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The ELISA Method

Overzicht

An enzyme-linked immunosorbent assay (ELISA) is typically performed to detect the presence and/or amount of a target protein of interest within an experimental sample. Detection of the target protein is made possible by antibodies, which make the ELISA an immunoassay. Through a series of incubation and washing steps, these antibodies, which are frequently linked, or conjugated, to an enzyme, will detect protein coating the bottom of a well on a microtiter plate. When exposed to a substrate, antibody-bound enzyme will cause a color change, thereby indicating the presence of the protein-of-interest in the sample.

In this video, the theory behind how ELISAs work is explained, including a discussion of both primary and secondary antibody binding and the importance of blocking steps. Theory is followed by practice, as the video progresses to an explanation of the step-by-step procedure. Finally, variations of the standard ELISA such as the sandwich and competitive ELISAs are introduced, and real world applications of this method, such as in over-the-counter pregnancy tests are explained.

Procedure

The ELISA, or enzyme-linked immunosorbent assay, is a widely used method for determining the presence or absence of a specific target protein.

Via a series of washing and binding steps, an antibody conjugated, or linked, to an enzyme will recognize a target protein at the bottom of a 96-well plate. When substrate is added to the sample, an enzymatic reaction will occur, causing a color change that allows the identification and quantification of the target protein.

Before we discuss

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Tags

ELISAEnzyme linked Immunosorbent AssayTarget ProteinWashing And Binding StepsAntibody ConjugatedEnzyme linked Antibody96 well PlateSubstrateEnzymatic ReactionColor ChangeIdentification And QuantificationEquipment And Reagents96 well Flat Bottom PlateExperimental SampleSpecific Target ProteinsPrimary AntibodyHigh Affinity BindingEpitopeUnlabeled AntibodyNonspecific Binding Sites

Ga naar...

0:00

Overview

0:43

ELISA: Components and Principles

4:03

Running an ELISA

7:18

Applications

9:53

Summary

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