Source : Whitney Swanson1,2, Frances V. Sjaastad2,3, et Thomas S. Griffith1,2,3,4
1 Département d’urologie, Université du Minnesota, Minneapolis, MN 55455
2 Center for Immunology, Université du Minnesota, Minneapolis, MN 55455
3 Microbiologie, Immunologie, et programme d’études supérieures de biologie de cancer, université du Minnesota, Minneapolis, MN 55455
4 Centre du cancer maçonnique, Université du Minnesota, Minneapolis, MN 55455
L’analyse immunosorbent enzymatique (ELISA) est fréquemment utilisée pour mesurer la présence et/ou la concentration d’un antigène, d’un anticorps, d’un peptide, d’une protéine, d’une hormone ou d’une autre biomolécule dans un échantillon biologique. Il est extrêmement sensible, capable de détecter de faibles concentrations d’antigènes. La sensibilité de l’ELISA est attribuée à sa capacité à détecter les interactions entre un seul complexe antigène-anticorps (1). De plus, l’inclusion d’un anticorps spécifique à l’antigène conjugué en zymatique permet la conversion d’un substrat incolore en un produit chromogénique ou fluorescent qui peut être détecté et facilement quanti par un lecteur de plaques. Par rapport aux valeurs générées par les quantités titrated d’un antigène connu d’intérêt, la concentration du même antigène dans les échantillons expérimentaux peut être déterminée. Différents protocoles ELISA ont été adaptés pour mesurer les concentrations d’antigènes dans une variété d’échantillons expérimentaux, mais ils ont tous le même concept de base (2). Le choix du type d’ELISA à effectuer, indirect, sandwich, ou compétitif, dépend d’un certain nombre de facteurs, y compris la complexité des échantillons à tester et les anticorps spécifiques à l’antigène disponibles à utiliser. L’ELISA indirecte est fréquemment utilisée pour déterminer les résultats d’une réponse immunologique, comme mesurer la concentration d’un anticorps dans un échantillon. Le sandwich ELISA est le mieux adapté pour analyser des échantillons complexes, tels que les supernatants de culture tissulaire ou les lysates tissulaires, où l’analyte, ou antigène d’intérêt, fait partie d’un échantillon mixte. Enfin, l’ELISA compétitif est le plus souvent utilisé lorsqu’il n’y a qu’un seul anticorps disponible pour détecter l’antigène d’intérêt. Les ELISA compétitifs sont également utiles pour détecter un petit antigène avec seulement un seul épitope d’anticorps qui ne peut pas accueillir deux anticorps différents en raison de l’entrave stérique. Le protocole décrira les procédures de base pour les essais indirects, sandwich, et compétitifs ELISA.
L’analyse indirecte ELISA est couramment utilisée pour mesurer la quantité d’anticorps dans le sérum ou dans le supernatant d’une culture hybridoma. La procédure générale pour l’action indirecte ELISA est la suivante :
L’extrait du sandwich ELISA diffère de l’extrait indirect ELISA en ce que la méthode n’implique pas d’enduire les plaques d’un antigène purifié. Au lieu de cela, un anticorps “capture” est utilisé pour enrober les puits de la plaque. L’antigène est “sandwiché” entre l’anticorps de capture et un deuxième anticorps conjugué enzymatique — où les deux anticorps sont spécifiques pour le même antigène, mais à des épitopes différents (3). En se liant au complexe anticorps/antigène de capture, l’anticorps de détection reste dans la plaque. Les anticorps monoclonaux ou les antiseras polyclonal peuvent être utilisés comme anticorps de capture et de détection. Le principal avantage du sandwich ELISA est que l’échantillon n’a pas besoin d’être purifié avant l’analyse. De plus, l’analyse peut être assez sensible (4). Beaucoup de kits ELISA disponibles dans le commerce sont de la variété de sandwich et l’utilisation testée, paires appariées d’anticorps. La procédure générale pour l’assay sandwich ELISA est:
La plupart des kits ELISA de sandwich disponibles dans le commerce sont livrés avec des anticorps de détection enzymatiques. Dans les cas où un anticorps de détection conjugué en zymatique n’est pas disponible, un anticorps secondaire conjugué enzymatique spécifique à l’anticorps de détection peut être utilisé. L’enzyme sur l’anticorps secondaire joue le même rôle, qui est de convertir le substrat incolore en un produit chromogénique ou fluorescent. L’anticorps secondaire conjugué aux enzymes mentionné ci-dessus aimerait plus être utilisé dans un sandwich « fait maison » ELISA développé par un chercheur qui a généré leurs propres anticorps monoclonaux, par exemple. Un inconvénient à l’utilisation d’un anticorps secondaire enzymatique conjugué est de s’assurer qu’il ne se lie qu’à l’anticorps de détection, et non pas l’anticorps de capture lié à la plaque. Cela se traduirait par un produit mesurable dans tous les puits, indépendamment de la présence ou l’absence d’antigène ou d’anticorps de détection.
