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20154
SARS-CoV-2 Spike Protein Serological IgG ELISA Kit
ELISA 试剂盒
ELISA 试剂盒

SARS-CoV-2 Spike Protein Serological IgG ELISA Kit #20154

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ELISA Image 1 - SARS-CoV-2 Spike Protein Serological IgG ELISA Kit

Patient Testing: Patient samples were tested using the SARS-CoV-2 Spike Protein Serological IgG ELISA Kit #20154. Serum or plasma was obtained from donors who had been diagnosed with SARS-CoV-2 (confirmed positive n=17) or from uninfected donors collected prior to the SARS-CoV-2 outbreak (confirmed negative n= 62). Samples were heat-inactivated (56°C for 30 min) and diluted 1:800 prior to running the assay, as described in the attached protocol. Samples were considered positive, negative, or inconclusive based on the criteria described in the “Data Analysis” section of the attached protocol. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated from these data.

ELISA Image 2 - SARS-CoV-2 Spike Protein Serological IgG ELISA Kit

Intra-Assay Precision: Three different serum samples were each tested in 16 replicates using a single assay kit of SARS-CoV-2 Spike Protein Serological IgG ELISA #20154. Intra-assay CV (%) was calculated for each sample, and each replicate was correctly identified as being positive or negative when compared to the Negative Control using the cutoff criteria described in the attached protocol.

ELISA Image 3 - SARS-CoV-2 Spike Protein Serological IgG ELISA Kit

Inter-Assay Precision: Six different assay kits from one lot of material were tested using 3 different serum samples run in duplicate wells and Positive and Negative Controls run in 4 replicate wells. Inter-assay CV (%) was calculated for each sample, and each assay kit correctly identified the samples as being positive or negative when compared to the Negative Control using the cutoff criteria described in the attached protocol.

购买 # 20154C
产品货号 规格 价格 库存
20154C
1 个试剂盒(96 次检测)

支持数据

反应性 H

应用缩写:

  • W-Western
  • IP-免疫沉淀法
  • IHC-免疫组织化学法
  • ChIP-染色质免疫沉淀法
  • IF-免疫荧光法
  • F-流式细胞术
  • E-P-ELISA 肽

物种交叉反应性缩写:

  • H-人
  • M-小鼠
  • R-大鼠
  • Hm- 仓鼠
  • Mk-猴子
  • Mi-水貂
  • C-鸡
  • Dm-黑腹果蝇
  • X-爪蟾
  • Z-斑马鱼
  • B-牛
  • DG-狗
  • PG-猪
  • Sc-酿酒酵母
  • Ce-秀丽隐杆线虫
  • Hr-马
  • All-预期所有物种
产品包括 体积 溶液颜色
Spike Protein Coated Microwells 96 次测试
Anti-Human IgG, HRP-linked Antibody (ELISA Formulated) 1 个 红色(冻干)
Sample Diluent A 25 ml
HRP Diluent 11 ml 红色
ELISA Wash Buffer (20X) 9801 25 ml
TMB Substrate 7004 11 ml
STOP Solution 7002 11 ml
Sealing Tape 2 ea
ELISA Kit #20154 Positive Control 1 个
ELISA Kit #20154 Negative Control 1 个

产品说明

The SARS-CoV-2 Spike Protein Serological IgG ELISA Kit is a solid phase ELISA that detects binding of human IgG to full-length SARS-CoV-2 spike protein (S-protein). Full-length spike protein has been coated onto microwells. After incubation with sample, human IgG specific for spike protein is captured on the plate. 然后洗涤微孔以除去未结合的物质。Anti-Human IgG, HRP-linked antibody is then used to recognize the bound IgG. 加入 HRP 底物 (TMB) 来显色。The magnitude of optical density for this developed color is proportional to the quantity of IgG specific for spike protein.

* 试剂盒中的*抗体为针对该试剂盒的定制制剂。

实验步骤

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SARS-CoV-2 Spike Protein Serological IgG ELISA Protocol

This ELISA Kit Is Intended For Research Use Only. Not For Use in Diagnostic or Clinical Procedures.

