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Immunology

Methods

Let’s open up the laboratory’s toolbox. Now that we understand the principles behind making antigen-antibody reactions visible, we can look at the specific, named methods that put those principles into practice. Each test is a unique instrument designed for a specific purpose—some are qualitative sledgehammers, perfect for a quick “yes” or “no,” while others are quantitative micrometers, capable of measuring incredibly small concentrations with high precision

Precipitation-Based Methods: Seeing Soluble Interactions

These are classic techniques that rely on forming a visible precipitate from the interaction of soluble antigens and antibodies in a gel medium. They are excellent for demonstrating fundamental immunologic principles

  • Ouchterlony Double Diffusion
    • The Goal: A qualitative technique used to compare different antigens and determine their relationship
    • The Setup: A flat, clear plate of agar gel is used as the “arena.” Small wells are punched into the agar. A known antibody solution is placed in a central well, and different antigen solutions are placed in the surrounding wells
    • The Process: The soluble antigens and antibodies diffuse out from their wells, moving towards each other through the gel. Where they meet in the zone of equivalence, they form a stable, visible line of white precipitate
    • The Interpretation: The shape and position of the precipitate lines tell a story about the antigens:
      • Pattern of Identity: If two different wells contain the exact same antigen, their precipitate lines will meet and fuse perfectly, forming a smooth arc. The antibody “sees” them as identical
      • Pattern of Non-identity: If the wells contain completely unrelated antigens, the two precipitate lines will form independently and cross over each other. The antibodies for each are specific and don’t interact
      • Pattern of Partial Identity: If the antigens are related but not identical (they share some epitopes but not all), the lines will merge but a small “spur” will point towards the simpler antigen. It shows that the antibody recognizes a shared epitope but also has a reaction specific to the more complex antigen
  • Radial Immunodiffusion (RID)
    • The Goal: A quantitative technique used to measure the concentration of a specific antigen
    • The Setup: Instead of having antibody in a well, the antibody is mixed uniformly throughout the entire agar gel before it’s poured. The patient’s sample (containing the unknown amount of antigen) is placed in a well cut into the gel
    • The Process: The antigen diffuses outward from the well in a 360-degree circle, creating a concentration gradient. As it diffuses, it binds with the antibody in the gel. A stable precipitate ring forms at the edge of the zone of equivalence
    • The Interpretation: The higher the initial concentration of the antigen in the well, the farther it will diffuse before it is all consumed. Therefore, the diameter of the precipitate ring is directly proportional to the antigen concentration. By running standards with known concentrations, we can create a standard curve and determine the exact concentration in the patient sample
    • Clinical Use: This was the classic method for quantifying serum immunoglobulins (IgG, IgA, IgM) and complement components. It has been largely replaced by faster, automated methods like nephelometry, but the principle is still foundational

Agglutination-Based Methods: Clumping for Clarity

These methods are fast, simple to perform, and incredibly versatile. They are based on the principle that antibodies can cross-link particulate antigens (or antigens bound to particles), creating visible clumping

  • Direct Agglutination
    • The antigen is a natural, intrinsic part of the particle’s surface
    • Classic Example: ABO blood typing. Reagent anti-A antibodies are mixed with patient red blood cells. If the patient’s cells have the A antigen on their surface, the antibodies will cause the cells to clump together. It’s a direct, visible result
  • Passive (or Indirect) Agglutination
    • Used when the antigen we want to detect is soluble. We can’t see a soluble antigen clump
    • The Trick: We take a soluble antigen and artificially attach it to the surface of a larger, inert carrier particle, like a latex bead. Now, when the patient’s antibody is present, it will clump the beads, giving us a visible reaction
    • Classic Example: The Rheumatoid Factor (RF) test. Latex beads are coated with human IgG. The RF in the patient’s serum is an IgM antibody that attacks IgG, so it aggressively clumps the coated beads
  • Reverse Passive Agglutination
    • We flip the script to detect an antigen in a patient’s sample
    • The Trick: The antibody is attached to the latex beads. When the patient sample is added, if the specific antigen is present, it will bind to the antibodies on the beads and cause them to clump
    • Classic Example: Rapid tests for detecting bacterial antigens (like Staphylococcus aureus or Group A Strep) from a culture swab or patient specimen
  • Agglutination Inhibition
    • This is the most conceptually tricky, because the result is inverted. It’s a competition-based assay
    • The Goal: To detect the presence of a specific soluble antigen in a patient sample
    • The Process (2 steps)
      1. The patient’s sample is mixed with a limited, known amount of reagent antibody. If the patient has the target antigen, it will bind to and neutralize the antibody
      2. Next, reagent latex beads coated with the same antigen are added to the mixture
    • The Interpretation (Inverse Logic)
      • Positive Test (Antigen is present): The patient’s antigen already used up all the reagent antibody in step 1. So, when the beads are added, there is no free antibody left to clump them. NO AGGLUTINATION = POSITIVE
      • Negative Test (Antigen is absent): The reagent antibody had nothing to bind to in step 1. When the beads are added, the antibody is free to cause clumping. AGGLUTINATION = NEGATIVE
    • Clinical Use: A classic method for detecting certain drugs of abuse in urine

Labeled Immunoassays: The Height of Sensitivity

These methods use a reporter molecule (a “label”) to generate a signal, allowing for incredible sensitivity and precise quantitation. They are the core of modern, automated immunology

  • Enzyme-Linked Immunosorbent Assay (ELISA)
    • The Goal: A highly versatile and sensitive method to detect and quantify either antigen or antibody
    • The Principle: An enzyme is used as the label. After the binding reaction, a substrate is added that the enzyme converts into a colored or chemiluminescent product. The signal intensity is measured and is proportional to the amount of antigen/antibody present
    • Common Formats
      • Indirect ELISA: Used to detect patient antibody. The well is coated with antigen. Patient serum is added. If antibody is present, it binds. Then, an enzyme-labeled secondary antibody (anti-human Ig) is added to detect the patient’s antibody
      • Sandwich (or Capture) ELISA: Used to detect patient antigen. The well is coated with antibody. Patient serum is added. If antigen is present, it gets “captured.” Then, a second, enzyme-labeled antibody is added that binds to a different epitope on the captured antigen, completing the “sandwich”
    • Clinical Use: The workhorse of the modern lab for everything from infectious disease screening (HIV, Hepatitis) to hormone measurement
  • Western Blot
    • The Goal: A highly specific, confirmatory test used to identify antibodies against individual proteins within a complex mixture of antigens
    • The Process (Multi-step)
      1. Electrophoresis Proteins from a pathogen (e.g., HIV) are separated by their molecular weight in a gel
      2. Blotting The separated proteins are transferred (“blotted”) from the gel onto a stable nitrocellulose membrane
      3. Immunoassay The membrane strip is incubated with patient serum. If the patient has antibodies, they will bind to their specific protein bands. The strip is then washed and an enzyme-labeled anti-human antibody is added, followed by a substrate that creates colored bands exactly where the patient’s antibodies have bound
    • Interpretation: A positive result requires that the patient’s serum reacts with a specific pattern of protein bands (e.g., p24 and gp41 for HIV), making it a very specific confirmatory test