ELISA Assay Protocol2018-01-08T08:53:26+00:00

ELISA Assays


ELISA Assay Definition

Enzyme Linked Immunosorbent Assay or ELISA is an immunoassay used to detect and quantitate peptides, proteins, antibodies and hormones in biological specimens (Analytes).


ELISA Assay Principle

An Antibody (Ab) or Antigen (Ag) is adsorbed to a solid surface and binds with a high affinity to its complementary reagent (Ag or Ab) that is typically linked to a labelling enzyme e.g. Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP). Adding an appropriate colorimetric substrate e.g. a HRP substrate such as TetraMethylBenzidine (TMB) or an AP substrate such as p-NitroPhenyl Phosphate (pNPP) to the bound enzyme conjugate results in a colored enzymatic reaction, which forms the basis for the detection and quantitation of the Analyte.

ELISA is a widely used method for diagnostic testing as it is sensitive, accurate and allows a rapid screening and quantitation of a large number of samples. Other techniques have been developed, but ELISA remains popular because of the above and the low cost.


Below is highlighted the most commonly used ELISA Protocols and ELISA Assay Types.


Sandwich ELISA Protocol

The most common type of ELISA Protocol for diagnostic use is sandwich ELISA being a powerful method for detection and quantification of diagnostic markers.

A Sandwich ELISA is built by different formats. It is typically a non-competitive Assay type where the immobilization of the Ag (Analyte) is accomplished via a capture Ab, so that the Analyte is bound between the capture Ab and a labelled secondary Ab. (hence the name “sandwich”). The detection method can be direct or indirect.

Kem-En-Tec Diagnostics provides the label on the secondary Ab: Typically HRP or AP

Kem-En-Tec Diagnostics provides the colorimetric substrate for detection and quantitation of the bound labeled secondary Antibody: Typically TMB or pNPP



The figures show the principle of a direct sandwich ELISA and an indirect sandwich ELISA respectively: the Analyte is immobilized using a capture Ab adsorbed to a Microcell plate.

In the direct sandwich ELISA Protocol a labeled secondary Ab is used to “sandwich” the Analyte. In an indirect sandwich ELISA Protocol a secondary Ab “sandwich” the Analyte and a labeled Ab specifically binds the secondary Ab.

Washing occurs between each step of the ELISA Protocol, allowing for only Analyte-specific complexes to remain bound to the plate.


A Competitive ELISA Assay Type

In a competitive ELISA Assay, it is essential for the Ab to be in limited quantities.  This allows for the reference Ag and the sample Ag (Analyte) to compete for the limited Ab binding sites.

A decrease in the colorimetric substrate signal indicates the presence of the Analyte compared to wells without the Analyte. This happens because the Analyte and the reference Ag compete for the same Ab binding site. Detection and Quantitation is obtained by generating a standard curve of the added reference Ag in different concentrations vs. Activity.

As shown from the Figure, the Activity of the standard curve is inversely related to the Ag concentration of the Analyte.




ELISA Assay Guide

In order to assist and inspire R&D employees, Kem-En-Tec Diagnostics has developed a comprehensive guide for ELISA Assay Development. The Guide takes the reader through the individual steps of an ELISA and provides Hint and Tips to each step.

The introduction presents the principle of the ELISA Assay, different basic types of ELISA Assays, modifications and combinations of these as well as describes the pro and cons of the different ELISA Assay protocols.

The last part includes an overview of suggestions to reagents with a view to optimize the ELISA Assay i.e. maximize ELISA Assay sensitivity, minimize ELISA Assay background and maximize ELISA Assay stability.

Please click at the link to download our ELISA Guide


Environmental considerations during assay development

IVD manufacturers are working towards more environmentally conscious and sustainable manufacturing. Much of these efforts have been focusing on replacement of toxic chemicals for the protection of health of the employees. Kem-En-Tec Diagnostics supports these requirements, recommending ECO-friendly colorimetric substrates and reagents with no toxic preservative formulations.

Now also of highest priorities are reducing usage of facility water and energy use, reducing facility waste, increasing facility recycling, and sourcing recycled/recyclable product packaging. Kem-En-Tec Diagnostics´ Eco-sensible options are available for the development and production of diagnostics assays without compromising assay quality and stability. Further, those options can reduce production cost.

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