ELISA Asas ：间接、三明治和竞争
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Overview
资料来源：惠特尼·斯旺森1，2，弗朗西斯·萨亚斯塔德2，3和托马斯·格里菲斯1，2，3，4
1明尼苏达大学泌尿科，明尼阿波利斯，MN 55455
2明尼苏达大学明尼阿波利斯分校免疫学中心，MN 55455
3明尼苏达大学明尼阿波利斯分校微生物学、免疫学和癌症生物学研究生课程，MN 55455
4共济会癌症中心，明尼苏达大学明尼阿波利斯分校，MN 55455
酶相关免疫吸附测定 （ELISA） 通常用于测量生物样品中抗原、抗体、肽、蛋白质、激素或其他生物分子的存在和/或浓度。它极其敏感，能够检测低抗原浓度。ELISA 的敏感性归因于其检测单个抗原-抗体复合物 （1） 之间的相互作用的能力。此外，加入酶结合的抗原特异性抗体，可以将无色基质转化为色化或荧光产物，由板读取器检测并轻松定量。与已知感兴趣的抗原的定子量生成值相比，可以确定实验样品中相同抗原的浓度。不同的ELISA方案已经适应了测量各种实验样品中的抗原浓度，但它们都具有相同的基本概念（2）。选择 ELISA 的类型执行，间接，三明治，或竞争，取决于许多因素，包括要测试的样品的复杂性和可用的抗原特异性抗体。间接 ELISA 通常用于确定免疫反应的结果，例如测量样品中抗体的浓度。三明治ELISA最适合分析复杂样品，如组织培养上生或组织解液，其中分析物或感兴趣的抗原是混合样品的一部分。最后，当只有一种抗体可用于检测感兴趣的抗原时，竞争ELISA最常使用。竞争的 ELISA 也可用于检测只有单个抗体表位的小型抗原，该表位由于阻抗而无法容纳两种不同的抗体。该协议将描述间接、三明治和竞争性ELISA检测的基本程序。
间接ELISA测定通常用于测量血清或杂交瘤培养液中抗体的含量。间接 ELISA 测定的一般程序是：
- 涂有抗原的油井
- 添加含有抗体的血清或杂交瘤培养上清液（原发性或1°抗体）
- 孵育和洗涤
- 添加二次（或2°）酶结合抗体
- 孵育和洗涤
- 添加基板
三明治ELISA测定法与间接ELISA测定不同，该方法不涉及涂覆纯化抗原的板。相反，”捕获”抗体用于涂覆板的孔。抗原被”夹在”捕获抗体和第二个”检测”酶结合抗体之间 – 两种抗体对同一抗原是特定的，但在不同的表位 （3）。通过与捕获抗体/抗原复合物结合，检测抗体留在板中。单克隆抗体或多克隆抗血清可用作捕获和检测抗体。三明治ELISA的主要优点是，样品在分析前不必经过纯化。此外，测定可能相当敏感 （4）。许多市售ELISA试剂盒都是三明治品种，并使用经过测试的匹配抗体对。三明治 ELISA 测定的一般程序是：
- 用捕获抗体涂井
- 添加含有抗原的测试样品
- 孵育和洗涤
- 添加酶结合检测抗体。
- 孵育和洗涤
- 添加基板
大多数市售三明治ELISA试剂盒都带有酶结合检测抗体。在无酶结合检测抗体的情况下，可以使用二次酶结合抗体，用于检测抗体。二次抗体上的酶的作用相同，即将无色基质转化为致色或荧光产物。例如，上述二次酶结合抗体更希望用于由一名已经产生自己单克隆抗体的调查员开发的”自制”三明治ELISA。使用二级酶结合抗体的一个缺点是，确保它只与检测抗体结合，而不是捕获抗体与板结合。这将导致在所有井中产生可测量的产品，无论是否存在抗原或检测抗体。
最后，利用竞争的ELISA检测检测可溶性抗原。它执行简单，但只有当纯化抗原有相对大量的可用时，它才适用。竞争 ELISA 测定的一般程序是：
- 涂有抗原的油井
- 孵育和洗涤
- 带酶结合原抗体的预孵化试验样品
- 将混合物加入好
- 孵育并洗去任何未结合的酶结合原抗体
- 添加基板
此检测中的”竞争”来自以下事实：步骤 3 中使用的测试样本中更多的抗原将导致可用于与油井抗原涂层结合的抗体较少。因此，测定结束时井中致色/含氟产物的强度与测试样品中存在的抗原量成反比。
任何类型的 ELISA 中的关键组成部分是已知浓度的定子标准，该标准将允许用户确定测试样品中存在的抗原浓度。通常，一系列水井被指定为创建标准曲线，其中已知数量的纯化重组蛋白以递减量添加到井中。当这些孔与测试样品同时处理时，用户可以从微板读取器获得一组已知蛋白质浓度的吸收率值参考集，以便与测试样品的吸光值一致。然后，用户可以计算一个标准曲线，测试样本可以进行比较，以确定感兴趣的蛋白质数量。标准曲线还可以确定用户稀释的精度。
最后，上面列出的每种 ELISA 类型中的最后一步要求添加基板。基材转化为产物的程度与井中酶的含量直接相关。马萝卜过氧化物酶 （HRP） 和碱性磷酸酶 （AP） 是发现与抗体结合的最常见酶。正如所料，有许多基质可用于产生色化或荧光产物的酶。此外，基材具有一系列灵敏度，可提高测定的整体灵敏度。在选择要使用的基板类型时，用户还必须考虑到在实验结束时可用于读取板的仪器类型，以及相应的酶结合抗体。
