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Indirect and Direct ELISA

Our protocol for traditional ELISA formats using indirect and direct detection.
Last edited Thu 17 Feb 2022

Stage 1 - Sample Preparation

ELISAs can be run on a number of sample types. Here we provide ways to prepare different sample formats.

Materials required

  • Your sample
  • Extraction buffer (for example ab260490)
  • Protease inhibitor cocktail (for example ab65251)
  • Phosphatase inhibitor cocktail (optional – for example ab201112)
  • Wash buffer (for example ab206977)
  • A BCA or Bradford assay kit (for example ab102536 or ab102536)

Steps

1

Prepare the extraction buffer as recommended by the manufacturer.

  • Be sure to add protease inhibitors if not included.
  • Add phosphatase inhibitors for phosphorylated proteins.
2

Isolate the cells and suspend them in the extraction buffer.

  • You should prepare a suspension with ~ 1 mL of buffer per 107 cells.
  • Adherent cells can be isolated by scraping directly into extraction buffer with PBS.
  • Suspension cells can be isolated by washing with PBS, spinning down in a centrifuge, and resuspending the pellet in extraction buffer.
3

Lyse the cells.

  • Agitate the cell suspension in lysis buffer for 15 – 30 min at 4°C.
4

Spin down the suspension in a centrifuge to pellet insoluble contents.

  • Centrifuge at 18,000 x g for 20 mins at 4 °C.
  • Keep the supernatant and discard the pellet.
5

Determine the concentration of protein in your extract using a Bradford or BCA assay.

6

Aliquot supernatant into several tubes.

  • If not using immediately, store samples at -80 °C.

Stage 2 - Antigen Coating and Blocking

Now your samples are prepared, the antigen needs to be attached to the wells of the microplate so that it can be recognized by detection antibodies.

Materials required

  • Your sample
  • Your controls and standards
  • Coating buffer containing carbonates (for example ab210899)
  • Blocking buffer (for example ab126587)
  • Wash buffer (for example ab206977)
  • Microplate (for example ab210903)
  • A cover for the microplate (some plates come with seals, or you can use adhesive plastic film)
  • Automatic wash system (optional)

Steps

1

Dilute your samples and standards in coating buffer to the desired concentration.

  • Ensure the antigen concentration is within the expected dynamic range of the assay. If using an Abcam ELISA kit, the dynamic range will be stated.
  • Generally the antigen concentration should be < 20 µg / mL to avoid high background.
2

Adsorb samples to the wells.

  • Add 100 µL of your diluted antigen to the wells and cover the plate.
  • Adsorb with gentle agitation for 2 h at room temperature, or at 4 °C overnight.
3

Wash each well three times with wash buffer.

  • If washing manually, remove the solution from wells after each wash by flicking over a sink.
  • Alternatively, use an automatic wash system.
4

Perform background blocking.

  • Add 200 µL of blocking buffer to each well and cover the plate.
  • Block with gentle agitation for 1 – 2 h at room temperature, or at 4°C overnight.
5

Wash each well three times with wash buffer.

  • If washing manually, remove the solution from wells after each wash by flicking over a sink.
  • Alternatively, use an automatic wash system.

Stage 3 - Antibody incubation

Now that antigens are adsorbed onto the plate, you're ready to add antibodies to detect them. This can be done through indirect or direct methods.

Materials required

  • Primary antibody
  • Conjugated secondary antibody
  • Blocking buffer (for example ab126587)
  • Wash buffer (for example PBST)
  • ELISA plate with your samples adsorbed

Steps

1

Dilute the primary and secondary antibodies in blocking buffer.

  • Optimum dilutions will often be suggested on the antibody datasheet.
2

Add primary antibody to the wells.

  • Add 100 µL of pre-diluted primary antibody to each well and cover the plate.
  • Incubate for 2 h at room temperature or overnight at 4 °C.
3

Wash each well three times with wash buffer.

  • If washing manually, remove the solution from wells after each wash by flicking over a sink.
  • Alternatively, use an automatic wash system.
4

Add secondary antibody to the wells

  • Add 100 µL of pre-diluted conjugated secondary antibody to each well and cover the plate.
  • Incubate for 2 h at room temperature or overnight at 4 °C.
5

Wash each well three times with wash buffer.

  • If washing manually, remove the solution from wells after each wash by flicking over a sink.
  • Alternatively, use an automatic wash system.

