ELISA: A Comprehensive Guide to Enzyme-Linked Immunosorbent Assay
- CLYTE research team
- May 30
- 8 min read
Updated: May 31
The Enzyme-Linked Immunosorbent Assay (ELISA) and broader Enzyme Immunoassays (EIA) are indispensable techniques in modern biological research and diagnostics. These powerful and versatile methods allow for the detection and quantification of a wide array of analytes—including proteins, peptides, antibodies, and hormones—with high sensitivity and specificity. Furthermore, specialized ELISA protocols extend this capability to analyze protein expression and modifications directly within cells. Whether you're a seasoned researcher or new to the lab, understanding the nuances of various ELISA protocols is crucial for generating reliable and reproducible data. This guide, drawing from established procedures such as those outlined by Sigma-Aldrich, will walk you through the essential steps and considerations for performing successful ELISAs, including Sandwich, Phosphorylation, general EIA concepts, and In-Cell ELISA procedures.
The Fundamental Principle: Antigen-Antibody Interaction
At its core, every ELISA or EIA leverages the highly specific binding interaction between an antigen and an antibody. Typically, one of these components is immobilized on a solid surface, often a microplate well. The other component, present in the sample, binds to the immobilized molecule. An enzyme, conjugated (linked) to either a primary or secondary antibody, then catalyzes a chromogenic, chemiluminescent, or fluorescent reaction when an appropriate substrate is added. The intensity of the resulting signal is directly proportional to the amount of analyte present, allowing for quantitative measurement.
Navigating the Different Types of ELISA and EIA
Several ELISA formats have been developed, each with its own advantages and specific applications. EIA is a broader term that encompasses various enzyme-mediated immunoassays, with ELISA being a prominent type. The choice of assay format depends on the analyte of interest, the availability of specific antibodies, and the required sensitivity and context (e.g., purified protein vs. cellular analysis).
The main types include:
Direct ELISA: The antigen coated on the plate is directly detected by an enzyme-conjugated primary antibody.
Indirect ELISA: An unconjugated primary antibody binds the antigen, followed by an enzyme-conjugated secondary antibody.
Sandwich ELISA: The antigen is "sandwiched" between a capture antibody (coated on the plate) and a detection antibody. This is highly specific and common.
Competitive ELISA: Measures analyte concentration by detecting interference in binding between labeled and unlabeled antigen to a limited amount of antibody.
Phosphorylation ELISA: A specialized assay (often Sandwich or In-Cell format) using antibodies specific for phosphorylated forms of proteins.
In-Cell ELISA: Allows for the detection and quantification of intracellular proteins directly within fixed and permeabilized cells cultured in microplates.
Detailed Enzyme-Linked Immunosorbent Assay Procedures
Here, we delve into the specific steps for some of the most commonly employed ELISA protocols.
1. General Sandwich ELISA Protocol
This protocol is widely applicable for quantifying soluble analytes and forms the basis for many commercial ELISA kits.
Plate Coating (Capture Antibody):
Dilute the capture antibody to its optimal concentration in coating buffer (e.g., PBS, carbonate-bicarbonate buffer).
Add 50-100 µL of diluted capture antibody to each well of a high-binding 96-well microplate.
Incubate for 1-2 hours at 37°C or overnight at 4°C.
Aspirate coating solution and wash wells 3-4 times with 200-300 µL/well of wash buffer (e.g., PBS with 0.05% Tween-20, PBS-T).
Blocking:
Add 200-300 µL of blocking buffer (e.g., 1-5% BSA in PBS-T, or a commercial blocker) to each well.
Incubate for 1-2 hours at room temperature or 37°C, or overnight at 4°C.
Aspirate blocking buffer and wash wells 3-4 times with wash buffer.
Sample and Standard Incubation:
Prepare serial dilutions of your standards and samples in assay diluent (often the blocking buffer or a specific reagent).
Add 100 µL of standards and samples to appropriate wells (run in duplicate or triplicate). Include blank wells.
Cover plate and incubate for 1-2.5 hours at room temperature or 37°C (or as recommended, sometimes overnight at 4°C), often with gentle shaking.
Aspirate samples/standards and wash wells 3-4 times.
Detection Antibody Incubation:
Dilute the (often biotinylated) detection antibody in assay diluent.
Add 100 µL of diluted detection antibody to each well.
