ELISA Principles 101

ELISA (Enzyme Linked Immuno-Sorbent Assay) is a highly sensitive immunoassay for in vitro quantification of soluble analytes, including: cytokines, CD antigens, apoptosis markers, hormones, metabolites, and growth factors. 

ELISA - The Basics

A key step in developing any ELISA assay is immobilization of the analyte or antigen of interest. This may be realized by direct adsorption to a solid-support e.g., an assay plate, or indirectly via a capture antibody that has been attached to the assay plate. 

Immobilizing the target allows for the separation of bound from non-bound material by washing, and makes ELISA a powerful tool for detecting and quantifying target analytes within a crude sample preparation. 

Regardless of the immobilization method or ELISA format used, the general workflow is similar:

  1. A primary antibody recognizes an epitope on the target analyte (or antigen) present in a sample.
  2. Depending on the assay format, a secondary antibody enzyme conjugate binds the primary antibody or the analyte itself, and reacts with a substrate to produce a signal that is detectable on a spectrophotometer, fluorometer or luminometer. 

The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP) and a large selection of substrates is available for each. The choice of substrate depends upon the required assay sensitivity and the availability of instrumentation for signal detection.

Sandwich ELISA Format

The sandwich ELISA is highly sensitive and robust and is probably the most widely used of the ELISA formats. In a sandwich ELISA, analyte immobilization is indirect as described below and shown in Figure 1.

  1. A capture antibody specific for the target analyte or antigen is coated onto the surface of a microtitre plate. 
  2. The test sample is applied, and any target analyte (A) present is bound by the capture antibody. 
  3. A second antibody (known as the detection antibody) is then applied, and this binds the target analyte that remains bound by the capture antibody. 
  4. In this way, the target becomes sandwiched between a pair of antibodies, each of which is specific for a different and non-overlapping region or epitope of the analyte. 
  5. A secondary antibody conjugate is added. This binds to the detection antibody and reacts with a substrate to produce a signal that is detectable on one of the instruments listed above.
Figure 1. Sandwich ELISA Schematic
Figure 1. Sandwich ELISA Schematic

Direct and Indirect ELISA

In the sandwich assay format described above, target analyte immobilization is indirect. The sandwich assay also uses indirect detection, since it relies on a secondary antibody conjugate to bind the detection antibody and produce a signal. 

When we refer to direct and indirect ELISA, it is important to know that we are referring to the mode of detection, and not the mode of target immobilization. In general, regardless of how the target is immobilized, the detection step will have the greatest influence on the sensitivity of an ELISA.

In the indirect ELISA format, the target analyte is adsorbed directly to the assay plate, and indirect detection is used (Figure 2). Indirect detection offers high sensitivity because each primary antibody contains several epitopes that can be bound by the secondary antibody conjugate, which leads to signal amplification.

Figure 2. Indirect ELISA Schematic
Figure 2. Indirect ELISA Schematic

Although indirect detection is the most widely used ELISA detection format, it is worth knowing that detection may also be direct. Direct detection occurs via an enzyme that is conjugated to the primary (capture) antibody, resulting in minimal, if any, signal amplification (Figure 3). 

Figure 3. Direct ELISA Schematic
Figure 3. Direct ELISA Schematic

Qualitative and Quantitative ELISA

ELISA can be run qualitatively or quantitatively. Qualitative ELISAs provide a straightforward 'yes' or 'no' about the presence or absence of the target analyte in a sample, where assay results are usually read according to a predefined statistical measure to define the cut-off for a so-called positive sample.

In practice however, quantitative ELISAs are much more commonplace. Here, the optical density (OD) of the output signal from every sample is compared to a standard curve that is included in every ELISA run. A standard curve is typically a serial dilution of a standard preparation of the target analyte, of which the starting concentration is known. For example, if a test sample yields an OD of 0.5, the concentration of the standard dilution that yielded OD = 0.5 must match the  analyte concentration in the test sample.

Alternative Formats - Competitive ELISA

A competitive ELISA, also known as an inhibition or blocking ELISA, is a variation on the ELISA format that is often used when the target analyte only has one epitope available for antibody binding. The general workflow for a competitive ELISA is as follows:

  1. The analyte present in the test sample competes for antibody binding with a fixed amount of biotin- or HRP-labeled reference analyte supplied with the ELISA kit. 
  2. The higher the target analyte concentration in the sample, the more it outcompetes the labeled analyte, thus reducing the output signal.
  3. In this way, unlike the other ELISA formats described above, the more target analyte present in the test sample, the weaker the output signal.

BioSite ELISA - Something For Everyone 

ELISA can be applied to diverse sample types, including cell culture supernatants, tissues, serum, plasma, and other bodily fluids. Nordic BioSite has ELISA kits for 1000's of unique targets, covering all sample types in the following species:

Note that other ELISA formats exist besides what is described in this article. Do get in touch with us at info@nordicbiosite.com to hear more about ELISA and find out if we have the ELISA kit to fit your needs.