Fonte: Whitney Swanson1,2, Frances V. Sjaastad2,3e Thomas S. Griffith1,2,3,4
1 Dipartimento di Urologia, Università del Minnesota, Minneapolis, MN 55455
2 Centro di Immunologia, Università del Minnesota, Minneapolis, MN 55455
3 Programma di laurea in microbiologia, immunologia e biologia del cancro, Università del Minnesota, Minneapolis, MN 55455
4 Masonic Cancer Center, Università del Minnesota, Minneapolis, MN 55455
Il saggio di immunoassorbimento enzimatico (ELISA) viene spesso utilizzato per misurare la presenza e/o la concentrazione di un antigene, anticorpo, peptide, proteina, ormone o altra biomolecola in un campione biologico. È estremamente sensibile, in grado di rilevare basse concentrazioni di antigene. La sensibilità di ELISA è attribuita alla sua capacità di rilevare le interazioni tra un singolo complesso antigene-anticorpo (1). Inoltre, l’inclusione di un anticorpo antigene-specifico coniugato enzimatico consente la conversione di un substrato incolore in un prodotto cromogenico o fluorescente che può essere rilevato e facilmente quantificato da un lettore di piastre. Se confrontati con i valori generati dalle quantità titolate di un antigene noto di interesse, è possibile determinare la concentrazione dello stesso antigene nei campioni sperimentali. Diversi protocolli ELISA sono stati adattati per misurare le concentrazioni di antigene in una varietà di campioni sperimentali, ma tutti hanno lo stesso concetto di base (2). La scelta del tipo di ELISA da eseguire, indiretto, sandwich o competitivo, dipende da una serie di fattori, tra cui la complessità dei campioni da testare e gli anticorpi antigene-specifici disponibili per l’uso. L’ELISA indiretto viene spesso utilizzato per determinare l’esito di una risposta immunologica, come la misurazione della concentrazione di un anticorpo in un campione. L’ELISA sandwich è più adatto per l’analisi di campioni complessi, come supernatanti di coltura tissutale o lasasità tissutale, in cui l’analita, o antigene di interesse, fa parte di un campione misto. Infine, l’ELISA competitivo viene spesso utilizzato quando è disponibile un solo anticorpo per rilevare l’antigene di interesse. Gli ELISA competitivi sono anche utili per rilevare un piccolo antigene con un solo epitopo anticorpale che non può ospitare due anticorpi diversi a causa dell’ostacolo sterico. Il protocollo descriverà le procedure di base per i test ELISA indiretti, sandwich e competitivi.
Il saggio ELISA indiretto è comunemente usato per misurare la quantità di anticorpi nel siero o nel surnatante di una coltura di ibridoma. La procedura generale per il saggio ELISA indiretto è:
Il test ELISA sandwich differisce dal test ELISA indiretto in quanto il metodo non prevede il rivestimento delle piastre con un antigene purificato. Invece, un anticorpo “cattura” viene utilizzato per rivestire i pozzetti della piastra. L’antigene è “inserito” tra l’anticorpo di cattura e un secondo anticorpo coniugato enzimatico “di rilevamento” – dove entrambi gli anticorpi sono specifici per lo stesso antigene, ma a epitopi diversi (3). Legandosi al complesso anticorpo/antigene di cattura, l’anticorpo di rilevamento rimane nella piastra. Gli anticorpi monoclonali o gli antisieri policlonali possono essere utilizzati come anticorpi di cattura e rilevamento. Il vantaggio principale del sandwich ELISA è che il campione non deve essere purificato prima dell’analisi. Inoltre, il test può essere abbastanza sensibile (4). Molti kit ELISA disponibili in commercio sono della varietà sandwich e utilizzano coppie di anticorpi testati e abbinati. La procedura generale per il test ELISA sandwich è:
La maggior parte dei kit ELISA sandwich disponibili in commercio sono dotati di anticorpi di rilevamento coniugati con enzimi. Nei casi in cui non è disponibile un anticorpo di rilevazione coniugato con enzima, può essere utilizzato un anticorpo secondario coniugato con enzima specifico per l’anticorpo di rilevamento. L’enzima sull’anticorpo secondario svolge lo stesso ruolo, che è quello di convertire il substrato incolore in un prodotto cromogenico o fluorescente. Il suddetto anticorpo secondario coniugato con enzima vorrebbe più essere utilizzato in un ELISA sandwich “fatto in casa” sviluppato da un investigatore che ha generato i propri anticorpi monoclonali, per esempio. Uno svantaggio dell’utilizzo di un anticorpo secondario coniugato con enzima è quello di essere sicuri che si leghi solo all’anticorpo di rilevamento e non all’anticorpo di cattura legato alla piastra. Ciò si tradurrebbe in un prodotto misurabile in tutti i pozzi, indipendentemente dalla presenza o dall’assenza di antigene o anticorpo di rilevamento.
