ELISAアッセイ:間接、サンドイッチ、競争力
English
Share
Overview
ソース: ホイットニー・スワンソン1,2, フランシス V. シャアスタッド2,3,トーマス・S・グリフィス1,2,3,4
ミネソタ大学泌尿器科1ミネアポリス、MN 55455
2ミネソタ大学免疫学センター,ミネアポリス,MN 55455
3ミネソタ大学、ミネアポリス、MN 55455
4ミネソタ大学、ミネアポリス、MN 55455
酵素結合免疫吸着アッセイ(ELISA)は、生体試料中の抗原、抗体、ペプチド、タンパク質、ホルモン、または他の生体分子の存在および/または濃度を測定するために頻繁に使用される。それは非常に敏感であり、低い抗原濃度を検出することができる。ELISAの感度は、単一の抗原抗体複合体(1)間の相互作用を検出する能力に起因する。さらに、酵素共役抗原特異的抗体を含めることで、無色基板を、プレートリーダーで検出して容易に定量できる発色または蛍光産物に変換することができます。目的の既知の抗原のツトレート量によって生成された値と比較すると、実験試料中の同じ抗原の濃度を決定することができる。異なるELISAプロトコルは、様々な実験サンプルで抗原濃度を測定するように適応されていますが、それらはすべて同じ基本的な概念を持っています(2)。実行するELISAの種類、間接的、サンドイッチ、または競争力のあるものを選択することは、試験対象のサンプルの複雑さや使用可能な抗原特異的抗体など、多くの要因に依存します。間接ELISAは、サンプル中の抗体の濃度を測定するなど、免疫学的応答の結果を決定するために頻繁に使用されます。サンドイッチELISAは、組織培養上清や組織リザーゼなどの複雑なサンプルの分析に最適です。最後に、競合するELISAは、目的の抗原を検出するために利用可能な抗体が1つしかない場合に最も頻繁に使用されます。競争力のあるELISAは、立体的なヒンサーのために2つの異なる抗体を収容できない単一の抗体エピトープのみを持つ小さな抗原を検出するのにも役立ちます。このプロトコルは、間接的、サンドイッチ、および競合するELISAアッセイの基本的な手順を説明します。
間接ELISAアッセイは、一般的に血清中またはハイブリドーマ培養の上清中の抗体の量を測定するために使用されます。間接 ELISA アッセイの一般的な手順は次のとおりです。
- 抗原で井戸を塗る
- 血清またはハイブリドーマ培養用上清を含有する抗体(原発性または1°抗体)を添加する
- インキュベートと洗浄
- 二次(または2°)酵素共役抗体を追加する
- インキュベートと洗浄
- 基板の追加
サンドイッチELISAアッセイは、この方法が精製抗原でプレートをコーティングすることを伴わないという点で間接的なELISAアッセイとは異なる。代わりに、「捕捉」抗体は、プレートの井戸をコーティングするために使用されます。抗原は、捕捉抗体と第2の「検出」酵素共役抗体との間に「挟まれた」ものであり、両方の抗体は同じ抗原に特異的であるが、異なるエピトープ(3)で行われる。捕捉抗体/抗原複合体に結合することにより、検出抗体はプレート内に残る。モノクローナル抗体またはポリクローナル抗セラのいずれかを捕捉および検出抗体として使用することができる。サンドイッチELISAの主な利点は、サンプルが分析前に精製される必要がない場合です。また、アッセイは非常に敏感になりうる(4)。多くの市販のELISAキットは、サンドイッチの品種であり、テストされた、一致した抗体のペアを使用しています。サンドイッチELISAアッセイの一般的な手順は次のとおりです。
- 捕獲抗体が付いているコート井戸
- 抗原を含む試験サンプルを追加する
- インキュベートと洗浄
- 酵素共役検出抗体を追加します。
- インキュベートと洗浄
- 基板の追加
