ELISA Principles, Formats & Streamlining

A short overview of the principles and different formats of ELISA is provided on this webpage. If you want to find out more, download our complete guide by filling out the form on the right-hand side.

What is ELISA?

ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based technique invented in the 1970s and evolved from the radioimmunoassay methods that were developed during the previous decade. As the name suggests, the principles of ELISA readout relies on the use of an antibody which has been linked to an enzyme. When an appropriate substrate is added the enzymatic activity produces a color change that can be measured, and this provides information about the presence and quantity of the target antigen in the sample material.

Sandwich_ELISA_indirect_detection

Schematic representation of a sandwich ELISA with indirect detection. The microplate is coated with a specific capture antibody, which binds the target antigen in the sample. A second specific antibody is then added and is detected with an enzyme-linked anti-species antibody. Following the addition of an appropriate substrate, the enzymatic activity produces a color change that can be measured.

ELISA formats

There are several ways in which an ELISA can be configured:

  • Antigen-down ELISA
  • Sandwich ELISA
  • Competitive ELISA

What are the main steps of an ELISA?

A typical sandwich ELISA with direct detection is performed as follows:

  • Immobilization of the capture antibody on the surface of the microplate
  • Plate washing to remove unbound antibody
  • Blocking to prevent non-specific reagent binding
  • Plate washing
  • Addition and incubation of the sample
  • Plate washing
  • Addition and incubation of the directly labeled detection antibody
  • Plate washing
  • Detection

Direct vs indirect ELISA

Although ELISAs have traditionally relied on the use of labeled secondary antibodies for detection, these reagents can be a source of background signal, resulting in decreased assay sensitivity. Background staining can occur due to non-specific binding of secondary antibodies and is often a problem in multiplexing experiments, during which secondary antibodies from different species are used simultaneously and can cross-react with one another.

Advantages of direct detection?

  • Non-specific binding is avoided since secondary antibodies are not used
  • Multiplexing is possible with antibodies from the same species
  • Faster since there is no secondary antibody incubation step and therefore fewer wash steps
  • Data quality is improved through assay simplification

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If you want to find out in detail about the principles, different types of ELISA, the main steps in the development and different ways of detection download the ELISA guide by filling out the form on this webpage.

You will also find answers to these questions:

  • What are common antibody labels?

  • How to label antibodies in 15 minutes?

  • How to save time and antibodies when developing an assay from scratch?

  • How to perform an ELISA in under 3 hours?

  • What are solutions for common problems in ELISA?

Download the ELISA guide by filling out the form below.

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