Ensayos ELISA: Indirecto, Sándwich y Competitivo
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Overview
Fuente: Whitney Swanson1,2, Frances V. Sjaastad2,3y Thomas S. Griffith1,2,3,4
1 Departamento de Urología, Universidad de Minnesota, Minneapolis, MN 55455
2 Centro de Inmunología, Universidad de Minnesota, Minneapolis, MN 55455
3 Programa de Posgrado en Microbiología, Inmunología y Biología del Cáncer, Universidad de Minnesota, Minneapolis, MN 55455
4 Centro De Cáncer Masónico, Universidad de Minnesota, Minneapolis, MN 55455
El ensayo inmunoabsorbente ligado a enzimas (ELISA) se utiliza con frecuencia para medir la presencia y/o concentración de un antígeno, anticuerpo, péptido, proteína, hormona u otra biomolécula en una muestra biológica. Es extremadamente sensible, capaz de detectar bajas concentraciones de antígenos. La sensibilidad de ELISA se atribuye a su capacidad para detectar las interacciones entre un único complejo antígeno-anticuerpo (1). Además, la inclusión de un anticuerpo específico de antígeno conjugado en enzimas permite la conversión de un sustrato incoloro en un producto cromogénico o fluorescente que puede ser detectado y fácilmente cuantificado por un lector de placas. En comparación con los valores generados por cantidades valoradas de un antígeno de interés conocido, se puede determinar la concentración del mismo antígeno en las muestras experimentales. Se han adaptado diferentes protocolos ELISA para medir las concentraciones de antígenos en una variedad de muestras experimentales, pero todos tienen el mismo concepto básico (2). La elección del tipo de ELISA para realizar, indirecta, sándwich o competitiva, depende de una serie de factores, incluyendo la complejidad de las muestras a probar y los anticuerpos específicos de antígeno disponibles para su uso. El ELISA indirecto se utiliza con frecuencia para determinar el resultado de una respuesta inmunológica, como la medición de la concentración de un anticuerpo en una muestra. El sándwich ELISA es el más adecuado para analizar muestras complejas, como sobrenatantes de cultivo de tejido o lysates de tejido, donde el analito, o antígeno de interés, es parte de una muestra mixta. Por último, el ELISA competitivo se utiliza con mayor frecuencia cuando sólo hay un anticuerpo disponible para detectar el antígeno de interés. Los ELISA competitivos también son útiles para detectar un pequeño antígeno con un solo epítopo de anticuerpos que no puede acomodar dos anticuerpos diferentes debido a la obstrucción estaica. El protocolo describirá los procedimientos básicos para los ensayos ELISA indirectos, sándwiches y competitivos.
El ensayo elSAY ELISA indirecto se utiliza comúnmente para medir la cantidad de anticuerpos en suero o en el sobrenadante de un cultivo de hibridato. El procedimiento general para el ensayo elAse indirecto el ISA es:
- Capa de pozos con antígenos
- Añadir sobrenadante de cultivo de suero o hibrioma que contenga anticuerpos (primarios o anticuerpos de 1o)
- Incubar y lavar
- Añadir anticuerpos secundarios (o 2o) conjugados enzimáticos
- Incubar y lavar
- Añadir sustrato
El ensayo el sándwich ELISA difiere del ensayo elISA indirecto en que el método no implica el recubrimiento de las placas con un antígeno purificado. En su lugar, se utiliza un anticuerpo “capturar” para recubrir los pocillos de la placa. El antígeno se “se intercala” entre el anticuerpo de captura y un segundo anticuerpo conjugado enzimático de “detección”, donde ambos anticuerpos son específicos para el mismo antígeno, pero en diferentes epítopos (3). Al unirse al complejo de anticuerpos/antígenos de captura, el anticuerpo de detección permanece en la placa. Los anticuerpos monoclonales o los antisueros policlonales se pueden utilizar como anticuerpos de captura y detección. La principal ventaja del sándwich ELISA es que la muestra no tiene que ser purificada antes del análisis. Además, el ensayo puede ser bastante sensible (4). Muchos kits ELISA disponibles comercialmente son de la variedad sándwich y utilizan pares de anticuerpos probados y emparejados. El procedimiento general para el ensayo ELISA sándwich es:
- Pozos de abrigo con anticuerpo de captura
- Añadir muestras de prueba que contengan antígeno
- Incubar y lavar
- Añadir anticuerpode detección conjugado enzimático.