A. 溶液和试剂

注意:用去离子水/纯净水或等效水制备溶液。仅制备实验当天所需的足够试剂。

  1. Spike Protein Coated Microwells: Bring all to room temperature before opening bag/use. 应将未使用的微孔板条放回可重复密封且含有干燥剂包的原装袋中并存储于 4°C 下。
  2. 1X ELISA 洗涤缓冲液:通过使用去离子水稀释 ELISA 洗涤缓冲液 (20X)(每个试剂盒中均有提供)至 1X 来制备。
  3. Sample Diluent A: Diluent provided for dilution of samples and for reconstitution of Positive and Negative Controls included in kit.
  4. HRP Diluent: Red colored diluent for reconstitution and dilution of the Anti-Human IgG, HRP-linked Antibody (11 mL provided).
  5. Anti-Human IgG, HRP-linked Antibody (ELISA Formulated): Supplied lyophilized as a red colored cake or powder. Add 1.0 mL of HRP Diluent (red solution) to yield a concentrated stock solution. 在室温下孵育 5 分钟,同时不时轻轻混合,以充分重新配制。To make the final working solution, add the full 1.0 mL volume of reconstituted HRP-linked Antibody to 10.0 mL of HRP Diluent in a clean tube and gently mix. For best results, use this working solution immediately. Unused working solution may be stored for up to 4 weeks at 4°C, although there may be some loss of signal compared to freshly made solution.
  6. Positive Control: Reconstitute the vial of lyophilized Positive Control with 1.0 mL Sample Diluent A. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the “Test Procedure” section. Positive Controls are recommended to be used immediately after reconstituting, however remaining material may be stored at -80°C (there may be some loss of the Positive Control signal if freeze/thawed). 阳性对照作为一种对照试剂提供,而不是作为一种绝对定量方法。
  7. Negative Control: Reconstitute the vial of lyophilized Negative Control with 1.0 mL Sample Diluent A. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the “Test Procedure” section. Negative Controls are recommended to be used immediately after reconstituting, however remaining material may be stored at -80°C (there may be some loss of the Negative Control signal if freeze/thawed).
  8. TMB 底物 (#7004):使用前请恢复至室温。
  9. STOP 溶液 (#7002):使用前请恢复至室温。

B. Test Procedure

:在运行检测之前,请将所有材料和已制备的试剂平衡至室温。

  1. 如上(第 A 部分)所述制备所有试剂。
  2. Human-sourced samples should be handled in accordance with accepted safety practices. Samples should be diluted at least 1:800 with Sample Diluent A and can be further serially diluted if relative quantification is needed by the user. Positive and Negative Controls do NOT need to be diluted after reconstitution. Refer to the datasheet which shows typical results observed for the Positive Control, Negative Control, serum from uninfected individuals, and serum from SARS-CoV-2 patients. When using the cutoff criteria described below to determine if a sample is positive for anti-CoV-2 Spike Protein antibodies, samples diluted 1:800 must be compared to the undiluted Negative Control.

    NOTE: Sample storage/handling, including heat-inactivation of samples, can potentially affect observed signals. Therefore, it is strongly recommended that in addition to the Positive and Negative Controls included with the kit, the user includes their own negative and positive patient samples as controls when running the assay in order to establish an appropriate cutoff value.

  3. Add 100 μL of each diluted sample, Positive Control, Negative Control, and blank (Sample Diluent A only) to the appropriate wells. Seal the plate with the supplied sealing tape and incubate for 1 hour at 37°C.
  4. 轻轻取下胶带并洗涤各孔:
    1. 将平板内容物弃入容器中。
    2. 用 1X ELISA 洗涤缓冲液洗涤 4 次,每孔每次 200 µl。每次洗涤后,将孔吸干或倒空。倒转平板并用干净的纸巾将其吸干,以便除去每个孔中的残留溶液,但无论何时都不得让孔完全干燥。
    3. 用无绒织物清洁全部孔的底面。
  5. Add 100 μL of reconstituted Anti-Human IgG, HRP-linked Antibody (ELISA Formulated). 用胶带密封,平板在 37°C 下孵育 30 分钟。
  6. Repeat wash procedure (Section B, Step 4).
  7. 向每个孔添加 100 μl TMB 底物。Seal with tape and incubate the plate in the dark for 10 min at 37°C.
  8. 向每个孔添加 100 μL STOP 溶液。温和振摇数秒。

