Fields »
ABOUT & HELP

THE HUMAN PROTEOME

THE HUMAN PROTEOME PROTEIN CLASSES PROTEIN EVIDENCE

LEARN

DICTIONARIES METHODS CELL LINES EVENTS

MEDIA & DATA

MEDIA DOWNLOADABLE DATA

THE HUMAN PROTEIN ATLAS

INTRODUCTION ASSAYS & ANNOTATION DATA QUALITY & SCORING DISCLAIMER RELEASE HISTORY DATA USAGE POLICY ANTIBODY AVAILABILITY HELP

ANTIBODY SUBMISSION

ANTIBODY SUBMISSION CONDITIONS

ABOUT US

THE PROJECT ORGANIZATION PUBLICATIONS LINKS CONTACT

METHODS

Method > ELISA

Enzyme-linked immunosorbent assay and microarrays


Introduction

Since the very first use of antibodies for the detection of antigens of interest, a plenitude of different technologies have been develop that make use of the antibodies' capability to bind to another molecule or substance. One of the most common applications today are measuring the quantity of a biomolecule in a sample by "enzyme-linked immunosorbent assay" (ELISA) that refers to the use of an enzyme that makes an interaction between an antibody and its binding partner visible (Gan & Patel, 2013). During the 1950s, the scientists Yalow and Berson developed a method where radioactivity is used to determine the amount of an analyte in the solution. This radioimmunoassay (RIA), for which Yarlow received the Nobel prize in 1977, was very sensitive for the detection of hormones but soon it became clear that working with radioactivity is not safe for a general use. Hence, an alternative was developed by linking enzymes to antibodies instead of a radioactive molecule, and in addition the process of adhering molecules to surfaces was further refined. This lay the foundation for Perlmann and Engvall in Sweden (Engvall & Perlmann, 1971) as well as Schuurs and van Weemen from the Netherlands (Van Weemen & Schuurs, 1971), to build assays with immobilized and enzyme-modified reagent in the early 1970s. Nowadays, scientists also use colored molecules (so called fluorophores) as reporters to visualize the interaction that occurs in the solution. Many variants of experimental procedures have been designed and developed, and it is common to build assays using more than one antibody (e.g. sandwich assays). To further enhance the possibilities offered by the immunoassay format, applications where more than one molecule is present in every reaction chamber has been developed (e.g. microarray assays).

Technology


Assay design

The use of antibodies allows designing experiments in many different ways. On one side, the different reagents, additives, and solutions may be changed and tested with regard to their concentration, incubation time, and number of washes to avoid unwanted interactions, which disturb the analysis. Moreover, the mode of detection can be modified in a number of ways as described in Figure 1.

Figure 1. Different ELISA setups.



In ELISAs the antibodies may (A) detect an immobilized antigen, (B) capture a labelled antigen, (C) capture an unlabeled antigen and use a second, labelled antibody to detect the captured antigen, or (D) use a third antibody for detection, or even use two antibodies for detection (E) (see Proximity Ligation Assay). Direct labeling of the antibody or antigen as in (A), (B), and (C) is the simplest and fastest method for detection, however, using a secondary detection method, as in (D) and (E), will increase the sensitivity. The method used in (D) also allows greater flexibility, whereas method (E) further increases the specificity as three antibodies must bind the antigen in order to produce a reporter molecule. Out of the presented assays, the most commonly used concepts are shown in (C) and (D).

Multiplexing

A new phase in the advancement of immunoassays came with the development of a technology called microarrays. The term microarray most commonly describes the ordered organization of small volume droplets that have dried on a small surface area. The reaction dimensions are miniaturized so that many assays can be performed in multiple samples in parallel, several thousands of different features may be presented to the surrounding solution. This means that you can investigate a large number of molecules with one single measurement. There is the possibility to use microscope glass slides and specialized robotics that leave very small drops of liquid (1 nl) on the glass in a ordered fashion leaving behind tiny spots (diameter of 0.15 mm). Another technique for multiplexing is to use even smaller and color-coded particles (diameter of 0.005 mm) that are coated with antibodies to fish out the analyte from the solution.

Sensitivity

In many applications it is important to measure very small amounts (sometimes only traces) of a molecule in a sample solution. In order to achieve a good sensitivity, the conditions of the experiment need to be adjusted to suit the antibodies, the detection system, and the samples. In addition, there is progress being made on using better colors, specialized lasers and filters, as well as miniaturization (Ekins & Edwards, 1997).


Specific examples

There are many examples of how ELISAS may be used in research and in diagnostics. One specific example is the sensitive sandwich-type enzyme immunoassay used to determine the quantity of biomarker protein prostate-specific antigen (PSA) in males in order to detect prostate cancer (Kuriyama et al., 1980).

Great attention has been given to microarray assays and their use in parallel analysis of DNA and RNA molecules. To translate these advantages to assays for analyzing proteins or for using antibodies, new protocols and routines had to be developed and established. This included techniques for measuring the abundance of proteins in different sample types (e.g. cell, serum, urine), techniques to determine how proteins are modified in biological processes (e.g. phosphorylation), or to determine specific protein-protein interactions. There is a wide range of applications that all have in common to measure many parameters in one reaction tube. One important application is the antibody binding analysis of purified antibodies used as research reagents, and another example is the analysis of antibodies present in blood plasma from patients with a disease. These protein microarrays can either consist of proteins, protein fragments, or small peptides to test the specificity of the binding reagent. Protein microarrays can reveal the interactions to entire proteins or larger protein fragments, while peptide microarrays show to which parts (epitopes) of the proteins the antibodies bind. A typical epitope mapping result is shown in Figure 2 (Edfors et al., 2014). Synthesizing millions of overlapping peptides with only one amino acid residue shift on one single array enables mapping of antibody binding regions at high resolution giving very detailed information of the linear epitopes recognized by the antibody. Peptide arrays may also be used for studies of antibody reactivity in plasma samples from patients with infectious and autoimmune diseases.

Figure 2. Epitope mapping of a polyclonal antibody on a peptide array where the result displays four distinct linear epitopes and the consecutive overlapping peptides which are bound. X-axis: peptides, Y-axis: mean fluorescence intensity (MFI). (Edfors et al., 2014)




References and Links
Enzyme-linked immunosorbent assay
Introduction
Technology
Assay design
Multiplexing
Sensitivity
Specific examples
References and Links



The Human Protein Atlas project is funded
by the Knut & Alice Wallenberg foundation.