample will be required.
    4. Prepare a mix containing the following per sample:
      0.5 µl KC57 conjugated to RD1 or FITC
      0.5 µl FLAQ beads
      39 µl FACS buffer 0.5% Triton X-100
    5. Transfer 160 µl of the standards and the viral supernatants to a 96 well V-bottom plate.
    6. Add 40 µl of the reagent mix to each well and mix.
    7. Incubate at 37 °C for 1 h.
    8. Centrifuge each plate at 2,000 rpm for 2 min. Do not use plate sealers at this step to avoid well-to-well contamination due to the low superficial tension of the FACS buffer supplemented with 0.5% Triton X-100.
    9. Discard the supernatants and wash beads once with 150 µl of FACS buffer.
    10. Resuspend the beads in each well in 100 µl of FACS buffer containing 1% PFA.
    11. Acquire samples on a flow cytometer, which will create files with an FCS extension. FCS files are flow cytometry standard files containing all the specifications needed to completely describe experimental data flow sets.
    12. Analyze the fcs files to obtain the median fluorescence intensities of the beads by the FITC or PE detectors for experiments with KC-57 conjugated with FITC or RD1, respectively.
    13. Plot the median fluorescence in the PE channel (x axis) against the concentration of p24Gag in the standard (y axis) in Excel. After adding the trendline, determine the equation that fits the standard curve the best and calculate the concentration (y value) of the unknown.

Representative data



Figure 1. Representative data. A. Principle of the FLAQ assay. B. Gating strategy. The population of beads is first gated on Forward Scatter area (FSC-A) and Side Scatter area (SSC-A). Overlay of the population of beads for the standard (10 ng/ml to 78.12 pg/ml). C. Standard Curve.

Notes

  1. We can reuse the antibody at least 5 times, generating 5 ml of beads. The median fluorescence is slightly lower for each reuse.
  2. The presence of triton ensures good dilution of viral preparations. Supernatants from transfected 293T should contain between 500 and 1,000 ng/ml of p24Gag protein while supernatants from infected cultures (SupT1 or PBMCs) range from 20 to 200 ng/ml. Usually three dilutions per sample are analyzed to ensure that the measurement will fall within the dynamic range of the assay.

Recipes

  1. Fluorescence-activated cell sorting (FACS) staining buffer
    1x phosphate-buffered saline (PBS)
    2% fetal bovine serum
    Stored at 4 °C

Acknowledgments

We would like to thank Hayden et al. for developing the FLAQ assay.

References

  1. Hayden, M. S., Palacios, E. H. and Grant, R. M. (2003). Real-time quantitation of HIV-1 p24 and SIV p27 using fluorescence-linked antigen quantification assays. AIDS 17(4): 629-631.

简介

荧光连接的抗原定量(FLAQ)测定允许HIV-1p24Gag抗原的快速定量。 将病毒上清液裂解并与涂覆有针对HIV-1p24 Gag的多克隆抗体的聚苯乙烯微球和与荧光染料缀合的检测抗体(图1)温育。 洗涤后,通过流式细胞术测量微球的荧光,并反映裂解物中抗原的丰度。 FLAQ测定的速度,简单性和宽动态范围对于在HIV-1研究实验室中进行的许多应用是最佳的。

关键字:病毒生产, 艾滋病咨询门诊, 上清, ELISA, p24gag

材料和试剂

  1. Sphero蛋白A聚苯乙烯颗粒(6-8μm)(Spherotech,目录号:PAP-60-5)
  2. 人抗p24抗Gag HIV-1 IIIB多克隆抗体(ImmunoDX,目录号2503)
  3. 正常人IgG(Sigma-Aldrich,目录号:I2511)
  4. p24 Gag 重组蛋白(Abcam,目录号:ab43037)
  5. 与荧光素异硫氰酸酯(FITC)或也称为RD1(Beckman Coulter,目录号:6604665和6604667)缀合的抗p24 Gag KC57克隆
  6. 1×磷酸盐缓冲盐水(PBS),不含钙和镁(Corning,目录号:21-031-CV)
  7. 牛血清白蛋白(BSA)(Axenia Biologix,目录号:S200)
  8. Triton X-100(Thermo Fisher Scientific,目录号:BP151-500)
  9. 16%多聚甲醛(PFA) 溶液(Electron Microscopy Sciences,目录号:15710)
  10. 荧光激活细胞分选(FACS)染色缓冲液(参见配方)

设备

  1. Eppendorf离心机
  2. 旋转搅拌器
  3. 96孔V型底板(可选)(Thermo Fisher Scientific,目录号:12-565-216)
  4. 用于96孔微量培养板(Thermo Fisher Scientific,目录号:14-245-53A)的Thermo Fisher盖子
  5. 板密封剂(VWR International,目录号:62402-921)
  6. Eppendorf管
  7. 多通道移液器(可选)
  8. Pipetman
  9. 提示(1 ml,200μl和10μl)
  10. 试剂库(可选)
  11. 用于Eppendorf管或96孔板(可选)的组织培养离心机
  12. 37℃孵育器
  13. 流式细胞仪
    注意:FLAQ需要配备蓝色激光激发(488 nm)和一个测量参数的流式细胞仪。 具有525/50 nm或585/42带通滤波器的光电倍增管(PMT)分别用于检测FITC或RD1(藻红蛋白)信号。