Cover plate and incubate for 1-2 hours at room temperature or 37°C with shaking.
Aspirate detection antibody and wash wells 3-4 times.
Enzyme Conjugate (e.g., Streptavidin-HRP) Incubation:
If using a biotinylated detection antibody, dilute enzyme-conjugated streptavidin (e.g., Streptavidin-HRP) in assay diluent.
Add 100 µL of diluted enzyme conjugate to each well.
Cover plate and incubate for 20 minutes to 1 hour at room temperature with shaking (protect from light if using HRP).
Aspirate enzyme conjugate and wash wells 3-5 times.
Substrate Development:
Add 100 µL of enzyme substrate (e.g., TMB for HRP) to each well.
Incubate at room temperature (e.g., 5-30 minutes for TMB), protected from light, until desired color develops.
Stopping the Reaction:
Add 50-100 µL of stop solution (e.g., 1 M H₂SO₄ or 0.5 M HCl for TMB/HRP) to each well. Color may change (e.g., TMB turns yellow).
Gently tap plate to ensure mixing.
Reading Absorbance:
Read absorbance within 30 minutes using a microplate reader at the appropriate wavelength (e.g., 450 nm for TMB). A reference wavelength (e.g., 570-630 nm) can be used for background subtraction.
2. Phosphorylation Assay Procedure
Phosphorylation ELISAs are designed to detect and quantify specific phosphorylated proteins. These assays are crucial for studying signal transduction pathways.
Principle: They typically utilize the Sandwich ELISA format. One antibody captures the total protein (phosphorylated and unphosphorylated), and a second antibody, specific for the phosphorylated epitope on the target protein, is used for detection. Alternatively, an In-Cell ELISA format can be used for analyzing phosphorylated proteins within cells.
Key Reagents: The critical components are highly specific antibodies:
A capture antibody that binds the target protein regardless of its phosphorylation state (for Sandwich ELISA).
A detection antibody that specifically recognizes the phosphorylated form of the target protein (this is often enzyme-conjugated or biotinylated).
For In-Cell ELISAs, primary antibodies target either the total protein or the phospho-specific form.
Procedure:
For Sandwich ELISA format: Follow the "General Sandwich ELISA Protocol" detailed above, ensuring the capture and detection antibodies are appropriate for the total and phosphorylated protein of interest, respectively. Pay close attention to lysis buffer recommendations if extracting proteins from cells/tissues, ensuring phosphatase inhibitors are included to preserve phosphorylation states.
For In-Cell ELISA format: Follow the "In-Cell ELISA Protocol" detailed below, using a primary antibody specific for the phosphorylated target. Often, results are normalized to the total amount of target protein, measured in parallel wells using an antibody to the non-phosphorylated protein or a housekeeping protein.
3. EIA Assay Considerations
Enzyme Immunoassay (EIA) is a broad term referring to any immunoassay that uses an enzyme-linked antibody or antigen to detect the target analyte. ELISA is the most common type of EIA performed in microplates.
Procedural Overlap: The specific steps for an "EIA" will depend on its format (e.g., direct, indirect, sandwich, competitive). Therefore, the "General Sandwich ELISA Protocol" or protocols for Direct/Indirect ELISAs (which involve similar coating, blocking, incubation, and detection steps, but with fewer antibody layers) are all examples of EIA procedures.
Key Elements Common to EIAs:
Immobilization of an antigen or antibody to a solid phase.
Binding of the sample analyte to the immobilized component.
Binding of an enzyme-conjugated antibody (or subsequent binding of an enzyme conjugate).
Addition of an enzyme substrate leading to a measurable signal.
Washing steps between incubations to remove unbound materials.
Essentially, the detailed ELISA protocols described herein (Sandwich, In-Cell) are specific instances of EIA procedures. The Sigma-Aldrich source primarily details these specific ELISA methodologies as its main EIA protocols.
4. In-Cell ELISA Assay Procedure
In-Cell ELISAs (ICE) allow for the quantification of intracellular protein levels (including phosphorylated proteins) directly in cells cultured in microplates, eliminating the need for cell lysis and protein extraction for some applications.
Cell Culture and Treatment:
Seed cells (e.g., 10,000-50,000 cells/well, depending on cell type) in 100-200 µL of culture medium in a 96-well tissue culture-treated microplate.