Stage 4 - Detection

ELISA typically uses antibodies conjugated with enzymes such as horseradish peroxidase (HRP). These react with a substrate in oxidizing conditions to produce either a colored or fluorescent product. The signal generated is proportional to the concentration of the protein of interest. This signal can be measured at several time points throughout the substrate incubation (kinetic mode), or at a defined point in time after the reaction is complete (end-point mode).

Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the two most widely used enzymes for detection in ELISA.

Consider that some biological materials have high levels of endogenous enzyme activity (such as high AP activity in alveolar cells or high peroxidase activity in red blood cells) that may result in a nonspecific signal. If necessary, perform an additional blocking treatment with levamisol (for AP) or 0.3% H2O2 in methanol (for peroxidase).

Materials required

  • Your samples and standards in a microplate
  • Plate shaker
  • Enzyme substrate (for example for HRP: TMB ab171523 or Stoplight Red)
  • Stop solution (for example TMB stop solutions: ab171529 or ab171531)
  • Plate reader

Steps

1

Set up your plate reader to observe the color change or fluorescence at the expected wavelength.

  • If using kinetic mode, configure the plate reader to detect at specific time intervals.
2

Bring all reagents to room temperature.

  • Allow around 10 min for all wells to equilibrate to room temperature.
3

Add the enzyme substrate solution to each well.

  • Add 50 – 100 µL of enzyme substrate to each well and incubate with gentle agitation on a plate shaker, as directed by the manufacturer. See below for specific substrates that can be used.
HRP substratesThe substrate for HRP is hydrogen peroxide. The cleavage of hydrogen peroxide is coupled to the oxidation of a hydrogen donor, which changes color during the reaction 
TMB (3,3’,5,5’-tetramethylbenzidine)Add TMB solution to each well, incubate for 15–30 min, add an equivalent volume of stopping solution (2 M H2SO4), and measure the optical density at 450 nm 
OPD (o-phenylenediamine dihydrochloride)Keep and store the substrate in the dark, as it is light-sensitive. Add the OPD solution and incubate for 10-30 min. The reaction can be stopped using sulfuric acid 2.5-3.0 M H2SO4. For stopped reactions, measure the reaction using a plate reader at 450 nm; for reactions that are not stopped, measure the reaction at 490 nm. 
ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt)
ABTS is an HRP substrate. Add ABTS solution to each well and incubate for 20 min on a rocker. If color generation occurs rapidly, then reduce the concentration of the conjugated antibody in the assay. Do not dilute this solution. This highly sensitive substrate produces a green product. The end product is measured at 405 nm. Always handle carefully and wear gloves as some enzyme substrates are considered hazardous (potential carcinogens) 
   
AP substrateP-Nitrophenyl-phosphate (pNPP) is the most widely used substrate for most applications. Measure the yellow color of nitrophenol at 405 nm after a 15–30 min incubation at room temperature and stop the reaction by adding an equivalent volume of 0.75 M NaOH. 
4

Add the stop solution to each well

  • Add 100 µL of stop solution to each well. 
  • Shake the plate on a shaker for 1 min to mix.
5

Read the signal development in the plate reader.

  • If reading in kinetic mode, measure the signal at the pre-defined time points during the reaction.
  • If reading in end-point mode, allow the reaction to proceed at room temperature and measure at the end of the time course.
  • For colorimetric detection, you can add a stop solution to terminate the reaction and stabilize the signal.

Stage 5 - Data Analysis

Here we have provided step-by-step best-practice guidelines to analyzing data for quantitative ELISAs.

Materials required

  • Plate reader with curve-fitting software

Tip: If the plate reader does not come with software, you can use a general statistical analysis tool such as GraphPad Prism

An in-depth guide on the analysis of data from ELISA can be found here.

Steps

1

Plot the standard curve from your standard controls using curve-plotting software.

  • Plot concentration of standards used on the x-axis.
  • Plot the signal minus any blanks on the y-axis.
2

Determine the curve fit and regression coefficient.

  • Take note of the regression coefficient (R2) and the equation generated from the curve-fitting model.
  • Acceptable R2  > 0.99.
3

Perform a spike recovery test.

  • Compare the signals of standards in buffer against standards spiked in sample matrix. The difference between the signals, expressed as a percentage, gives sample recovery.
  • Acceptable recovery range = 80 – 120 % 
4

Calculate the coefficient of variation.

  • Calculate the mean (µ) and standard deviation (σ) of replicates. Use the following formula to determine the coefficient of variation (CV%) = σ / µ.
  • Acceptable intra-assay CV < 10%.
  • Acceptable inter-assay CV < 15%.
5

Calculate the sample concentration.

  •  Use the equation from the curve-fit generated in Stage 5, Step 2 to determine the concentration of samples.