ほとんどの市販のサンドイッチELISAキットには、酵素共役検出抗体が付属しています。酵素共役検出抗体が利用できない場合には、検出抗体に特異的な二次酵素共役抗体を使用することができます。二次抗体上の酵素は、無色基板を発色性または蛍光産物に変換するのと同じ役割を果たす。上記の二次酵素共役抗体は、例えば、独自のモノクローナル抗体を生成した研究者によって開発された「自家製」サンドイッチELISAに使用されることをより望むでしょう。二次酵素共役抗体を使用する場合の欠点の1つは、プレートに結合した捕捉抗体ではなく、検出抗体に結合することを確認することです。これは、抗原または検出抗体の有無にかかわらず、すべてのウェルで測定可能な製品をもたらすでしょう。
最後に、競合するELISAアッセイは可溶性抗原を検出するために使用される。実行するのは簡単ですが、精製抗原が比較的多量に利用可能な場合にのみ適しています。競争力のあるELISAアッセイの一般的な手順は次のとおりです。
- 抗原で井戸を塗る
- インキュベートと洗浄
- 酵素共役一次抗体を使用したプインキュベート試験試料
- よく混合物を追加
- 結合性のない酵素結合一次抗体をインキュベートして洗い流す
- 基板の追加
このアッセイにおける「競合」は、ステップ3で使用される試験試料中の抗原が多いほど、ウェルをコーティングする抗原に結合するために利用できる抗体が少なくなるという事実から来ている。したがって、アッセイの末端にあるウェル内の発色性/フルオロゲン生成物の強度は、試験試料中に存在する抗原の量に反比例する。
ELISAの任意のタイプの重要なコンポーネントは、ユーザーが試験サンプルに存在する抗原濃度を決定することを可能にする既知の濃度の一値化された基準です。通常、一連のウェルは標準曲線を作成するために指定され、そこでは精製された組換えタンパク質の既知の量が減少量でウェルに添加されます。これらのウェルが試験試料と同時に処理されると、ユーザーは、マイクロプレートリーダーから得られた吸光度値の基準セットを持ち、既知のタンパク質濃度が試験試料の吸光度値と一緒に行くことができる。ユーザーは、存在するタンパク質の量を決定するために試験サンプルを比較できる標準曲線を計算することができます。標準曲線は、ユーザーの希釈作成の精度の程度を決定することもできます。
最後に、上記の各 ELISA タイプの最後のステップでは、基板の追加が必要です。基板から製品への変換の程度は、ウェル内に存在する酵素の量に直接関係する。ホースラディッシュペルオキシダーゼ(HRP)およびアルカリホスファターゼ(AP)は、抗体に結合して見つかった最も一般的な酵素である。予想通り、発色性または蛍光産物を産生する酵素に特異的に利用可能な基板が多数存在する。さらに、基板はアッセイの全体的な感受性を高めることができる感度の範囲で利用できる。ユーザはまた、使用する基板の種類を選択する際に実験の最後にプレートを読み取るために利用可能な器械使用の種類と、対応する酵素共役抗体を考慮する必要があります。
HRPで一般的に使用される発色基質には、2,2′-アジノビス[3-エチルベンゾアゾリン-6-スルホン酸]-ジアモニウム塩(ABTS)および3,3′,5′-テトラメチルベンジジン(TMB)が含まれますが、P-ニトロフェニルホスフェース(PN)はApBTに使用されます。水溶性の緑と青色の反応製品をそれぞれ製造します。緑色のABTSプロダクトに2つの主要な吸光度のピークがある、青いTMBプロダクトは370および652 nmで最もよく検出される間、410および650 nm。ABTSとTMBの色は、450 nmで読み取るのに最適な酸性停止溶液を添加すると黄色に変化します。ABTS の色の開発は遅いですが、TMB では高速です。TMBはABTSよりも敏感であり、酵素反応が長すぎると、より高いバックグラウンド信号が生成されることがあります。PNPPは、405 nmで光を吸収するAP変換後に黄色の水溶性製品を生成します。
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.