Culture cells until desired confluency.
Apply experimental treatments (e.g., drug compounds, growth factors) as required. Include appropriate controls.
Cell Fixation:
Remove culture medium.
Add 100-150 µL of fixative (e.g., 4% formaldehyde in PBS) to each well.
Incubate for 15-20 minutes at room temperature.
Aspirate fixative. Wash cells 3-5 times with 200 µL/well of wash buffer (e.g., PBS). Ensure cells are not allowed to dry out.
Cell Permeabilization & Blocking (Often Combined):
Add 100-150 µL of permeabilization/blocking buffer (e.g., PBS with 0.1-0.5% Triton X-100 and 1-5% BSA or serum) to each well.
Incubate for 30-60 minutes at room temperature.
Aspirate buffer. Wash cells 3-5 times with wash buffer (or PBS with 0.1% Tween-20).
Primary Antibody Incubation:
Dilute primary antibody (specific for the target protein or its phosphorylated form) in antibody dilution buffer (often the permeabilization/blocking buffer or a specialized diluent).
Add 50-100 µL of diluted primary antibody to each well.
Incubate for 1-3 hours at room temperature or overnight at 4°C with gentle agitation.
Aspirate primary antibody. Wash cells 3-5 times extensively.
Secondary Antibody Incubation:
Dilute enzyme-conjugated secondary antibody (e.g., HRP-conjugated anti-mouse IgG) in antibody dilution buffer.
Add 50-100 µL of diluted secondary antibody to each well.
Incubate for 1 hour at room temperature with gentle agitation, protected from light if HRP-conjugated.
Aspirate secondary antibody. Wash cells 3-5 times extensively.
Substrate Development:
Add 100 µL of enzyme substrate (e.g., TMB for HRP) to each well.
Incubate at room temperature, protected from light, until sufficient color develops (typically 5-30 minutes).
Stopping the Reaction:
Add 50-100 µL of stop solution (e.g., 1 M H₂SO₄) to each well.
Reading Absorbance:
Read absorbance as per the Sandwich ELISA protocol (e.g., 450 nm for TMB).
Normalization (Optional but Recommended): To account for cell number variations, results can be normalized. This can be done by:
Staining cells with a total protein stain (e.g., Janus Green) after absorbance reading, then eluting the stain and reading its absorbance.
Parallel wells processed with an antibody against a housekeeping protein.
Data Analysis and Interpretation
For all ELISA types yielding quantitative data, a standard curve is generated by plotting the absorbance values of the standards against their known concentrations (for Sandwich/Competitive ELISA) or by normalizing signals (for In-Cell ELISA, often relative to controls or total protein). This curve is then used to interpolate the concentrations or relative expression levels of the analyte in the unknown samples. Various curve-fitting models (e.g., linear, log-log, four-parameter logistic) should be chosen based on the assay's characteristics.
Key Considerations for Optimal ELISA Performance
Reagent Quality & Specificity: Use high-quality, validated antibodies and reagents. For phosphorylation or In-Cell ELISAs, antibody specificity is paramount.
Temperature Control: Consistency in incubation temperatures is key.
Pipetting Accuracy: Crucial for reproducibility, especially with small volumes.
Washing Technique: Thorough washing is vital to minimize background. For In-Cell ELISAs, be gentle to avoid detaching cells.
Cell Handling (for In-Cell ELISA): Maintain cell viability until fixation. Ensure consistent cell seeding and avoid cell layer disruption during washes.
Controls: Always include appropriate controls: blanks, standards, positive/negative experimental controls. For In-Cell ELISAs, include untreated cells, and consider wells with secondary antibody only to check for non-specific binding.
Optimization: Incubation times, antibody concentrations, and blocking reagents may need optimization for your specific system.
The Enduring and Evolving Value of ELISA/EIA
ELISA and EIA techniques, in their diverse formats from traditional Sandwich assays to sophisticated In-Cell analyses, remain indispensable tools in research and clinical settings. Their sensitivity, specificity, and adaptability make them suitable for a vast range of applications. By understanding the underlying principles and diligently following optimized protocols like those discussed, researchers can harness the full potential of these immunoassays to generate accurate, meaningful data, advancing scientific knowledge, drug discovery, and health outcomes.
References
Sigma-Aldrich. ELISA Protocols. https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/elisa/elisa-protocols