Enfin, l’analyse ELISA compétitive est utilisée pour détecter les antigènes solubles. Il est simple à réaliser, mais il n’est approprié que lorsque l’antigène purifié est disponible dans une quantité relativement importante. La procédure générale pour l’analyse ELISA compétitive est la suivante :
La «concurrence» dans cet essai vient du fait que plus d’antigène dans l’échantillon d’essai utilisé à l’étape 3 se traduira par moins d’anticorps disponibles pour se lier à l’antigène revêtement du puits. Ainsi, l’intensité du produit chromogénique/fluorogénique dans le puits à la fin de l’essai est inversement liée à la quantité d’antigène présente dans l’échantillon d’essai.
Un composant clé dans tout type d’ELISA est les normes titrated des concentrations connues qui permettront à l’utilisateur de déterminer la concentration d’antigène présente dans les échantillons d’essai. Typiquement, une série de puits sont désignés pour créer une courbe standard, où des quantités connues d’une protéine recombinante purifiée sont ajoutées aux puits en quantités décroissantes. Lorsque ces puits sont traités en même temps que les échantillons d’essai, l’utilisateur peut alors disposer d’un ensemble de valeurs d’absorption de référence obtenues auprès d’un lecteur de microplaques pour des concentrations de protéines connues pour aller de pair avec les valeurs d’absorption pour les échantillons d’essai. L’utilisateur peut ensuite calculer une courbe standard à laquelle les échantillons d’essai peuvent être comparés pour déterminer la quantité de protéines d’intérêt présente. La courbe standard peut également déterminer le degré de précision de la fabrication de dilution de l’utilisateur.
Enfin, la dernière étape de chacun des types ELISA énumérés ci-dessus prévoit l’ajout d’un substrat. Le degré de conversion du substrat en produit est directement lié à la quantité d’enzymes présente dans le puits. La peroxidase de raifort (HRP) et la phosphatase alcaline (AP) sont les enzymes les plus communes trouvées conjuguées aux anticorps. Comme prévu, il existe un certain nombre de substrats disponibles spécifiques pour l’une ou l’autre enzyme qui produisent un produit chromogénique ou fluorescent. En outre, les substrats sont disponibles dans une gamme de sensibilités qui peuvent augmenter la sensibilité globale de l’analyse. L’utilisateur doit également prendre en considération le type d’instrumentation disponible pour la lecture de la plaque à la fin de l’expérience lors de la sélection du type de substrat à utiliser, ainsi que son anticorps correspondant enzymatique.
Les substrats chromogéniques couramment utilisés pour hrP incluent 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) et 3,3′,5′-tetramethylbenzidine (TMB), tandis que p-Nitrophenyl produire des produits de réaction verts et bleus solubles dans l’eau, respectivement. Le produit vert ABTS a deux pics d’absorption majeurs, 410 et 650 nm, tandis que le produit tMB bleu est mieux détecté à 370 et 652 nm. Les couleurs de L’ABTS et du TMB changent en jaune à l’ajout d’une solution d’arrêt acide, qui est mieux lue à 450 nm. Le développement des couleurs pour ABTS est lent, alors qu’il est rapide pour TMB. TMB est plus sensible que ABTS, et peut produire un signal de fond plus élevé si la réaction enzymatique se produit trop longtemps. PNPP produit un produit jaune soluble dans l’eau après la conversion AP qui absorbe la lumière à 405 nm.
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.
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.
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.