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Immunology

Testing

At the heart of clinical immunology is a single, invisible event: the specific binding of an antibody to its antigen. As Medical Laboratory Scientists, our job is to act as molecular detectives, using a variety of clever techniques to make this “handshake” visible, measurable, and diagnostically useful. Every serology test, from the simplest slide test to the most complex automated assay, is built upon a core principle of visualizing this interaction

Part 1: Principles - How We “See” the Handshake

There are two fundamental strategies for turning a molecular binding event into a readable result. The choice of strategy depends on the test’s purpose, the required sensitivity, and the nature of the antigen being detected

Unlabeled Methods: Observing the Natural Consequences

These classic methods rely on the inherent ability of antibodies to physically alter their environment once they bind to their target. We aren’t adding any reporters; we are simply observing the secondary effects of the antigen-antibody binding

  • Precipitation: This principle applies when both the antigen and antibody are soluble. In the right concentrations (the zone of equivalence), antibodies cross-link the antigen molecules into a massive, insoluble lattice that falls out of solution and becomes visible as a precipitate. This is the foundation of gel diffusion techniques like Ouchterlony and Radial Immunodiffusion (RID)

  • Agglutination: This is one of the most widely used principles. It occurs when antibodies cross-link particulate antigens, such as whole bacteria, red blood cells, or antigens that have been artificially coated onto latex beads. This cross-linking creates visible clumping, providing a clear, often rapid, positive result. This principle is central to blood banking, syphilis screening (RPR), and many rapid latex agglutination kits

Labeled Methods: Tagging the Evidence for High Sensitivity

Modern serology largely relies on labeled immunoassays for their superior sensitivity and quantitation. In these methods, we covalently attach a reporter molecule (a “label”) to either an antibody or antigen. The signal generated by this label is what we measure, and it is directly proportional to the amount of binding that occurred

  • Enzyme Immunoassays (EIA or ELISA): The workhorse of the modern lab. An enzyme is used as the label. When its specific substrate is added, it produces a measurable color change. This highly sensitive and easily automated principle is used for everything from HIV screening to hormone testing

  • Fluorescent Immunoassays (FIA): A fluorochrome is used as the label. When excited by light of a specific wavelength, it emits light of a different wavelength, which can be visualized with a special microscope. The classic Antinuclear Antibody (ANA) test for autoimmune disease uses this method

  • Chemiluminescent Immunoassays (CLIA): The label is a molecule that produces light via a chemical reaction. This method is exceptionally sensitive, making it the technology of choice for most high-throughput, automated immunoassay platforms that measure low-concentration analytes like cardiac markers and tumor markers

Part 2: Methods - The Toolbox in Action

These named methods are the practical application of the principles discussed above. Each is a distinct procedure designed to answer a specific clinical question

Diffusion & Precipitation Methods

  • Ouchterlony Double Diffusion: A qualitative gel technique used to compare antigens. Antigens and antibodies diffuse towards each other from wells in an agar plate. The pattern of the resulting precipitate lines reveals whether the antigens are identical, non-identical, or partially identical
  • Radial Immunodiffusion (RID): A quantitative gel technique. Antigen from a patient sample diffuses out from a well into an agar gel that contains a uniform concentration of antibody. The diameter of the precipitate ring that forms is directly proportional to the concentration of the antigen in the sample

Agglutination Methods

  • Passive Agglutination: Used to detect antibody in a patient’s serum. Inert latex beads are coated with a known antigen. If the patient’s serum contains the specific antibody, it will cause the beads to clump
  • Reverse Passive Agglutination: Used to detect antigen in a patient’s sample. The latex beads are coated with a known antibody. If the sample contains the specific antigen, it will cause the beads to clump
  • Agglutination Inhibition: A competitive assay where no agglutination is a positive result. Patient antigen, if present, neutralizes a limited amount of reagent antibody, preventing it from clumping the antigen-coated beads that are added in the second step

Labeled Immunoassay Methods

  • ELISA (Enzyme-Linked Immunosorbent Assay): A highly versatile and sensitive method performed on a solid phase, like a microtiter well. The most common formats are the Indirect ELISA (to detect patient antibody) and the Sandwich ELISA (to capture and detect patient antigen)
  • Western Blot: A highly specific, confirmatory method used to identify antibodies to individual proteins. Pathogen proteins are first separated by size via electrophoresis, then blotted onto a membrane. The patient’s serum is applied, and an enzyme-labeled secondary antibody reveals which specific protein bands the patient’s antibodies have bound to
  • Nephelometry: An automated, quantitative method that measures antigen-antibody complex formation in a solution, not on a solid phase. It quantifies the concentration of an analyte by measuring the amount of light that is scattered by the forming immune complexes