    注意:阳性反应的初始颜色为蓝色,一旦添加 STOP 溶液,就变为黄色。

  9. 读取结果:
    1. 目测:在添加 STOP 溶液后 30 分钟内读取。
    2. 分光光度法测定:用无绒薄布擦拭各孔底部。添加 STOP 溶液后 30 分钟内,读取在 450 nm 处的吸光度。
  10. Data Analysis:
    1. Subtract “blank” well (Sample Diluent A only) absorbance 450 nm values from sample, Positive, and Negative Control values.
    2. Positive Control Values should be > 1.5 and Negative Control Values should be < 0.75.
    3. Samples (1:800 dilution) are considered positive if they are greater than 4.1 x Negative Control absorbance 450 nm value.
    4. Samples (1:800 dilution) are considered negative if they are less than 3 x Negative Control absorbance 450 nm value.
    5. Samples (1:800) are considered inconclusive if they are greater than the 3 x Negative Control absorbance 450 nm value and less than 4.1 x Negative Control absorbance 450 nm value.
    6. Limitations: Experimental cutoffs were determined by assaying a set of confirmed SARS-CoV-2 positive samples and uninfected donor serum collected prior to the SARS-CoV-2 pandemic. Researchers can establish or modify this cutoff using additional samples. Positive or negative results from this assay should not be the sole basis for determining the infection status of a sample. A negative result can occur in SARS-CoV-2 patient samples due to:
      • improper sample handling/storage
      • timing of sample collection post-infection
      • patients having impaired immune function

实验步骤编号:2024

特异性/敏感性

The SARS-CoV-2 Spike Protein Serological IgG ELISA Kit detects endogenous levels of human IgG directed to full-length SARS-CoV-2 spike protein (S-protein).

物种反应性:

背景

COVID-19 疫情的病因是一种新型的高致病性冠状病毒,称为 SARS-CoV-2(严重急性呼吸系统综合症冠状病毒 2)。SARS-CoV-2 是冠状病毒家族的成员(1)。SARS-CoV-2 的基因组与其他冠状病毒相似,并且由四个关键结构蛋白组成:S,刺突蛋白;E,包膜蛋白;M,膜蛋白;N,核衣壳蛋白(2)。冠状病毒刺突蛋白是 I 类融合蛋白,具有胞外域、跨膜域和胞内尾巴(3,4)。高度糖基化的胞外域从病毒包膜表面突出,并促进与宿主细胞质膜的附着和融合。胞外域可进一步细分为宿主受体结合域(RBD)(S1)和膜融合(S2)亚基,它们是由宿主蛋白酶在 S1/S2 和 S2' 位点进行蛋白水解产生的。S1 和 S2 亚基在切割后仍保持结合并组装成冠状三聚体(2,4)。在人类中,SARS-CoV 和 SARS-CoV-2 刺突蛋白均利用血管紧张素转化酶2(ACE2)蛋白作为进入细胞的受体(5-7)。刺突蛋白亚基代表冠状病毒毒粒的关键抗原特征,因此代表疫苗、新型治疗性抗体和小分子抑制剂(8,9)的重要靶标。

  1. Zhou, P. et al. (2020) Nature 579, 270-3.
  2. Tortorici, M.A. and Veesler, D. (2019) Adv Virus Res 105, 93-116.
  3. Li, F. et al. (2006) J Virol 80, 6794-800.
  4. Li, F. (2016) Annu Rev Virol 3, 237-61.
  5. Shang, J. et al. (2020) Nature 581, 221-4.
  6. Wrapp, D. et al. (2020) Science 367, 1260-3.
  7. Yan, R. et al. (2020) Science 367, 1444-8.
  8. Yuan, Y. et al. (2017) Nat Commun 8, 15092.
  9. Amanat, F. and Krammer, F. (2020) Immunity 52, 583-9.

通路和蛋白

探索与本品相关的通路 + 蛋白。

仅供研究使用。不得用于诊断流程。

Cell Signaling Technology 是 Cell Signaling Technology, Inc. 的商标。

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