HRP常用的致色基质包括2，2′-Azinobis [3-乙苯甲酰胺-6-硫酸]-二氧化硅盐（ABTS）和3，3’，5，5’四甲基苯甲胺（TMB），而p-硝基磷酸（PNPP）用于AP。分别生产水溶性绿色和蓝色反应产物。绿色 ABTS 产品具有两个主要吸光峰值，410 和 650 nm，而蓝色 TMB 产品在 370 和 652 nm 时最佳检测。ABTS 和 TMB 的颜色在添加酸性停止溶液后变为黄色，最好在 450 nm 处读取。ABTS 的颜色开发速度很慢，而 TMB 的颜色开发速度很快。TMB 比 ABTS 更敏感，如果酶反应过长，可能会产生更高的背景信号。PNPP 在 AP 转换后产生黄色水溶性产品，在 405 nm 处吸收光线。
Procedure
Results
In the following example of an indirect ELISA, the presence of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice was determined. C57Bl/6 mice were infected with influenza A virus (A/PR/8; 105 PFU in 100 µL PBS i.p.) and serum was collected 28 days later. To quantitate the amount of IAV-specific IgG in the serum, 96-well ELISA plates were coated with purified A/PR/8 Influenza A virus (50 µL/well of 2 mg/ml PBS virus) overnight at 4°C. Coated plates were blocked for 1 hour at room temperature with 5% normal donkey serum in PBS, followed by incubation with diluted serum samples from IAV-challenged mice overnight at 4°C. The serum was initially diluted 1:12.5, followed by 1:4 dilutions (dilution range – 1:12.5 to 1:204,800). After washing, plates were incubated with an alkaline phosphatase (AP)-conjugated donkey anti-mouse IgG for 1 h. The plates were washed, and then p-Nitrophenyl Phosphate (PNPP; 1 mg/mL, 100 µL/well) was added. The colorless PNPP solution turns to a yellow color when AP is present. After 5-10 min, the enzymatic reaction was stopped by adding 100 µL/well 2N H2SO4. The plate was read on a microplate reader at 405 nm. The results obtained are shown in Table 1 and Figure 1.
| Sample | Wells | OD405 | Mean |
| Serum 1:12.5 | A1 | 2.163 | 2.194 |
| B1 | 2.214 | ||
| C1 | 2.204 | ||
| Serum 1:50 | A1 | 1.712 | 1.894 |
| B1 | 2.345 | ||
| C1 | 1.624 | ||
| Serum 1:200 | A1 | 1.437 | 1.541 |
| B1 | 1.73 | ||
| C1 | 1.456 | ||
| Serum 1:800 | A1 | 1.036 | 0.957 |
| B1 | 0.912 | ||
| C1 | 0.923 | ||
| Serum 1:3200 | A1 | 0.579 | 0.48 |
| B1 | 0.431 | ||
| C1 | 0.429 | ||
| Serum 1:12800 | A1 | 0.296 | 0.281 |
| B1 | 0.312 | ||
| C1 | 0.236 | ||
| Serum 1:51200 | A1 | 0.308 | 0.283 |
| B1 | 0.299 | ||
| C1 | 0.243 | ||
| Serum 1:204800 | A1 | 0.315 | 0.303 |
| B1 | 0.298 | ||
| C1 | 0.297 |
Table 1: Indirect ELISA assay data. Serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-infected mice containing IAV-specific IgG, optical density (OD) (405 nm) values and mean OD405 values.
