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Introduction to Immunoassays

• Immunoassays are used to detect and measure antigens or antibodies.
• Label-free assays detect antibody-antigen complexes by their ability to scatter light.
• Visible complexes form due to Ab-Ag interaction.
• A constant concentration of Ab can be used to measure specific Ag.
• Alternatively, coated cells or particles bind to the target Ab or Ag, causing precipitation or agglutination.

Learning Objectives

• Visualizing antibody-antigen (Ab-Ag) complexes
• Understanding label-free assays
• Defining pre-zone, equivalence-zone, and post-zone
• Understanding the principles of immunodiffusion assays
• Providing examples of immunodiffusion assays
• Describing the Latex agglutination assay (LAA) procedure

Label-Free Assays - Equivalence

• Pro zone: Antibody excess, little precipitation.
• Zone of equivalence: Optimal amount of Ag and Ab, maximum precipitation.
• Post zone: Antigen excess, little precipitation.

Immunodiffusion Assays

• Ouchterlony Immunodiffusion Assay (Double Diffusion):

  • Antibody (Ab) placed in center well, antigens in surrounding wells.
  • Ab-Ag complexes form precipitin lines
  • Patterns indicate similarities or differences between Ag.
  • Patterns include identity, partial identity, and non-identity.

• Single Radial Immunodiffusion (sRID) or Mancini Technique:

  • The precipitin ring size depends on antigen concentration, antibody concentration, and sample volume.
  • Rings form when Ag concentration is within the zone of equivalence.
  • Precipitate diameter is proportional to antigen concentration (linear plot).

Activity - sRID Data

• Example data tables for X-179A and X-187
• Results for sRID assays including untreated, acid, etc

Immunodiffusion Assays - Electroimmunodiffusion

• Rocket Immunodiffusion (Laurell Technique):
  • Electrophorosis of the antigen(Ag) in an agarose gel containing antibody(Ab).
  • The negatively charged antigen migrates towards a positive charge.
  • Rocket height is proportional to the antigen concentration.

Immunodiffusion Assays - Immunoelectrophoresis

• Investigation of proteins in a complex mixture to identify immunological characteristics.
• Separating the protein samples by electrophoresis in circular wells.
• Antiserum is placed in a central slot, antibodies precipitate proteins during diffusion.

Crossed Immunoelectrophoresis

• First dimension: Separating proteins by electrophoresis.
• Second dimension: Separated proteins further electrophoresed into a gel containing antibodies against the protein.
• Precipitates form, peak or rocket shapes, area proportional to amount of Ag.

Latex Agglutination Assay (LAA)

• Quick alternative to traditional immunodiffusion.
• Samples (blood, saliva, cerebrospinal fluid (CSF))
• Latex beads coated with antigen for antibody capture.
• Steps include: preparation of standard and samples, serial dilutions, preparation of beads, sample loading, and spectrophotometer reading.
• Total duration is two hours

The Hook Effect

• High analyte concentrations inhibit complex formation, restricting sensitivity.
• Leading to lower detection limits.

This quiz explores the basics of immunoassays, including the detection and measurement of antigens and antibodies. It covers label-free assays, the principles of immunodiffusion, and specific procedures like the Latex agglutination assay. Test your understanding of antibody-antigen interactions and the factors affecting precipitation.