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METHODS

Method > ELISA

Enzyme-linked immunosorbent assay and microarrays


Introduction

Since the very first use of antibodies for the detection of antigens of interest, many different technologies have been developed that make use of the antibodies' capability to bind to molecules or substances. One of the most common applications today are measuring the quantity of a biomolecule in a sample by "enzyme-linked immunosorbent assay" (ELISA). This term refers to the use of an enzyme to report an interaction between an antibody and its binding partner (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 by adhering molecules to surfaces. 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 reagents in the early 1970s. Nowadays, scientists also use colored molecules (so called fluorophores) that upon excitation re-emit light to visualize antibody-antigen interactions. Many variants of experimental procedures have been developed, and it is common to build assays using more than one antibody to detect a target of interest (see Figure 1 C-D). To further enhance the possibilities offered by the immunoassay format, applications called microarrays have been developed where more than one molecule can be measured in a single reaction chamber (see below).

Technology


Assay design

The use of antibodies allows designing experiments in many different ways for the intended analysis. To achieve the best possible results from the analysis, different reagents, additives, and solutions have to be tested for their optimal combination and concentration, incubation times and the number of wash cycles need to be evaluated and adjusted. This is to avoid unwanted interactions, which disturb the analysis from detecting the target of interest. Moreover, the mode of how a target is identified and detection can be performed in a number of ways, as described in Figure 1.

Figure 1. Different setups for ELISA and other immunossays.



In ELISA assays, the antibodies may (A) detect an immobilized antigen, (B) capture a labeled antigen, (C) capture an unlabeled antigen and use a second, labeled 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. Using a secondary antibody as detection method, as shown in (D) and (E), will further increase the sensitivity and selectivity of the analysis. 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 = 0.000000001 liter) 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. 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 type of 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 ELISA assays may be used in basic research and in clinical diagnostics. One specific example is the sensitive sandwich-type enzyme-linked immunoassay used to determine the amount of the protein prostate-specific antigen (PSA), which is a biomarker used to detect prostate cancer (Kuriyama et al., 1980).

Microarray assays have previously received a lot of attention for their use in parallel analysis of DNA and RNA molecules. To translate their advantages to assays for the analysis of proteins with antibodies, new protocols and routines had to be developed and established. Nowadays, there are techniques for measuring the amount of proteins in different sample types (e.g. cells, blood serum, urine), to determine how proteins are modified in biological processes (e.g. phosphorylation), or to describe specific protein-protein interactions. Another example is the analysis of antibodies circulating in blood from patients. Microarray applications have also been built for purified antibodies and to study the antibody binding characteristics – an important aspect when using binding reagents as research reagents. Such 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 particular 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 such arrays enables the mapping of antibody binding regions at high resolution. This gives very detailed information of the linear (continuous) epitopes recognized by an antibody. Just like with proteins, protein fragments or other antigens, the assembly of peptides on 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.