Figure 1: Indirect ELISA assay scatter plot of mean OD405 values(+ S. D.) and serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice. The OD405 values can be inversely correlated to the serum dilutions.
In the following example of a sandwich ELISA, a 1:2.5 dilution of recombinant human TNFα standards (starting at a concentration of 75 pg/mL) was added to the indicated wells of a 96-well flat-bottom plate. These standards led to a corresponding 2.5-fold change in the absorbance readings.
| Sample | Concentration (pg/mL) | Wells | Values | Mean Value | Back Concentration Calculation | Average |
| Standard 1 | 75 | A1 | 1.187 | 1.169 | 76.376 | 75.01 |
| A2 | 1.152 | 73.644 | ||||
| Standard 2 | 30 | B1 | 0.534 | 0.52 | 30.827 | 29.962 |
| B2 | 0.506 | 29.098 | ||||
| Standard 3 | 12 | C1 | 0.23 | 0.217 | 12.838 | 12.105 |
| C2 | 0.204 | 11.372 | ||||
| Standard 4 | 4.8 | D1 | 0.09 | 0.084 | 5.055 | 4.726 |
| D2 | 0.078 | 4.398 | ||||
| Standard 5 | 1.92 | E1 | 0.033 | 0.031 | 1.941 | 1.86 |
| E2 | 0.03 | 1.778 | ||||
| Standard 6 | 0.768 | F1 | 0.009 | 0.011 | 0.626 | 0.764 |
| F2 | 0.014 | 0.901 | ||||
| Standard 7 | 0.307 | G1 | 0.002 | 0.004 | 0.238 | 0.377 |
| G2 | 0.007 | 0.516 |
Table 2: TNFα Sandwich ELISA standard curve data. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL), OD (450 nm) values, mean OD450 values, back concentration calculations and their averages.
Figure 2: Standard Curve for TNFα sandwich ELISA. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL) was analyzed using sandwich ELISA.The OD450 values can be directly correlated to the standard dilution concentrations. The amount of TNFα protein in the test sample was determined using the standard curve, which corresponds to a concentration of 38.72 pg/mL.
Once the standard curve was generated, the amount of TNFα protein in the test sample was determined. In this sandwich ELISA example, the test samples gave OD450 readings of 0.636 and 0.681, which give an average of 0.6585. When plotting this OD450 reading on the above chart, this corresponds to a TNFα concentration of 38.72 pg/ml.
Applications and Summary
As demonstrated, a range of immunoassays (with slight variation in protocols) fall within the ELISA technique family. Determining which version of ELISA to use depends on a number of factors, including what antigen is being detected, the monoclonal antibody available for a particular antigen, and the desired sensitivity of the assay (5). Some strengths and weaknesses of the different ELISAs described herein are:
| ELISA | Strengths | Weaknesses |
| Indirect | 1) High sensitivity due to the fact that multiple enzyme-conjugated secondary antibodies can bind to the primary antibody | 1) High background signal may occur because the coating of the antigen of interest to the plate is not specific (i.e., all proteins in the sample will coat the plate) |
| 2) Many different primary antibodies can be recognized by a single enzyme-conjugated secondary antibody giving the user the flexibility of using the same enzyme-conjugated secondary antibody in many different ELISA (regardless of the antigen being detected) | ||
| 3) Best choice when only a single antibody for the antigen of interest is available | ||
| Sandwich | 1) The use of antigen-specific capture and detection monoclonal antibody increases the sensitivity and specificity of the assay (compared to the indirect ELISA) | 1) Optimizing the concentrations of the capture and detection monoclonal antibodies can be difficult (especially for non-commercial kits) |
| 2) Best choice for detecting a large protein with multiple epitopes (such as a cytokine) | ||
| Competitive | 1) Impure samples can be used | 1) Requires a large amount of highly pure antigen to be used to coat plate |
| 2) Less sensitivity to reagent dilution effects | ||
| 3) Ideal for detecting small molecules (such as a hapten) |
Table 3: Summary. A summary of the strengths and weaknesses of the different ELISA techniques.
While a simple and useful technique, there are also some drawbacks to any ELISA. One is the uncertainty of the amount of the protein of interest in the test samples. If the amount is too high or too low, the absorbance values obtained by the microplate reader may fall above or below the limits of the standard curve, respectively. This will make it difficult to accurately determine the amount of protein present in the test samples. If the values are too high, the test sample can be diluted prior to adding to the wells of the plate. The final values would then need to be adjusted according to the dilution factor. As mentioned, homemade kits often require careful optimization of the antibody concentrations used to yield a high signal-to-noise ratio.
