Enzyme-Linked Immunosorbent Assay (ELISA)

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What is ELISA?

ELISA (enzyme-linked immunosorbent assay) is a method used to quantitatively detect an antigen within a sample. An antigen is a toxin or other foreign substance, for example a flu virus or environmental contaminant, that causes the vertebrate immune system to mount a defensive response. The range of potential antigens is vast, so ELISAs are used in many areas of research and testing to detect and quantify antigens in a wide variety of sample types. Cell lysates, blood samples, food items, and more can be analyzed for specific substances of interest using ELISAs.

There are four major types of ELISAs: direct, indirect, competitive and sandwich. Each type is described below with a diagram illustrating how the analytes and antibodies are bonded and used.

Steps to run a sandwich ELISA assay

Most sandwich ELISAs are run in microplates, with the bottom of the plate wells serving as the solid surface to which antibodies and other reagents bind. A microplate washer is used to wash away non-specific material in the wells, and an absorbance ELISA microplate reader detects the color change produced when target antigen is present. And, a plate reader software is used to plot standard curves and calculate results.

The illustration below shows a workflow for a typical sandwich ELISA assay:

ELISA Assay Workflow \

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Step 1: Capture antibody binds to ELISA plate wells

Step 2: Add sample to well – antigen within the sample binds to the capture antibody.

Step 3: Wash microplate – Unbound material is washed away, leaving only the antigen of interest

Step 4: Add detection antibody – Enzyme-conjugated detection antibody binds to a second site on the antigen of interest

Step 5: Wash microplate – Unbound antibodies are washed away, leaving only those specific for the target of interest

Step 6: Add substrate – Substrate is converted by the enzyme on the detection antibody, producing a color change

Step 7: Read plate – The microplate reader detects the colored reaction product and outputs optical density (OD) values

Step 8: Calculate results – The amount of antigen in each sample is calculated and analyzed

While an ELISA is easy to set up, the assay procedure is time-consuming and labor-intensive. Laboratory automation for high-throughput plate-based assay workflows can help with providing walkaway time, increasing throughput, effectiveness and efficiency of the assay procedure, and reproducibility.

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ELISA assays and applications

Enzyme-linked immunosorbent assay is a commonly used analytical technique performed in many research and biotech labs. Below is a collection of application notes, research and technology related to significant ELISA assays and applications.

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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.

Resources for ELISA