Principles of Immunoassays

Immunoassays are bioanalytical methods used to detect and quantify specific substances within biological samples through highly selective antigen-antibody interactions. The fundamental principle of immunoassay is that antibodies bind specifically to their target antigens, allowing laboratories to accurately identify and measure analytes such as hormones, proteins, antibodies, and infectious agents.

They primarily operate through a competitive binding process, where a fixed amount of labelled analyte competes with varying amounts of unlabelled sample analyte for a finite number of binding sites on a specific anti-analyte antibody.

In this guide, we explore the principles of immunoassays, the different immunoassay techniques used in modern laboratories, and why quality assurance is essential for reliable testing.

Why are Immunoassays important?

Immunoassays have been used in many important areas, including:

- Diagnosis of diseases
- Therapeutic drug monitoring
- Clinical studies in drug discovery

The widespread use of immunoassay methods in pharmaceutical analysis are due to their inherent specificity, high-throughput capability, and high sensitivity. These characteristics allow for the analysis of a wide range of analytes in biological samples.

Immunoassays can detect antigens at extremely low concentrations, which other techniques cannot measure. Their speed allows clinicians to diagnose diseases quickly and accurately.

How Does an Immunoassay Work?

Although there are many different immunoassay formats, they all rely on the same fundamental principle: the highly specific binding between an antigen and an antibody.

A typical immunoassay contains three core components:

- The target analyte being measured

- An antibody that selectively binds to that analyte

- A detection system that produces a measurable signal, such as fluorescence, chemiluminescence or enzyme activity

Once binding occurs, the signal generated is proportional (or in competitive assays, inversely proportional) to the concentration of the analyte, allowing laboratories to accurately quantify substances within a sample.

Historical Background and Evolution of Immunoassays 

Rosalyn Sussman Yalow and Solomon Berson are credited with the development of the first immunoassays in the 1950s. Their work resulted in a Nobel Prize in Medicine in 1977. Since then, immunoassays have evolved greatly.

In the 1980s, a professor at Cardiff University replaced the radioactive iodine used in immunoassays with an acridinium ester, a substance that produces its own light through chemiluminescence. This type of immunoassay is now used in approximately 100 million clinical tests annually worldwide, enabling clinicians to measure a range of proteins, pathogens, and other analytes in blood samples.

Basic Principles of Immunoassays

The core principle of immunoassays is the use of an antibody that specifically binds to the target antigen. The antibodies that are used in the immunoassay must have a high affinity for the antigen, and they can be either monoclonal or polyclonal antibodies.

Monoclonal antibodies only bind to specific antigens and therefore give more accurate results, but can be more expensive. Polyclonal, on the other hand, are less expensive but can recognise multiple epitopes on an antigen or multiple antigens, so are less specific.

Another key feature of all immunoassays is the ability to produce a measurable signal in response to the antigen-antibody binding. Most immunoassays achieve this by chemically linking antibodies or antigens to a detectable label. Various labels are used in modern immunoassays, enabling detection through different methods. The reliability of these detection methods contributes to the overall analytical performance of an immunoassay. Combined with appropriate assay validation and ongoing quality assessment, this helps laboratories produce accurate and reproducible results.

Depending on the immunoassay format, the resulting signal may be measured using colour change, fluorescence, radioactivity, chemiluminescence (light emission), or light scattering. Modern immunoassay analysers are designed to detect these signals and convert them into quantitative results, allowing laboratories to process large numbers of patient samples efficiently and consistently.

Types of Immunoassays

There are several immunoassay techniques used in clinical laboratories, each based on the same immunoassay principle but differing in how the antigen-antibody interaction is detected and measured:

Turbidimetry and Nephelometry Immunoassays

Turbidimetric immunoassay, also known as immunoturbidimetry or turbidimetry, measures the turbidity of a sample to determine the level of an analyte. This method is often used to quantify antigen-antibody complexes, as their formation increases the sample's turbidity. When light passes through the reaction solution, some of it is scattered by the sample, some is absorbed, and the rest passes through. The concentration of protein antigen in the sample is determined by measuring the light absorbance.

Nephelometric immunoassay is a method related to immunoturbidimetry and is also used to quantify antigen-antibody complexes by measuring the sample's turbidity. However, nephelometry measures turbidity by detecting light scattered at a fixed angle.

Enzyme Immunoassays (EIA)

Enzyme immunoassays, including ELISAs, are straightforward, and widely used examinations based on specific interactions between antibodies and antigens. They harness the catalytic abilities of enzymes present in a sample to identify and measure immunological responses accurately.

ELISAs represent a distinct category within enzyme immunoassays, used for clinical analyses. Their purpose is to precisely identify and measure antibodies, proteins, and hormones.

Like all immunoassays used in clinical laboratories, ELISAs benefit from ongoing external quality assessment to help verify analytical performance and maintain confidence in patient results.

Radio Immunoassays (RIA) 

A radioimmunoassay (RIA) employs antigens labelled with radioisotopes to gauge the concentrations of substances in bodily fluids like blood and saliva. RIA's are sensitive and specific, and often used to detect a single analyte in samples. However, it's important to use extra precautions due to the radioactive substances involved.

Fluoroimmunoassay (FIA)

Fluorescent immunoassays (FIA) employ a fluorescent compound as the detection reagent for detecting and quantifying various compounds. Widely utilised in the in vitro diagnostics (IVD) sector, FIAs offer rapid detection and high sensitivity, surpassing many alternative methods.

Chemiluminescent Immunoassay (CLIA)

Chemiluminescence immunoassays are a type of enzyme immunoassay. While the principles of CLIA are similar to those of ELISA or fluoroimmunoassay, the type of reporter used is different. CLIA reporter systems rely on chemiluminescence, which is the emission of light resulting from a specific chemical reaction. CLIA has a fast reaction time, and is highly stable compared to FIA and RIA. Therefore, CLIA is used in a range of different fields including disease diagnosis, food safety and environmental monitoring.

Because of its excellent sensitivity, wide analytical range, and suitability for automation, CLIA is now one of the most widely used immunoassay technologies in clinical laboratories. Many laboratories use immunochemistry techniques such as CLIA within routine diagnostic testing and participate in EQA programmes to benchmark performance against peer laboratories.

Why External Quality Assessment is Important for Immunoassays

We've explored the principles of immunoassays and their importance in laboratory medicine. However, even highly sensitive immunoassays require ongoing performance monitoring to ensure results remain accurate, reliable, and comparable between laboratories.

External Quality Assessment (EQA) plays a crucial role in maintaining and improving the quality of immunoassay testing. It ensures that laboratories produce reliable, accurate, and standardised results, which are essential for effective patient care and research outcomes.

At UK NEQAS IIA, we provide EQA programmes that help laboratories monitor the performance of immunoassays used across immunochemistry, allergy, autoimmunity and oncology testing.

Our EQA schemes enable laboratories to benchmark performance, identify analytical variation, and support continuous quality improvement and accreditation requirements.

Alongside our EQA programmes, the Digital Academy provides webinars, publications, technical presentations and educational resources to help laboratory professionals stay informed about immunoassay principles, assay performance, and best practice.

Explore our EQA programmes or visit the Digital Academy to continue your professional development.