References
- Porstmann, T. and Kiessig S.T. Enzyme immunoassay techniques. An overview. Journal of Immunological Methods. 150 (1-2), 5-21 (1992).
- Suleyman Aydin. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4-15 (2015).
- Gan. S. D. and Patel K. R. Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. Journal of Investigative Dermatology, 133 (9), 1-3 (2013).
- Kohl, T. O. and Ascoli C.A. Immunometric Antibody Sandwich Enzyme-Linked Immunosorbent Assay. Cold Spring Harbor Protocols, 1 (6), (2017).
- Sakamoto, S., Putalun, W., Vimolmangkang, S., Phoolcharoen, W., Shoyama, Y., Tanaka, H., and Morimoto S. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. Journal of natural medicines, 72 (1), 32-42 (2018).
Transcript
Enzyme-linked Immunosorbent Assay, or ELISA is a highly sensitive quantitative assay commonly used to measure the concentration of an analyte like cytokines and antibodies in a biological sample. The general principle of this assay involves three steps: starting with capture, or immobilization, of the target analyte on a micro plate, followed by the detection of the analyte by target-specific detection proteins, and lastly, enzyme reaction, where a conjugated enzyme converts its substrate to a colored product. Based on different methods of capture and detection, ELISA can be of four types: direct, indirect, sandwich, and competitive.
For direct ELISA, the target antigen is first bound to the plate, and is then detected by a specific detection antibody. This method is commonly used for screening antibodies for a specific antigen. Indirect ELISA is used for detecting antibodies in a sample in order to quantify immune responses. The plate is first coated with a specific capture antigen, which immobilizes the target antibody, and this antigen-antibody complex is then detected using a second antibody.
In the case of sandwich ELISA, the target analyte is an antigen, which is captured on the plate using a capture antibody and then detected by the detection antibody, hence forming an antibody-antigen-antibody sandwich. This method is useful for measuring the concentration of an antigen in a mixed sample.
Competitive ELISA is used when only one antibody is available for a target antigen of interest. The plate is first coated with the purified antigen. Meanwhile, the sample containing the antigen is pre-incubated with the antibody and then added to the plate, to allow any free antibody molecules to bind to the immobilized antigen. The higher the signal from the plate, the lower the antigen concentration in the sample. In all of the four types of ELISA, direct, indirect, sandwich, and competitive, the detection antibody is either directly conjugated to the enzyme or can be indirectly linked to it through another antibody or protein.
The enzymes commonly used for the reaction are horseradish peroxidase or alkaline phosphatase with their respective substrates, both producing a soluble, colored product that can be measured and quantified using a plate reader. In this video, you will observe how to perform indirect ELISA, sandwich ELISA, and competitive ELISA, followed by examples of quantification of the target analyte from the indirect and sandwich ELISA methods.
The first experiment will demonstrate how to use indirect ELISA to determine the presence of anti-influenza virus antibodies in serum obtained from influenza-infected mice.
To begin, add 50 microliters of purified antigen – in this case, 2 milligrams per milliliter of purified A/PR/8 Influenza A virus- to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive cover and incubate it overnight at 4 degrees celsius to allow the antigen to bind to the plate. The following day, remove the coating solution by flicking the plate over a sink. Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of a blocking buffer- here, 5% donkey serum in 1X PBS- to each well. Leave the plate to incubate for at least 2 hours at room temperature. Following the incubation, remove the blocking buffer and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink once more to remove the wash.
Then, prepare the test samples by adding 460 microliters of PBS to a fresh tube, and then adding 40 microliters of serum to make a 1 to 12.5 dilution. Then, add 300 microliters of PBS to a second tube, and then add 100 microliters of the first dilution. Continue this serial dilution range until obtaining a final sample with a dilution of 1 to 204,800. Add the serially diluted serum samples in triplicate to the wells. Cover the plate with an adhesive cover and incubate at room temperature for an hour. Next, remove the samples by flicking the plate into the sink and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Once again, flick the plate to remove the wash.
Now, add 100 microliters of an enzyme-conjugated secondary antibody, which in this experiment is a horseradish peroxidase, or HRP, conjugated donkey anti-mouse secondary, to each well. Incubate the plate for one hour at room temperature, and flick the plate to remove any excess liquid. Wash the plate with 1X PBS containing 1% Tween-20 and then apply 100 microliters of the indicator substrate at a concentration of one milligram per milliliter to each well. Incubate the plate with the substrate for 5 to 10 minutes at room temperature. In this example, the colorless 3,3′, 5,5′ – tetramethylbenzidine, or TMB, substrate turns a blue color when HRP is present. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid. The samples will turn a yellow color.
