Indirect and direct ELISA

This indirect and direct ELISA protocol provides a detailed guide for researchers aiming to detect and quantify proteins using enzyme-linked immunosorbent assays. This protocol outlines sample preparation, coating, blocking, detection, and analysis for both direct and indirect ELISA formats. It supports a wide range of sample types, including cell lysates, tissue extracts, and culture media. With clear instructions and recommended reagents, the protocol ensures reproducibility and sensitivity. Whether you're new to ELISA or optimizing your workflow, this guide helps streamline your assay setup and improve data quality. It’s ideal for applications in biomarker detection, antibody screening, and protein expression analysis.

Introduction

ELISA (enzyme-linked immunosorbent assay) is a cornerstone technique in immunology and molecular biology for detecting specific proteins or antigens. This protocol focuses on two common ELISA formats: direct and indirect. These methods differ in how the target is detected but share core steps such as antigen immobilization and signal generation. The protocol is designed to help researchers achieve high specificity and sensitivity in their assays. It includes practical tips for sample handling, reagent preparation, and plate washing.

Background and principles

ELISA works by immobilizing an antigen on a solid surface, typically a microplate, and detecting it using antibodies linked to enzymes. In direct ELISA, a labeled primary antibody binds directly to the antigen. In indirect ELISA, an unlabeled primary antibody binds the antigen, followed by an enzyme-conjugated secondary antibody. The enzyme reacts with a substrate to produce a measurable signal, often a color change. This signal correlates with the amount of antigen present. The indirect format offers signal amplification and greater flexibility, while the direct format is faster and reduces background. Both rely on high-affinity antibody-antigen interactions for specificity.

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

Prepare the extraction buffer as recommended by the manufacturer.

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

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.

Lyse the cells.

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

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.

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

Aliquot supernatant into several tubes.

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

Materials required

• Your sample in cell culture
• Microcentrifuge

Steps

Collect the cell culture media, spin down in a centrifuge and remove the pellet.

• Centrifuge at 1,000 – 10,000 x g for 10 min at 4°C.
• Keep the supernatant and discard the pellet.

Aliquot supernatant into several tubes.

• Aliquots should have a minimum volume of 50 µL.
• If not using immediately, store samples at -80°C.

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

Prepare the extraction buffer as recommended by the manufacturer.

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

Dissect the tissue with clean tools on ice, as quickly as possible to prevent degradation by proteases.

Add extraction buffer to the dissected tissue.

• For a ~ 5 mg piece of tissue, add 300 µL of extraction buffer.

Homogenize the suspension with an electric homogenizer.

Lyse the suspension.

• Agitate for 2 h at 4°C.

Spin down suspension in a centrifuge to pellet insoluble contents.

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

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

Aliquot extract into several tubes.

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

Materials required

• Your sample
• An assay-validated anti-coagulant (such as sodium citrate, EDTA or heparin)
• Microcentrifuge

Steps

Collect blood samples in tubes with anti-coagulant.

• Anti-coagulant should be diluted to a final concentration of 0.1 M.

Spin down samples in a centrifuge and remove the pellet.

• Centrifuge at 1,000 – 10,000 x g for 10 min at 4°C.
• Keep the supernatant and discard the pellet.

Aliquot the supernatant into several tubes.

• Aliquots should have a minimum volume of 50 µL.
• If not using immediately, store samples at -80°C.

Materials required

• Your sample
• Microcentrifuge

Steps

Collect samples in untreated tubes and leave undisturbed at room temperature for 20 min.

Spin down samples in a centrifuge and remove the pellet.

• Centrifuge at 1,000 – 10,000 x g for 10 min at 4°C.
• Keep the supernatant and discard the pellet.

Aliquot the supernatant into several tubes.

• Aliquots should have a minimum volume of 50 µL.
• If not using immediately, store samples at -80°C.

Materials required

• Your samples
• Microcentrifuge

Steps

Collect the samples, spin down in a centrifuge and remove the pellet.

• Centrifuge at 1,000 – 10,0000 x g for 2 min at 4°C.
• Keep the supernatant and discard the pellet.

Aliquot supernatant into several tubes.

• Aliquots should have a minimum volume of 50 µL.
• 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

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.

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.

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.

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.

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

Dilute the primary and secondary antibodies in blocking buffer.

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

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.

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.

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.

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.

Materials required

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

Steps

Dilute the primary antibody in blocking buffer.

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

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.

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

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.

Bring all reagents to room temperature.

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

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.

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.

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

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.

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.

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%

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%.

Calculate the sample concentration.

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

Comparison to other methods

Direct and indirect ELISA are simpler than sandwich ELISA and require fewer antibodies. However, sandwich ELISA offers higher specificity and sensitivity, especially for complex samples. Western blotting provides protein size information but is more time-consuming and less quantitative. Immunohistochemistry allows spatial localization of proteins but lacks the throughput of ELISA. Flow cytometry enables multi-parameter analysis but requires specialized equipment. Direct ELISA is faster but less sensitive than indirect ELISA, which benefits from signal amplification. Overall, direct and indirect ELISA strike a balance between simplicity, cost, and performance, making them ideal for many routine protein detection tasks.

Applications

Direct and indirect ELISA are widely used in biomedical research, diagnostics, and drug development. Common applications include quantifying cytokines, hormones, and antibodies in biological samples. These methods are also used for vaccine development, allergen detection, and monitoring immune responses. In clinical settings, ELISA helps diagnose infections, autoimmune diseases, and cancer biomarkers. In research, it supports protein expression studies, epitope mapping, and antibody screening. The protocol’s flexibility allows adaptation to various sample types, including serum, plasma, cell lysates, and tissue extracts. Its compatibility with high-throughput formats makes it suitable for large-scale screening and reproducible quantitative analysis.

Limitations

While direct and indirect ELISA are versatile, they have limitations. Direct ELISA may suffer from lower sensitivity due to the lack of signal amplification. Indirect ELISA, while more sensitive, can introduce background noise from secondary antibodies. Both formats require high-quality antibodies and careful optimization of blocking and washing steps to reduce non-specific binding. They are less suitable for detecting low-abundance targets in complex matrices compared to sandwich ELISA. Additionally, ELISA does not provide information on protein size or localization. Cross-reactivity and matrix effects can also impact accuracy. Despite these challenges, proper protocol adherence can mitigate most issues.

Troubleshooting

Common ELISA issues include weak or no signal, high background, and inconsistent results. Weak signals may result from insufficient antigen coating, degraded antibodies, or incorrect incubation times. High background often stems from inadequate blocking, poor washing, or non-specific antibody binding. Ensure all reagents are fresh and stored properly. Use recommended buffers and follow incubation times precisely. If variability occurs, check pipetting accuracy and plate uniformity. For indirect ELISA, verify the specificity of both primary and secondary antibodies. Running controls and optimizing antibody concentrations can help resolve most problems. Abcam’s protocol includes tips to improve assay performance and reproducibility.