ELISA (enzyme-linked immunosorbent assay) is a plate-based technique for detecting and quantifying a specific protein, antibody, hormone, or other biomolecule in a liquid sample. It is one of the most widely used assays in a research lab because it is relatively cheap, scales to 96 or 384 samples at once, and doesn't require specialized imaging equipment beyond a plate reader. This guide walks through the four common ELISA formats, what you need before you start, and a step-by-step protocol you can adapt to any of them.
What ELISA Measures and When to Use It
ELISA works by immobilizing either an antigen (the molecule you're testing for) or a capture antibody on a solid surface — almost always the wells of a polystyrene microplate — and then using an enzyme-linked antibody to generate a signal proportional to how much of the target is present. The enzyme (commonly horseradish peroxidase or alkaline phosphatase) converts a colorless substrate into a colored, fluorescent, or chemiluminescent product, and a plate reader measures the signal in each well.
Researchers reach for ELISA when they need to quantify a soluble analyte across many samples — measuring a cytokine concentration across dozens of serum samples, screening hybridoma supernatants for antibody titer, or confirming seroconversion in an infection or vaccination study. It is not the right tool when you need to confirm a protein's molecular weight or check for degradation products; for that, see Western Blot Protocol Basics.
The Four Main ELISA Formats
The steps below vary slightly depending on which format you run. All four use the same core logic — immobilize, incubate, wash, detect — but differ in what's coated on the plate and how the signal is generated.
- Direct ELISA: the antigen is coated directly on the plate, and an enzyme-linked primary antibody binds it directly. Fastest format (no secondary antibody step) but the least sensitive, since there's no signal amplification.
- Indirect ELISA: the antigen is coated on the plate, an unlabeled primary antibody binds it, and an enzyme-linked secondary antibody (raised against the primary's host species) generates the signal. More sensitive than direct ELISA because multiple secondary antibodies can bind each primary, and it's the standard format for detecting a patient or animal's antibody response (e.g., serology testing).
- Sandwich ELISA: a capture antibody is coated on the plate, the sample is added and the target antigen binds it, then a second, enzyme-linked detection antibody binds a different epitope on the same antigen. The most sensitive and specific format because the analyte is recognized twice, but it requires two antibodies against non-overlapping epitopes of the same target.
- Competitive ELISA: the sample and a known, labeled antigen compete for a limited number of antibody binding sites; signal is inversely proportional to the amount of analyte in the sample. Used for small molecules (hormones, drugs, toxins) too small to be bound by two antibodies at once.
Materials and Equipment
- 96-well (or higher-density) ELISA microplate, typically high-binding polystyrene
- Coating antigen or capture antibody, diluted in coating buffer (commonly carbonate-bicarbonate buffer, pH ~9.6, or PBS)
- Blocking buffer (BSA, non-fat dry milk, or a commercial blocker in PBS/TBS)
- Primary and, if indirect/sandwich, enzyme-conjugated secondary or detection antibody
- Wash buffer (PBS or TBS with 0.05% Tween-20)
- Enzyme substrate (TMB is standard for HRP conjugates; pNPP for alkaline phosphatase)
- Stop solution (commonly diluted sulfuric or hydrochloric acid for TMB)
- Multichannel pipette and a plate washer or squeeze bottle for consistent, low-variance washing — see Micropipette Types and Uses if you're choosing equipment
- Plate reader capable of reading absorbance at the substrate's detection wavelength (450 nm for TMB after stopping)
Reagent dilutions for coating buffer, wash buffer, and standards all come down to accurate molar and percentage solution prep — see Molarity and Solution Calculations for the Lab for the underlying math, and Serial Dilution Technique for building a standard curve.
Step-by-Step Protocol
1. Plate Coating
Dilute the antigen or capture antibody to the manufacturer- or literature-recommended concentration in coating buffer, and add a consistent volume (typically 50–100 µL) to each well. Incubate either at room temperature for 1–2 hours or, more commonly for better reproducibility, at 4°C overnight. Include enough wells for a standard curve (a dilution series of a known-concentration standard) alongside your unknowns, plus blank wells with no coating antigen to establish background.
2. Blocking
Remove the coating solution and add blocking buffer (typically 200–300 µL per well) to occupy any remaining unbound surface on the plate. Skipping or under-blocking is one of the most common causes of high background signal, because antibodies added later can stick nonspecifically to the bare plastic. Incubate 1–2 hours at room temperature, or per your kit's protocol.
