ELISA-Test: Indirekt, Sandwich und kompetitiv
English
Share
Overview
Quelle: Whitney Swanson1,2, Frances V. Sjaastad2,3, und Thomas S. Griffith1,2,3,4
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455
2 Zentrum für Immunologie, University of Minnesota, Minneapolis, MN 55455
3 Mikrobiologie, Immunologie und Krebsbiologie Graduate Program, University of Minnesota, Minneapolis, MN 55455
4 Freimaurerkrebszentrum, University of Minnesota, Minneapolis, MN 55455
Enzymgebundener Immunsorbent-Assay (ELISA) wird häufig verwendet, um das Vorhandensein und/oder die Konzentration eines Antigens, Antikörpers, Peptids, Proteins, Hormons oder eines anderen Biomoleküls in einer biologischen Probe zu messen. Es ist extrem empfindlich, in der Lage, niedrige Antigenkonzentrationen zu erkennen. Die Empfindlichkeit von ELISA wird auf seine Fähigkeit zurückgeführt, die Wechselwirkungen zwischen einem einzelnen Antigen-Antikörper-Komplex zu erkennen (1). Darüber hinaus ermöglicht die Aufnahme eines enzymkonjugierten antigenspezifischen Antikörpers die Umwandlung eines farblosen Substrats in ein chromogenes oder fluoreszierendes Produkt, das von einem Plattenleser erkannt und leicht quantisiert werden kann. Im Vergleich zu den Werten, die durch titrated Mengen eines bekannten Antigens von Interesse erzeugt werden, kann die Konzentration des gleichen Antigens in den experimentellen Proben bestimmt werden. Verschiedene ELISA-Protokolle wurden angepasst, um Antigenkonzentrationen in einer Vielzahl von Versuchsproben zu messen, aber sie alle haben das gleiche Grundkonzept (2). Die Wahl des Typs von ELISA, der indirekt, Sandwich oder wettbewerbsfähig ist, hängt von einer Reihe von Faktoren ab, einschließlich der Komplexität der zu testenden Proben und der antigenspezifischen Antikörper, die zur Verwendung zur Verfügung stehen. Der indirekte ELISA wird häufig verwendet, um das Ergebnis einer immunologischen Reaktion zu bestimmen, wie z. B. die Messung der Konzentration eines Antikörpers in einer Probe. Der Sandwich-ELISA eignet sich am besten zur Analyse komplexer Proben, wie Gewebekulturüberstand oder Gewebelysate, bei denen der Analyt oder Antigen von Interesse Teil einer gemischten Probe ist. Schließlich wird der wettbewerbsfähige ELISA am häufigsten verwendet, wenn nur ein Antikörper zur Verfügung steht, um das Antigen von Interesse zu erkennen. Wettbewerbsfähige ELISAs sind auch nützlich, um ein kleines Antigen mit nur einem einzigen Antikörper-Epitop zu erkennen, das aufgrund von sterischer Behinderung zwei verschiedene Antikörper nicht aufnehmen kann. Das Protokoll wird die grundlegenden Verfahren für die indirekten, Sandwich- und wettbewerbsfähigen ELISA-Assays beschreiben.
Der indirekte ELISA-Assay wird häufig verwendet, um die Menge an Antikörpern im Serum oder im Überstand einer Hybridomkultur zu messen. Das allgemeine Verfahren für den indirekten ELISA-Test ist:
- Mantelbrunnen mit Antigenen
- Hinzufügen von Serum- oder Hybridomkultur-Überstand-Antikörpern (Primär- oder 1°-Antikörper)
- Inkubieren und waschen
- Sekundärer (oder 2°) enzymkonjugierter Antikörper hinzufügen
- Inkubieren und waschen
- Substrat hinzufügen
Der Sandwich-ELISA-Assay unterscheidet sich vom indirekten ELISA-Assay dadurch, dass das Verfahren keine Beschichtung der Platten mit einem gereinigten Antigen beinhaltet. Stattdessen wird ein “Capture”-Antikörper verwendet, um die Brunnen der Platte zu beschichten. Das Antigen wird zwischen dem Capture-Antikörper und einem zweiten “detektions”-Enzymkonjugatierten Antikörper “sandwiched” – wobei beide Antikörper spezifisch für das gleiche Antigen sind, jedoch an unterschiedlichen Epitopen (3). Durch Bindung an den Capture-Antikörper/Antigen-Komplex verbleibt der Detektionsantikörper in der Platte. Als Aufnahme- und Nachweisantikörper können entweder monoklonale Antikörper oder polyklonale Antisera verwendet werden. Der Hauptvorteil des Sandwich-ELISA besteht darin, dass die Probe nicht vor der Analyse gereinigt werden muss. Darüber hinaus kann der Test sehr empfindlich sein (4). Viele handelsübliche ELISA-Kits sind von der Sandwich-Vielfalt und verwenden getestete, abgestimmte Paare von Antikörpern. Das allgemeine Verfahren für den Sandwich ELISA Assay ist:
- Mantelbrunnen mit Capture-Antikörper
- Hinzufügen von Testproben mit Antigen
- Inkubieren und waschen
- Fügen Sie enzymkonjugierten Detektionsantikörper hinzu.