软件

  1. Forecyt(Intellicyt)
  2. FlowJoX软件(Tree Star)或其他FACS分析软件

程序

  1. 制备用p24 Gag HIV-1 IIIB多克隆抗体包被的FLAQ珠子
    1. 吸取1 ml Sphero蛋白A聚苯乙烯颗粒(以下称为珠)到离心管中。
    2. 在12,000rpm离心2分钟以沉淀珠粒。
    3. 除去上清液。
    4. 加入1毫升1×PBS的珠粒,离心12,000 rpm,2分钟
    5. 用80μl的商业小瓶重悬珠粒 人类抗p24 Gag HIV-1 IIIB多克隆抗体 浓度1mg/ml。 吸移三次上下,孵育15   min,在室温下在旋转混合器上
    6. 在12,000rpm离心2分钟以沉淀珠粒。
    7. 取出并保留含抗体的上清液。
    8. 加入1毫升1×PBS的珠粒。
    9. 在12,000rpm离心2分钟。
    10. 再加1ml 1×PBS。
    11. 在12,000rpm离心2分钟。
    12. 添加到珠粒100μl含有4.8的1x PBS溶液 mg/ml人正常IgG封闭试剂。 通过吹吸和向上充分混合   下降3次。
    13. 在室温下在旋转混合器上孵育15分钟
    14. 在12,000rpm离心2分钟以沉淀珠粒。
    15. 弃去上清液。
    16. 用1ml 10mg/ml BSA的PBS洗涤珠两次,每次以12,000rpm旋转2分钟。
    17. 将珠子重悬在1ml的10mg/ml BSA的PBS中,并将这些"FLAQ珠子"保存在4℃。
    18. 使用含有步骤7(注1)的p24 Gag HIV-1 IIIB多克隆抗体的上清液重复步骤A1-17。

  2. 测量病毒制剂中的p24 Gag 含量
    1. 以8μg/ml重悬在FACS缓冲液中的Abcam p24Gag蛋白。 等分和冻结。
    2. 通过连续制备200μl的Abcam p24 Gag 蛋白标准品 在具有0.5%Triton-X100的FACS缓冲液中稀释1/2。 通常我们执行8   系列稀释度范围为10ng/ml至78.12pg/ml。
    3. 收成 并在补充的FACS缓冲液中制备稀释液 用0.5%Triton-X100(注2)。 每个样品需要160μl。
    4. 准备每个样品包含以下物质的混合物:
      0.5μl与RD1或FITC结合的KC57 0.5μlFLAQ珠子
      39μlFACS缓冲液0.5%Triton X-100
    5. 转移160微升标准品和病毒上清液到96孔V型底板
    6. 向每个孔中加入40μl试剂混合物并混合
    7. 在37℃孵育1小时
    8. 将每个板在2,000rpm离心2分钟。 不要使用板 密封剂在此步骤中避免由于低的良好对污染 补充有0.5%Triton的FACS缓冲液的表面张力 X-100。
    9. 弃去上清液,并用150μlFACS缓冲液洗涤珠子一次
    10. 将每个孔中的珠子重悬在100μl含有1%PFA的FACS缓冲液中
    11. 在流式细胞仪上采集样品,这将创建文件 FCS扩展。 FCS文件是包含流式细胞仪标准文件   所有的规格需要完全描述实验数据 流集。
    12. 分析fcs文件以获取中值 通过FITC或PE检测器的珠的荧光强度 用分别与FITC或RD1缀合的KC-57进行的实验。
    13. 绘制PE通道(x轴)中的荧光中位数 Excel中标准(y轴)中p24 的浓度。 添加后 趋势线,确定适合标准曲线的方程 最好并计算未知物的浓度(y值)。

代表数据



图1.代表性数据 A. FLAQ测定的原理。 B.门控策略。珠子群首先在前向散射区(FSC-A)和侧向散射区(SSC-A)上门控。珠粒群体对于标准品(10ng/ml至78.12pg/ml)的覆盖。 C.标准曲线。

笔记

  1. 我们可以重复使用抗体至少5次,产生5毫升珠。每次重复使用时,中值荧光略低。
  2. triton的存在确保了病毒制剂的良好稀释。来自转染的293T的上清液应含有500至1,000ng/ml的p24 Gag蛋白,​​而来自受感染培养物(SupT1或PBMC)的上清液的范围为20至200ng/ml。通常分析每个样品的三个稀释度以确保测量将落入测定的动态范围内

食谱

  1. 荧光激活细胞分选(FACS)染色缓冲液
    1×磷酸盐缓冲盐水(PBS)
    2%胎牛血清 储存在4°C

致谢

我们要感谢Hayden等人开发FLAQ测定法。

参考文献

  1. Hayden,M.S.,Palacios,E.H。和Grant,R.M。(2003)。 使用荧光染料实时定量HIV-1 p24和SIV p27, linked antigen quantification assays。 AIDS 17(4):629-631。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gesner, M., Maiti, M., Grant, R. and Cavrois, M. (2014). Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24Gag . Bio-protocol 4(24): e1366. DOI: 10.21769/BioProtoc.1366.
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Seung-Yong Jung
Gladstone Institutes
Like all other quantitative analysis with fluorescence, it needs to carefully decide the proper dilution rates of the samples in order to increase the accuracy in the linear intensity-concentration range.
3/9/2017 6:23:24 PM Reply
Marielle Cavrois
Gladstone Institutes, and University of California, San Francisco (UCSF), USA

Yes, the FlaQ assay is designed to quantify p24Gag in solutions ranging from 10 ng/ml to 78.12 pg/ml.

3/9/2017 9:15:57 PM