Enzyme-Linked Immunosorbent Assay (ELISA)
An overview of ELISA principles, assay formats, and plate‑based workflows
What is ELISA?
ELISA is a plate-based immunoassay used to detect and quantify specific antigens or antibodies. It uses antibody-antigen binding and an enzyme-linked signal measured by a microplate reader.
How does ELISA work?
In an enzyme-linked immunosorbent assay (ELISA), antibodies bind to a target antigen or an antigen binds to antibodies in the sample, depending on the assay format. An enzyme-linked detection system then produces a colorimetric, fluorescent, or luminescent signal. The intensity of that signal is measured with a microplate reader and used to determine the presence or concentration of the target analyte.
ELISA is widely used in research, clinical, biopharmaceutical, food-safety, and environmental-testing laboratories to measure proteins, antibodies, hormones, toxins, and other analytes. Samples may include cell lysates, serum, plasma, food products, and environmental specimens.
The four primary ELISA formats are direct, indirect, sandwich, and competitive ELISA. Each uses a different antibody-binding strategy and offers different considerations for sensitivity, specificity, assay complexity, and antibody requirements. The diagrams below illustrate how the target analyte and antibodies interact in each format.
ELISA data can be acquired using microplate readers and analyzed with SoftMax® Pro Software, which supports assay setup, standard-curve generation, data visualization, quantitative analysis, and reporting.
What are the four types of ELISA?
The four main types of ELISA are direct, indirect, sandwich, and competitive ELISA. They differ in how antibodies and antigens interact, the number of antibodies required, and how the signal relates to the amount of target present.
Example workflow: steps in a sandwich ELISA assay
Most sandwich ELISAs are performed in microplates, where the bottom of each well serves as a solid surface for antibody binding. During the assay, wash steps remove non-specific material, and an enzyme-linked detection reaction produces a measurable signal when the target antigen is present. Results are typically measured using a microplate reader and analyzed to generate standard curves and calculate antigen concentrations.
The illustration below shows an example workflow for a typical sandwich ELISA assay.
The steps below outline a typical sandwich ELISA workflow used in plate‑based laboratory assays. Specific protocols may vary depending on the assay design and reagents used.
Step 1: Capture antibody binds to the microplate well surface.
Step 2: Add sample — antigen present in the sample binds to the capture antibody.
Step 3: Wash microplate — unbound material is removed, leaving only the antigen of interest.
Step 4: Add detection antibody — an enzyme‑conjugated detection antibody binds to a second site on the antigen.
Step 5: Wash microplate — unbound antibodies are removed, leaving only antigen‑specific complexes.
Step 6: Add substrate — the enzyme on the detection antibody converts the substrate, producing a detectable signal.
Step 7: Read microplate — a microplate reader detects the signal and measures optical density (OD).
Step 8: Calculate results — antigen concentration is determined using a standard curve and data analysis tools commonly integrate with microplate reader software.
While ELISA workflows are relatively straightforward to perform, they typically involve multiple incubation and wash steps that can be time‑intensive. In laboratory settings where higher sample volumes are required, automation is often used to support plate‑based ELISA workflows by reducing manual handling and improving consistency across assays.
ELISA assays and applications
ELISA (enzyme-linked immunosorbent assay) is a widely used analytical technique in research and testing laboratories for detecting and quantifying specific analytes. The resources below provide examples of ELISA assay types, workflows, and application areas, along with related application notes, educational content, and technology overviews.
FAQs
How do I choose the right ELISA format?
The best ELISA format depends on the target analyte, available antibodies, sensitivity requirements, and assay complexity. Sandwich ELISAs are commonly used when high specificity and sensitivity are required, while competitive ELISAs are often used for small molecules or targets with limited antibody options. Once the assay format is selected, Molecular Devices SpectraMax® microplate readers can support common ELISA readout workflows depending on the detection method.
Which microplate reader is best for ELISA assays?
The ideal microplate reader depends on the assay detection method. Absorbance readers are commonly used for colorimetric ELISAs, while multimode readers support absorbance, fluorescence, and luminescence detection for labs that need broader assay flexibility. Molecular Devices SpectraMax® microplate readers support a range of ELISA workflows and can be paired with SoftMax® Pro Software for acquisition, analysis, and reporting.
What is the difference between colorimetric, fluorescent, and luminescent ELISA detection?
Colorimetric ELISAs generate a color change measured by absorbance, fluorescent ELISAs use fluorescent labels for increased sensitivity, and luminescent ELISAs produce light through a chemical reaction. The right method depends on assay sensitivity, dynamic range, and instrumentation needs. SpectraMax® readers can support ELISA workflows across common detection methods when configured for the required read mode.
