Precipitation Reactions in Immunology

Precipitation Reactions

• Involves the combination of soluble antigens with soluble antibodies to form visible, insoluble complexes.
• Affinity refers to the initial force of attraction between a single Fab site on an antibody molecule and a single epitope on the corresponding antigen.
  • Attraction results from various bonds: Ionic, hydrophobic, hydrophilic, and Van der Waals forces.
• Avidity is the sum of all attractive forces between an antigen and an antibody, representing the overall strength of the antigen-antibody binding.
• Law of Mass Action: All antigen-antibody binding is reversible and governed by the law of mass action, where free reactants are in equilibrium with bound reactants.

Precipitation Curve

• Three zones of antigen-antibody ratios exist, impacting precipitation reaction results:
  • Pro-zone: Antibody excess can lead to false negative reactions. Remedy: Perform dilution.
  • Post-zone: Antigen excess leads to false negatives. Remedy: Repeat the test after a week.
  • Zone of equivalence: Optimum precipitation with an equal number of multivalent sites of antigen and antibody.

Types of Precipitation Reactions

Precipitation in a Fluid Medium

• Turbidimetry: Measures the turbidity (cloudiness) of a solution by determining light reduction caused by reflection, absorption, or scattering. Measurement units are absorbance, reflecting the ratio of incident to transmitted light.
• Nephelometry: Measures scattered light at a specific angle from the incident beam as it passes through a suspension. Light scattering is proportional to the solution's concentration.

Precipitation by Passive Immunodiffusion

• No electrical current is used to accelerate this process.
• Support medium: Agar, agarose, and gel.
• Diffusion rate depends on particle size, temperature, gel viscosity, and hydration.
• Oudin Single Diffusion: Antibody is incorporated into agarose within a test tube. Antigen is layered on top, causing precipitation as antigen moves down the tube, proportional to the antigen concentration.
• Radial Immunodiffusion: Antibody is uniformly distributed within the support gel. Antigen is placed in a well. Precipitation occurs as antigen diffuses outward, forming a stable lattice network in the zone of equivalence.

Agglutination

• A process where particulate antigens, such as cells, aggregate to form larger complexes when a specific antibody is present.

Steps of Agglutination

• Sensitization Phase: Initial antigen-antibody combination through single antigenic determinants on the particle surface.
• Lattice Formation: Represents the sum of interactions between the antibody and multiple antigenic determinants on a particle. Depends on environmental conditions and relative antigen and antibody concentrations.

Types of Agglutination Reactions

• Direct Agglutination: Antigen is directly bound to a particle. Useful for identifying antigens or antibodies.
• Passive Agglutination: Antigen or antibody is attached to an inert carrier particle (e.g., latex beads). Enhanced sensitivity and broader applications.
• Reverse Passive Agglutination: Antibody is attached to particles, while the antigen is free in the sample. Rapid diagnosis, often used in clinical laboratories.
• Agglutination Inhibition: Based on competition between particulate and soluble antigens for limited antibody-combining sites. Lack of agglutination indicates a positive reaction.
  • Hemagglutination Inhibition: RBCs are the indicator particles. Used to detect antibodies for viruses like rubella, mumps, measles, influenza, and others.
• Antiglobulin-mediated Agglutination (Coomb's Test or AHG): Detects non-agglutinating antibodies by coupling with a second antibody.
  • Direct Antiglobulin Test (DAT): Investigates in vivo RBC sensitization (e.g., hemolytic disease of the newborn, autoimmune hemolytic anemia).
  • Indirect Antiglobulin Test (IAT): Investigates in vitro RBC sensitization (e.g., cross-matching, antibody detection, identification, and phenotyping).

Errors in Agglutination

• False Positives: Can occur due to over-centrifugation, contaminated glassware, autoagglutination, saline storage in glass bottles, cross-reactivity, rheumatoid factor, heterophile antibodies, and delayed reading.
• False Negatives: Can occur due to under-centrifugation, inadequate washing, inactive reagents, delay in testing, prozone/post-zone phenomenon, incorrect incubation temperatures, insufficient incubation time, failure to add antiglobulin reagent, etc.

Sources of Error in Electrophoresis

• Incorrect current direction, pH of buffer, electrophoresis time, concentration of antigen and antibody, and amount of current applied can all lead to inaccurate results.

Characteristics of Different Precipitation Methods:

• Oudin's Test: Single diffusion, Single Dimension.
• Oakley and Fulthrope (Modified Oudin): Double diffusion, Single Dimension.
• Rocket Immunoelectrophoresis: Single diffusion, Single Dimension.
• Radial Immunodiffusion: Single diffusion, Double Dimension.
• Ouchterlony: Double diffusion, Double Dimension.
• Countercurrent Immunoelectrophoresis: Double diffusion, Single Dimension.