Within 30 minutes of adding the stop solution, insert the plate into a microplate reader and read the plate at the appropriate wavelength for the substrate to determine the absorbance of the wells.
To begin the sandwich ELISA, the plate must be coated with purified capture antibody. To do this, add 100 microliters of the capture antibody at a concentration within the 1-10 microgram per milliliter range, to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive plate cover and then incubate the plate overnight at 4 degrees celsius. After the incubation, remove the coating solution by flicking the plate over a sink.
Now, block the remaining protein- binding sites in the coated wells by adding 200 microliters of 5% nonfat dry milk to the wells. Incubate the plate at room temperature for at least 2 hours. Next, remove the blocking buffer, and then wash the wells with 1X PBS containing 1% Tween-20. Remove the wash by flicking the plate over the sink. Now, add 100 microliters of the test sample to the wells, seal the plate with an adhesive cover, and then incubate it at room temperature for 2 hours. After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink to remove the wash and then add 100 microliters of enzyme-conjugated detection antibody to the wells.
Seal the plate with an adhesive cover. Leave the plate to incubate at room temperature for 2 hours. After the incubation, remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Next, add 100 microliters of the indicator substrate at a concentration of 1 milligram per milliliter, and incubate the plate for 5 to 10 minutes at room temperature. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid to the wells and then read the plate within 30 minutes of adding the stop solution in a microplate reader.
To perform a competitive ELISA, first coat the wells of a 96-well ELISA plate with 100 microliters of purified antigen at a concentration of 1-10 micrograms per milliliter. Cover the plate with an adhesive plate cover and then incubate overnight at 4 degrees celsius. Following this, remove the unbound antigen solution from the wells by flicking the plate over a sink.
Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of blocking buffer to each well- here, 5% nonfat dry milk in PBS. Incubate the plate for at least 2 hours at room temperature. While blocking the wells, prepare the antigen-antibody mixture in a 1. 5 milliliter tube by adding 150 microliters of sample antigen to 150 microliters of primary antibody for each well in the assay. Incubate this mixture for 1 hour at 37 degrees celsius. Now, remove the blocking buffer from the wells by flicking the plate over a sink. Then, wash the wells with 1X PBS containing Tween 20 and then add 100 microliters of the sample antigen- primary antibody mixture.
Leave the plate to incubate at 37 degrees celsius for one hour. Next, remove the sample mixture by flicking the plate over a sink and then wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody. Add 100 microliters of an enzyme-conjugated secondary antibody to each well and incubate the plate for one hour at 37 degrees celsius. Following this, wash the plate with 1X PBS containing 1% Tween-20 and then add 100 microliters of the substrate solution to each well. Wait for 5-10 minutes. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid and then measure the absorbance in a microplate reader within 30 minutes of adding the stop solution.
For the semi-quantitative indirect ELISA assay, the presence of influenza A virus antibodies in serially diluted samples of serum from influenza A- infected mice was determined by reading the absorbance of each well at 405 nanometers in a plate reader. This raw data is exported to a spread sheet for calculation purposes. In this experiment, the serially diluted serum samples, which range from 1 – 12.5, to 1 – 204,800, were repeated in triplicate.
To analyze the data, the mean absorbance value is therefore calculated for each set of triplicates by adding all the values for each dilution and dividing the sum by 3. Once the mean for each set of triplicates is determined, the mean OD450 readings are plotted against the serial dilutions. The OD readings decrease as the serum is diluted, indicating that less antibodies are found in the more diluted samples. In the quantitative sandwich ELISA, dilutions of known standard, in this case recombinate Human TNFalpha, were added to a 96-well plate and read along with the unknown samples.
To create the standard curve, the mean absorbance value for each set of readings of the known concentrations was calculated. Then, the mean absorbance value was plotted on the y-axis, against the known protein concentrations on the x-axis. A best fit curve is added through the points in the graph.
Once the standard curve is generated, the amount of TNFalpha protein in the test sample can be determined by first calculating the mean absorbance value for the test sample. In this example, the test samples gave OD450 readings of 0.636 and 0. 681. Adding these values and dividing the sum by 2 gives an average of 0.659. From the y-axis on the standard curve graph, extend a horizontal line from this absorbance value to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding concentration which, in this test sample, corresponds to a TNFalpha concentration of 38.72 picograms per milliliter.