Can ELISA be automated?
Yes. ELISA workflows can be automated with liquid handling systems, plate washers, incubators, and microplate readers. Automation can help improve throughput, consistency, and reproducibility while reducing manual handling. Molecular Devices SpectraMax® readers can fit into automated or semi-automated ELISA workflows depending on the lab setup and integration needs.
When should researchers use ELISA instead of other immunoassays?
ELISA is often used when researchers need a sensitive, scalable, and quantitative method for measuring specific analytes across many samples. The best assay choice depends on study goals, throughput needs, sensitivity requirements, and available instrumentation. For labs running plate-based immunoassays, SpectraMax® microplate readers and SoftMax® Pro Software can support ELISA data acquisition and quantitative analysis workflows.
Latest Resources
Workflow of an ELISA protocol
The workflow of a typical sandwich ELISA protocol has multiple reagent addition, incubation and wash steps. Here we’ve highlighted each step and the instrumentation and tools needed to conduct the ELISA assay including a microplate washer, absorbance ELISA plate reader and software.
Workflow: ELISA Assay Protocol
CAPTURE ANTIBODY BINDS TO WELLS
First, the capture antibody is bound to the bottom of the microplate well.
ELISA Plate
Most ELISAs are run in 96- or 384- well microplates, a 96-well plate being the most common and sometimes referred to as an ELISA plate. The bottom of the microplate wells serve as the solid surface to which antibodies and other reagents attach. Microplates are typically included in an ELISA kit.
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ADD SAMPLE
Sample is added to the well, and antigen within the sample binds to the capture antibody.
WASH MICROPLATE
Unbound material is washed away, leaving only the antigen ofinterest and minimizing the potential for high backgroundsignal.
https://vids.moleculardevices.com/watch/db2tLDgnn3Bk9GpUSQ4VzQ
Microplate Washer
The MultiWash+™ Microplate Washer is a 96- and 384-well automated washer–compact, quiet, and efficient washer with 20 different wash protocol options.
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Save time and money with a SpectraMax Microplate Reader, SoftMax Pro Software, and an AquaMax Microplate Washer bundle.
ADD DETECTION ANTIBODY
Enzyme-conjugated detection antibody binds to a second site on the antigen of interest, providing the means to detect the antigen.
WASH MICROPLATE
Unbound antibodies are washed away, leaving only those specific for the target of interest and again minimizing the potential for background signal.
https://vids.moleculardevices.com/watch/vGfoof4ysIDAd4XGwxqokQ
ELISA Plate Washer
The AquaMax Microplate Washer: Aspiration and dispensing of 96- and 384-wells occur simultaneously in all wells leading to high-precision assays and faster microplate processing without mechanical plate indexing or quadrant processing.
ADD SUBSTRATE
Substrate is converted by the enzyme on the detection antibody, producing a color change, with intensity proportional to the amount of antigen present.
Depending on the enzyme and substrate used, the readout can also be fluorescent or luminescent.
READ PLATE
The microplate reader detects the colored reaction product and outputs optical density (OD) values that indicate how much light is absorbed by the contents of each well.
https://vids.moleculardevices.com/watch/2tKEp18eNj6v1e31bfJwAe
ELISA Plate Reader
An ELISA plate reader, like the SpectraMax ABS Plus Absorbance ELISA Microplate Reader, detects the color change produced when target antigen is present. It does so by measuring how much of the light passed through the wells of the microplate is absorbed by the material within the wells. The more antigen is present, the higher the absorbance value.
https://vids.moleculardevices.com/watch/2tKEp18eNj6v1e31bfJwAe
ELISA Plate Reader Software
An ELISA plate reader software, like our SoftMax Pro data analysis software, is used to plot standard curves and calculate results from the absorbance values provided by the microplate reader.
A standard curve is run so that the amount of antigen in each sample can be accurately calculated. A preconfigured protocol, helps save time by calculating results automatically.
CALCULATE RESULTS
The amount of antigen in each sample is calculated, and different samples—for example, cells subjected to different treatment conditions—can be compared.
Automate your workflow
We offer scalable, automation-ready workcells ranging from entry-level to advanced, and can be customized to meet your specific research needs
https://vids.moleculardevices.com/watch/EsYUk5mwGne1KefZnMGZjq
Automation-ready ELISA workcells
Automating an ELISA workflow can take the tedious steps of sample prep, reagent pipetting, and microplate washing out of your hands and can improve the reproducibility of your results. An automated ELISA workflow can be paired with control software and components, creating scalable, high-throughput workcells.