- Incubar y lavar
- Añadir sustrato
La mayoría de los kits ELISA sándwiches disponibles comercialmente vienen con anticuerpos de detección conjugados enzimáticos. En los casos en que no se dispone de un anticuerpo de detección conjugado enzimático, se puede utilizar un anticuerpo secundario conjugado enzimático específico para el anticuerpo de detección. La enzima del anticuerpo secundario desempeña la misma función, que consiste en convertir el sustrato incoloro en un producto cromogénico o fluorescente. Al mencionado anticuerpo secundario conjugado enzimática le gustaría más ser utilizado en un sándwich “casero” ELISA desarrollado por un investigador que ha generado sus propios anticuerpos monoclonales, por ejemplo. Un inconveniente del uso de un anticuerpo secundario conjugado enzimático es asegurarse de que sólo se une al anticuerpo de detección, y no al anticuerpo de captura unido a la placa. Esto resultaría en un producto medible en todos los pozos, independientemente de la presencia o ausencia de antígeno o anticuerpo de detección.
Por último, el ensayo ELISA competitivo se utiliza para detectar antígenos solubles. Es fácil de realizar, pero sólo es adecuado cuando el antígeno purificado está disponible en una cantidad relativamente grande. El procedimiento general para el ensayo ELISA competitivo es:
- Capa de pozos con antígeno
- Incubar y lavar
- Muestra de prueba de preincubación con anticuerpos primarios conjugados enzimáticos
- Agregue la mezcla al pozo
- Incubar y lavar cualquier anticuerpo primario conjugado en enzimas sin ataduras
- Añadir sustrato
La “competencia” en este ensayo proviene del hecho de que más antígeno en la muestra de prueba utilizada en el paso 3 resultará en menos anticuerpos disponibles para unir se adhieren al antígeno que recubre el pozo. Por lo tanto, la intensidad del producto cromogénico/fluorogénico en el pozo al final del ensayo está inversamente relacionada con la cantidad de antígeno presente en la muestra de ensayo.
Un componente clave en cualquier tipo de ELISA son los estándares valorados de concentraciones conocidas que permitirán al usuario determinar la concentración de antígeno presente en las muestras de ensayo. Típicamente, una serie de pozos se designan para crear una curva estándar, donde las cantidades conocidas de una proteína recombinante purificada se agregan a los pozos en cantidades decrecientes. Cuando estos pozos se procesan al mismo tiempo que las muestras de prueba, el usuario puede tener un conjunto de referencia de valores de absorbancia obtenidos de un lector de microplacas para que las concentraciones de proteínas conocidas vayan junto con los valores de absorción para las muestras de prueba. A continuación, el usuario puede calcular una curva estándar a la que se pueden comparar las muestras de prueba para determinar la cantidad de proteína de interés presente. La curva estándar también puede determinar el grado de precisión de la fabricación de dilución del usuario.
Por último, el último paso en cada uno de los tipos ELISA enumerados anteriormente requiere la adición de un sustrato. El grado de conversión del sustrato al producto está directamente relacionado con la cantidad de enzima presente en el pozo. La peroxidasa de rábano picante (HRP) y la fosfatasa alcalina (AP) son las enzimas más comunes encontradas conjugadas con los anticuerpos. Como era de esperar, hay una serie de sustratos disponibles específicos para cualquiera de las enzimas que producen un producto cromogénico o fluorescente. Además, los sustratos están disponibles en una gama de sensibilidades que pueden aumentar la sensibilidad general del ensayo. El usuario también debe tener en cuenta el tipo de instrumentación disponible para leer la placa al final del experimento al recoger el tipo de sustrato a utilizar, junto con su correspondiente anticuerpo conjugado enzimático.
Los sustratos cromogénicos de uso común para HRP incluyen 2,2′-Azinobis [3-etilbenzotiazolina-6-ácido sulfónico]-sal de diamonio (ABTS) y 3,3′,5,5′-tetrametilbenzidina (TMB), mientras que p-fosfato de nitrofenilo (PNPP) se utiliza para AP. producir productos de reacción de color verde y azul solubles en agua, respectivamente. El producto verde ABTS tiene dos picos de absorbancia principales, 410 y 650 nm, mientras que el producto azul TMB se detecta mejor a 370 y 652 nm. Los colores de ABTS y TMB cambian a amarillo tras la adición de una solución de parada ácida, que se lee mejor a 450 nm. El desarrollo de color para ABTS es lento, mientras que es rápido para TMB. TMB es más sensible que ABTS, y puede producir una señal de fondo más alta si la reacción enzimática continúa demasiado tiempo. PNPP produce un producto amarillo soluble en agua después de la conversión de AP que absorbe la luz a 405 nm.
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.