Infine, il test ELISA competitivo viene utilizzato per rilevare antigeni solubili. È semplice da eseguire, ma è adatto solo quando l’antigene purificato è disponibile in una quantità relativamente grande. La procedura generale per il test ELISA competitivo è:
La “competizione” in questo test deriva dal fatto che più antigene nel campione di prova utilizzato nella fase 3 si tradurrà in meno anticorpi disponibili per legarsi all’antigene che riveste il pozzo. Pertanto, l’intensità del prodotto cromogenico/fluorogenico nel pozzo alla fine del test è inversamente correlata alla quantità di antigene presente nel campione di prova.
Un componente chiave in qualsiasi tipo di ELISA sono gli standard titolati di concentrazioni note che consentiranno all’utente di determinare la concentrazione di antigene presente nei campioni di prova. Tipicamente, una serie di pozzi sono designati per la creazione di una curva standard, in cui quantità note di una proteina ricombinante purificata vengono aggiunte ai pozzetto in quantità decrescenti. Quando questi pozzetto vengono elaborati contemporaneamente ai campioni di prova, l’utente può quindi avere un set di riferimento di valori di assorbanza ottenuti da un lettore di micropiasche per le concentrazioni proteiche note da associare ai valori di assorbanza per i campioni di prova. L’utente può quindi calcolare una curva standard a cui è possibile confrontare i campioni di prova per determinare la quantità di proteina di interesse presente. La curva standard può anche determinare il grado di precisione della diluizione dell’utente.
Infine, l’ultimo passaggio in ciascuno dei tipi ELISA sopra elencati richiede l’aggiunta di un substrato. Il grado di conversione del substrato in prodotto è direttamente correlato alla quantità di enzima presente nel pozzo. La perossidasi di rafano (HRP) e la fosfatasi alcalina (AP) sono gli enzimi più comuni coniugati agli anticorpi. Come previsto, ci sono un certo numero di substrati disponibili specifici per entrambi gli enzimi che producono un prodotto cromogenico o fluorescente. Inoltre, i substrati sono disponibili in una gamma di sensibilità che possono aumentare la sensibilità complessiva del test. L’utente deve anche prendere in considerazione il tipo di strumentazione disponibile per la lettura della piastra alla fine dell’esperimento quando sceglie il tipo di substrato da utilizzare, insieme al corrispondente anticorpo coniugato con enzima.
I substrati cromogenici comunemente usati per l’HRP includono 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) e 3,3′,5,5′-tetramethylbenzidine (TMB), mentre p-Nitrophenyl Phosphate (PNPP) è usato per AP. ABTS e TMB producono rispettivamente prodotti di reazione di colore verde e blu solubili in acqua. Il prodotto ABTS verde ha due picchi di assorbanza principali, 410 e 650 nm, mentre il prodotto TMB blu è meglio rilevato a 370 e 652 nm. I colori di ABTS e TMB cambiano in giallo con l’aggiunta di una soluzione di arresto acido, che è meglio leggere a 450 nm. Lo sviluppo del colore per ABTS è lento, mentre è veloce per TMB. TMB è più sensibile di ABTS e può produrre un segnale di fondo più alto se la reazione enzimatica procede troppo a lungo. PNPP produce un prodotto solubile in acqua giallo dopo conversione AP che assorbe la luce a 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.