3. Sample and Antibody Incubation
Wash the plate (see step 4) and add diluted samples and standards. For indirect ELISA, follow with a primary antibody incubation; for sandwich ELISA, the sample step and detection-antibody step are separate additions. Incubate for the time and temperature specified for your assay — commonly 1–2 hours at room temperature or overnight at 4°C for maximum sensitivity. If your protocol calls for an enzyme-linked secondary or detection antibody, add and incubate that next, after washing away the previous unbound reagent.
4. Washing
Wash between every incubation step — typically 3–5 washes with wash buffer, fully aspirating or decanting between each wash. Inconsistent, incomplete, or too-few washes are the single most common source of both high background and poor plate-to-plate reproducibility; a plate washer removes most of the well-to-well variability that manual pipetting introduces.
5. Substrate Development and Stop
Add the enzyme substrate and incubate in the dark for the time specified (commonly 5–30 minutes for TMB), watching for visible color development in the standard curve's highest wells. Add stop solution once color development is adequate but before the highest-concentration standards saturate the reader's linear range. Stopping too late is a common cause of a flattened, non-linear standard curve.
6. Reading and Analyzing Results
Read absorbance immediately after stopping (TMB read at 450 nm). Subtract the blank-well average from every reading, plot the standard curve (commonly a four-parameter logistic fit), and interpolate unknown concentrations from it. Run standards and samples in duplicate or triplicate whenever the plate layout allows — a single well doesn't let you catch a pipetting or edge-well artifact.
Common Troubleshooting Issues
- High background across all wells: insufficient blocking, too-concentrated antibody, or inadequate washing. Increase block time/concentration, dilute antibodies further, and add wash steps.
- Weak or no signal: expired or degraded substrate, antigen/antibody concentration too low, or a reagent added in the wrong order. Confirm reagent activity with a known-positive control before troubleshooting the whole plate.
- Edge effect (outer wells reading differently from inner wells): usually evaporation or temperature variation during long incubations. Use a humidified chamber, avoid placing critical samples only in outer wells, or pre-warm plates before starting.
- Non-linear or compressed standard curve: substrate development stopped too late (saturating the reader) or standards diluted incorrectly. Re-check the dilution series math and stop timing.
ELISA vs. Western Blot: When to Use Which
ELISA and Western blot both use an antibody to detect a protein, which is why they’re often confused, but they answer different questions. ELISA is a plate-based, high-throughput method for quantifying how much of a soluble analyte is present across many samples; it doesn’t tell you the molecular weight of the target or whether the antibody is binding something else of a similar size. Western blot separates proteins by size first, so it confirms identity and size but is lower-throughput and only semi-quantitative. Many labs use both: an ELISA to screen or quantify across a sample set, and a Western blot to confirm specificity for a subset of those samples. See Western Blot Protocol Basics: Step-by-Step Walkthrough for the companion protocol.
Frequently Asked Questions
What is ELISA used for?
ELISA is used to detect and quantify a specific antigen, antibody, hormone, cytokine, or other biomolecule in a liquid sample — common applications include serology (antibody-response) testing, cytokine quantification, hybridoma antibody-titer screening, and diagnostic testing for infectious disease markers.
How long does an ELISA take to run?
A same-day ELISA (room-temperature coating and antibody incubations) typically takes 4–6 hours from plate coating to reading. Protocols using an overnight 4°C coating or primary-antibody step (common for maximum sensitivity) effectively span two days, though the active hands-on time is similar.
What’s the difference between direct, indirect, sandwich, and competitive ELISA?
Direct ELISA uses one enzyme-linked antibody against a plate-bound antigen; indirect ELISA adds an unlabeled primary antibody plus an enzyme-linked secondary for signal amplification; sandwich ELISA captures the antigen between two antibodies for the highest specificity and sensitivity; competitive ELISA measures analyte by how much it displaces a labeled competitor from limited antibody binding sites, and is used for small molecules that can’t be bound by two antibodies simultaneously.
Why is my ELISA background so high?
The most common causes are insufficient blocking (too short, too dilute, or skipped entirely), antibody concentrations that are too high, and incomplete washing between steps. Start troubleshooting by extending the block step and adding an extra wash cycle before changing antibody concentrations.
Can ELISA be automated?
Yes — liquid handlers and automated plate washers are common in labs running ELISA at scale, and reduce the well-to-well pipetting and wash-timing variability that causes much of the plate-to-plate inconsistency in a manual protocol. For lower-throughput research use, a calibrated multichannel pipette and a basic plate washer cover most needs; see Pipette Calibration: How and When to Calibrate Lab Pipettes to keep manual results reproducible.
