Immunoassays

What is an immunoassay?

An immunoassay is a biochemical test that measures the presence of a substance typically in a liquid or the or the concentration of a specific component in a solution primarily through the use of an antibody or an antigen. The molecule detected by the immunoassay is often referred to as an “analyte” and is often a protein or an antibody, although it may be other kinds of molecules, as long as the proper antibodies that have the required properties for the assay are present. 

What are immunoassays used for?

Immunoassays are frequently used for several purposes such as:

  • research
  • diagnostics 

where immunoassays typically are used to detect analytes in biological liquids such as detection of various components in serum, cerebrospinal fluids or urine. 

 

Which types of immunoassays exist?

Immunoassays come in many different formats and variations. Some immunoassays are be carried out by mixing reagents and samples following a physical measurement, whereas other immunoassays are carried out in multiple steps with reagents being added and washed away. Multi-step assays are often referred to as separation immunoassays or heterogeneous immunoassays, whereas one step assays typically are called homogeneous immunoassays or  non-separation immunoassays.

Five types of immunoassays are currently available

  • Radioimmunoassay (RIA)
  • Counting Immunoassay (CIA)
  • Enzyme Immunoassays (EIA) or Enzyme-linked immunosorbent assays (ELISA)
  • Fluoroimmunoassay (FIA)
  • Chemiluminescenceimmunoassay (CLIA)

Radioimmunoassays were one of the first types of immunoassays developed, which typically is very sensitive. In this assay, a radioisotope-labelled antigen of interest interacts its complementary antibody. Then a sample with the antigen to be measured is added, which competes with the radioactive antigen. After washing away unbound antigens, the radioactivity of the sample is measured. The amount of radioactive signal is inversely related to the amount of target antigen. Although very sensitive, this assay is only seldom used, due to the health hazards of using radioactive substances. 

Counting immunoassays are applying polystyrende beads, which are coated with antibodies of different specificities to the target component. During incubation the beads bind to multiple antigens. Ultimately, the solution is passed through a cell counter and only the unbound beads are counted. The number of unbound beads is inversely proportional to the amount of antigen present in the sample.

Enzyme-linked immunosorbent assays employ enzyme-linked antibodies. After incubation with the analyte unbound antibodies are washed away. Bound enzyme-linked antibody-antigen complexes are detected by adding a substrate to the solution. The enzyme catalyzes a chemical reaction of the substrate to produce a quantifiable color change.  

Four types of enzyme-linked immunosorbent assays exist: 

  • Direct ELISA
  • Sandwich ELISA
  • Competitive ELISA
  • Reverse ELISA

Fluoroimmunoassay applies antibodies labeled with fluorescent probes. Following incubation with antigen, the antigen-antibody complexes are isolated and the fluorescent intensity is measured.

Chemiluminescentimmunoassays employ the same principles as enzyme-linked immunosorbent assay or fluoroimmunoassays, only the reporter is different. Luminescence is based on the release of light due to an electron being kicked up to a higher energy state and emitting a photon as it relaxes down, which basically is the same principle as fluorescence. However, in chemiluminescence this is achieved by a chemical reaction, whereas this is achieved by certain frequencies of light in fluorescence.