- Inkubieren und waschen
- Substrat hinzufügen
Die meisten handelsüblichen Sandwich-ELISA-Kits sind mit enzymkonjugierten Nachweisantikörpern ausgestattet. In Fällen, in denen kein enzymkonjugierter Detektionsantikörper verfügbar ist, kann ein sekundärer enzymkonjugierter Antikörper verwendet werden, der speziell für den Nachweisantikörper ist. Das Enzym auf dem sekundären Antikörper erfüllt die gleiche Rolle, d.h. das farblose Substrat in ein chromogenes oder fluoreszierendes Produkt umzuwandeln. Der oben erwähnte sekundäre enzymkonjugierte Antikörper würde eher in einem “hausgemachten” Sandwich-ELISA verwendet werden, das von einem Forscher entwickelt wurde, der beispielsweise eigene monoklonale Antikörper erzeugt hat. Ein Nachteil bei der Verwendung eines sekundären enzymkonjugierten Antikörpers besteht darin, sicher zu sein, dass er nur an den Nachweisantikörper bindet und nicht an den an die Platte gebundenen Capture-Antikörper. Dies würde zu einem messbaren Produkt in allen Brunnen führen, unabhängig vom Vorhandensein oder Fehlen von Antigen oder Detektionsantikörper.
Schließlich wird der wettbewerbsfähige ELISA-Test verwendet, um lösliche Antigene zu erkennen. Es ist einfach durchzuführen, aber es ist nur geeignet, wenn das gereinigte Antigen in einer relativ großen Menge verfügbar ist. Das allgemeine Verfahren für den wettbewerbsfähigen ELISA-Test ist:
- Mantelbrunnen mit Antigen
- Inkubieren und waschen
- Präinkubent-Testprobe mit enzymkonjugierten Primärantikörpern
- Mischung gut hinzufügen
- Inkubieren und waschen Sie alle ungebundenen enzymkonjugierten Primärantikörper
- Substrat hinzufügen
Der “Wettbewerb” in diesem Test kommt aus der Tatsache, dass mehr Antigen in der Testprobe in Schritt 3 verwendet wird in weniger Antikörper zur Verfügung, um an die Antigenbeschichtung des Brunnens zu binden führen. Somit hängt die Intensität des chromogenen/fluorogenen Produkts im Brunnen am Ende des Tests umgekehrt mit der in der Testprobe vorhandenen Antigenmenge zusammen.
Eine Schlüsselkomponente in jeder Art von ELISA sind die titrierten Standards bekannter Konzentrationen, die es dem Benutzer ermöglichen, die antigenische Konzentration in den Testproben zu bestimmen. Typischerweise sind eine Reihe von Brunnen für die Erstellung einer Standardkurve vorgesehen, bei der bekannte Mengen eines gereinigten rekombinanten Proteins in abnehmenden Mengen zu den Brunnen hinzugefügt werden. Wenn diese Brunnen gleichzeitig mit den Testproben verarbeitet werden, kann der Anwender einen Referenzsatz von Absorptionswerten erhalten, die von einem Mikroplattenleser für bekannte Proteinkonzentrationen erhalten werden, um mit den Absorptionswerten für die Testproben zu gehen. Der Anwender kann dann eine Standardkurve berechnen, mit der die Testproben verglichen werden können, um die Menge des vorhandenen Proteins zu bestimmen. Die Standardkurve kann auch den Grad der Präzision der Verdünnungsherstellung des Benutzers bestimmen.
Schließlich erfordert der letzte Schritt in jedem der oben aufgeführten ELISA-Typen die Zugabe eines Substrats. Der Grad der Umwandlung des Substrats in das Produkt steht in direktem Zusammenhang mit der Menge des Enzyms, das im Brunnen vorhanden ist. Meerrettichperoxidase (HRP) und alkalische Phosphatase (AP) sind die häufigsten Enzyme, die konjugiert mit Antikörpern gefunden werden. Wie erwartet, gibt es eine Reihe von Substraten, die spezifisch für Enzyme verfügbar sind, die ein chromogenes oder fluoreszierendes Produkt produzieren. Darüber hinaus sind Substrate in einer Reihe von Empfindlichkeiten erhältlich, die die Gesamtempfindlichkeit des Assays erhöhen können. Der Anwender muss auch die Art der Instrumentierung berücksichtigen, die für das Ablesen der Platte am Ende des Experiments zur Verfügung steht, wenn er den zu verwendenden Substrattyp sowie den entsprechenden enzymkonjugierten Antikörper wählt.
Häufig verwendete chromogene Substrate für HRP umfassen 2,2′-Azinobis [3-Ethylbenzothiazolin-6-Sulfonsäure]-Diammoniumsalz (ABTS) und 3,3′,5,5′-Tetramethylbenzidin (TMB), während p-Nitrophenylphosphat (PNPP) für AP verwendet wird. ABTS und TMB wasserlösliche grüne bzw. blau gefärbte Reaktionsprodukte herstellen. Das grüne ABTS-Produkt hat zwei Hauptabsorptionsspitzen, 410 und 650 nm, während das blaue TMB-Produkt am besten bei 370 und 652 nm nachgewiesen wird. Die Farben von ABTS und TMB ändern sich nach Zugabe einer sauren Stop-Lösung in Gelb, die am besten bei 450 nm gelesen wird. Die Farbentwicklung für ABTS ist langsam, während sie für TMB schnell ist. TMB ist empfindlicher als ABTS und kann ein höheres Hintergrundsignal erzeugen, wenn die enzymatische Reaktion zu lange dauert. PNPP produziert ein gelbes wasserlösliches Produkt nach DER AP-Umwandlung, das Licht bei 405 nm absorbiert.
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.