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Immunology

Principles

We’ve established that the antigen-antibody “handshake” is a real, specific, and powerful event. But there’s a problem: it’s completely invisible to the naked eye. As Medical Laboratory Scientists, our entire job is to take this invisible molecular interaction and make it visible, measurable, and clinically meaningful. Every serological test you will ever perform is, at its heart, a clever strategy to visualize this handshake

Think of it this way: we are detectives trying to prove that a meeting took place between our two suspects, Antigen and Antibody. We need a method to get a “receipt” of their interaction. The principles of serologic testing are all about the different ways we can generate and read that receipt

There are two fundamental approaches we use to achieve this: unlabeled methods, where we observe the natural consequences of the binding, and labeled methods, where we intentionally “tag” one of the participants with a reporter molecule

Unlabeled Approach: Seeing the Consequences

These methods are the classic, foundational techniques of serology. They don’t require fancy labels or tags. Instead, they rely on the fact that when enough antibodies and antigens bind, they can form massive, visible complexes. We are observing a secondary phenomenon that results from the primary antigen-antibody binding event

  • Precipitation Reactions
    • Principle: This occurs when a soluble antigen reacts with its corresponding antibody (called a precipitin). If the concentrations are right (in the zone of equivalence), the antibodies cross-link the antigen molecules, forming an enormous, insoluble lattice that falls out of solution as a visible precipitate
    • In the Lab: This is the principle behind techniques like Radial Immunodiffusion (RID), where we measure the concentration of an antigen by the size of the precipitate ring it forms in an antibody-containing gel, and Ouchterlony Double Diffusion, used to compare the identity of different antigens
  • Agglutination Reactions
    • Principle: This is one of the most widely used principles in the lab. Agglutination is similar to precipitation, but in this case, the antigen is particulate (e.g., a whole bacterium or a red blood cell) or has been attached to the surface of a larger carrier particle (like a latex bead). When the antibody binds, it cross-links these large particles, causing visible clumping or agglutination
    • Types of Agglutination
      • Direct Agglutination: The antigen is naturally part of the particle’s surface. The classic example is ABO blood typing, where antibodies in patient plasma clump the A or B antigens on the reagent red blood cells
      • Passive (or Indirect) Agglutination: A soluble antigen has been artificially attached to a carrier particle, like a latex bead. A common example is a latex agglutination test for Rheumatoid Factor
    • In the Lab: Rapid, easy to perform, and the basis for countless tests, from rapid strep screens to syphilis testing (RPR)
  • Complement Fixation
    • Principle: This is a clever, two-part test that detects the presence of an antibody by seeing if it “uses up” or fixes complement
    • Stage 1: Patient serum (the potential antibody source) is mixed with a known antigen. A measured amount of complement (from guinea pig serum) is added. If the patient has the antibody, it will bind the antigen and “fix” (consume) the added complement
    • Stage 2: An indicator system is added—sheep red blood cells coated with anti-sheep antibodies. If the complement was used up in stage 1, there is none left to lyse the indicator cells. No lysis = a positive test. If the patient did not have the antibody, the complement was not fixed and is free to lyse the indicator cells. Lysis = a negative test. This inverse relationship is key!

Labeled Approach: Tagging the Evidence

Modern serology relies heavily on labeled immunoassays. Here, we don’t wait to see a secondary consequence like clumping. Instead, we directly detect the binding by covalently attaching a “reporter molecule” or label to either the antigen or the antibody. These assays are generally more sensitive, quantitative, and easily automated

  • Enzyme Immunoassays (EIA or ELISA)
    • Principle: This is the king of modern serology. The label used is a stable enzyme (like horseradish peroxidase or alkaline phosphatase). When the specific substrate for that enzyme is added, the enzyme catalyzes a reaction that produces a measurable color change. The amount of color is directly proportional to the amount of bound antibody or antigen
    • In the Lab: Used for everything from HIV screening and hepatitis testing to measuring hormone levels. It is the core technology inside most automated immunoassay analyzers
  • Fluorescent Immunoassays (FIA)
    • Principle: The label is a fluorochrome, a molecule that absorbs light at one wavelength and emits it at a different, longer wavelength. This emitted light can be detected with a fluorescent microscope or a specialized instrument
    • Types of FIA
      • Direct Fluorescent Antibody (DFA): The primary antibody that recognizes the patient antigen is directly labeled with the fluorochrome
      • Indirect Fluorescent Antibody (IFA): The primary antibody is unlabeled. A labeled secondary anti-human antibody is then used to detect the binding of the primary antibody. This method amplifies the signal
    • In the Lab: The classic example is the Antinuclear Antibody (ANA) test for autoimmune diseases like lupus, which uses the IFA method
  • Chemiluminescent Immunoassays (CLIA)
    • Principle: The label is a substance that emits light as the result of a chemical reaction. No external light source is needed to excite it. This process is extremely efficient, producing a large signal from a small amount of binding
    • In the Lab: This is the technology that drives most modern, high-throughput automated immunoassay platforms. Its incredible sensitivity allows for the detection of very low concentrations of analytes, like cardiac markers (troponin) and specific tumor markers
  • Radioimmunoassay (RIA)
    • Principle: The original labeled immunoassay. The label is a radioisotope (like Iodine-125). The amount of bound antigen or antibody is measured by counting the radioactivity with a gamma counter
    • In the Lab: Largely replaced by EIA and CLIA due to the expense, safety concerns, and waste disposal issues associated with radioactive materials. However, it’s a historically important technique that established the principles for all other labeled assays