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
What wavelength should I use to measure ELISA results?
The correct wavelength depends on the enzyme and substrate used in your ELISA assay. For colorimetric ELISAs using horseradish peroxidase (HRP) with TMB substrate, absorbance is typically measured at 450 nm after adding a stop solution. Some protocols also recommend a reference wavelength, such as 570 or 620 nm, to help correct for optical imperfections. Always follow your assay manufacturer’s recommended settings to ensure accurate measurements.
How do I calculate ELISA concentrations from absorbance values?
ELISA concentrations are typically calculated by comparing the absorbance values of unknown samples against a standard curve generated from known concentrations of the target analyte. Curve-fitting models, such as four-parameter logistic (4PL) or five-parameter logistic (5PL) regression, are commonly used to determine sample concentrations. SoftMax® Pro Software simplifies this process by supporting standard-curve generation, automated calculations, and data analysis, reducing the need for manual calculations or transferring data between programs.
What causes high background in an ELISA assay?
High background signals can result from insufficient washing, nonspecific antibody binding, excessive antibody concentrations, or improper blocking conditions. These factors can make it difficult to distinguish true signals from background noise, potentially affecting assay sensitivity and accuracy. To reduce background, optimize washing steps, verify reagent concentrations, and follow the recommended blocking and incubation procedures. Consistent plate handling and appropriate data analysis can also help improve assay reproducibility.
Why are my ELISA results inconsistent?
Inconsistent ELISA results can arise from pipetting variability, uneven washing, differences in incubation times or temperatures, reagent preparation, or plate-handling errors. These factors can introduce variability between wells, plates, or experiments. Following standardized assay protocols, using appropriate controls, and maintaining consistent measurement conditions can help improve reproducibility. Microplate readers paired with data analysis software, such as SpectraMax® readers and SoftMax Pro Software, can also help establish repeatable acquisition and analysis workflows.
What is a standard curve in ELISA, and why is it important?
An ELISA standard curve shows the relationship between known concentrations of an analyte and the corresponding assay signal. Researchers use this relationship to estimate the concentration of the analyte in unknown samples. A well-performing standard curve is essential for reliable quantitative results. Factors such as standard preparation, assay conditions, and the selected curve-fitting model can influence accuracy. SoftMax Pro Software supports standard-curve generation, curve fitting, and sample concentration calculations to help researchers analyze their ELISA data efficiently.
What is the difference between an ELISA plate reader and a spectrophotometer?
Both instruments can measure absorbance, but they are designed for different workflows. A conventional cuvette-based spectrophotometer typically measures samples individually, while an ELISA microplate reader measures multiple samples arranged in a microplate, commonly in 96- or 384-well formats. Microplate readers are particularly useful for ELISA because they support simultaneous analysis of multiple samples, standards, and controls while reducing the need for individual sample handling. Absorbance readers, such as the SpectraMax® ABS Plus, are designed for colorimetric assays, while multi-mode readers offer additional detection capabilities for laboratories performing a broader range of applications.
Can I use the same microplate reader for ELISA and other assays?
Yes. Many microplate readers support multiple assay types, allowing laboratories to perform ELISA alongside applications such as protein quantification, nucleic acid quantification, cell viability, and microbial growth assays. For laboratories primarily performing colorimetric ELISAs, an absorbance reader may provide the necessary capabilities. Laboratories requiring fluorescence, luminescence, or more advanced detection methods may benefit from a multi-mode reader, such as the SpectraMax® iD3s or iD5e. Choosing a reader that supports both current and anticipated applications can help laboratories adapt as research needs evolve.
How can I improve the reproducibility of my ELISA results?
Improving ELISA reproducibility starts with consistent sample preparation, accurate pipetting, proper washing, controlled incubation conditions, and standardized assay protocols. Using appropriate controls and evaluating variability between replicate measurements can also help identify potential issues. Consistent data acquisition and analysis are equally important. Using a microplate reader with integrated analysis software can help researchers apply the same measurement settings, calculations, and reporting methods across experiments, reducing avoidable variability in their workflows.
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 best microplate reader for ELISA depends on your detection method, sample throughput, and the other applications your laboratory performs. For routine colorimetric ELISAs, an absorbance microplate reader, such as the SpectraMax® ABS Plus, provides the capabilities needed to measure optical density and quantify assay results. Laboratories that also perform fluorescence- or luminescence-based assays may benefit from a multi-mode reader, such as the SpectraMax® iD3s or iD5e. Consider factors such as supported wavelengths, microplate formats, assay flexibility, and data analysis capabilities when selecting an instrument. SpectraMax readers integrate with SoftMax® Pro Software to support data acquisition, standard-curve 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 assays be automated?
Yes. ELISA workflows can be automated to reduce repetitive manual steps, improve consistency, and support higher sample throughput. Depending on laboratory requirements, automation may include liquid handling, reagent dispensing, plate washing, incubation, and microplate reading. Automated workflows can be particularly valuable for laboratories processing large sample volumes or performing repetitive assays. Molecular Devices offers microplate readers and automation solutions that can support automated or semi-automated ELISA workflows, helping researchers reduce hands-on time and maintain more consistent processes.
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.