Precipitation Reactions in Immunology
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Precipitation Reactions in Immunology

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Questions and Answers

What is a precipitation reaction in immunology?

It is the combination of soluble antigen with soluble antibody, producing visible insoluble complexes.

Define affinity in the context of antibody-antigen interactions.

Affinity refers to the initial force of attraction between a single Fab site on an antibody and a corresponding antigen epitope.

What does avidity signify in antigen-antibody binding?

Avidity represents the sum of all attractive forces between an antigen and an antibody, indicating the overall strength of their binding.

Explain the Law of Mass Action in relation to antigen-antibody reactions.

<p>The Law of Mass Action states that the binding of antigens and antibodies is reversible and that free reactants are in equilibrium with bound reactants.</p> Signup and view all the answers

What could be the consequence of applying current in the wrong direction during electrophoresis?

<p>It can result in ineffective separation of biomolecules, leading to incorrect analytical results.</p> Signup and view all the answers

Describe the Pro-zone phenomenon in precipitation reactions.

<p>The Pro-zone occurs when there is an excess of antibodies, potentially leading to false negative results.</p> Signup and view all the answers

What happens during the Post-zone phenomenon?

<p>The Post-zone occurs when there is an excess of antigen, which can also cause a false negative reaction.</p> Signup and view all the answers

How does the incorrect pH of the buffer affect the electrophoresis process?

<p>An incorrect pH can alter the charge of the molecules, affecting their migration rate and overall separation efficiency.</p> Signup and view all the answers

What is the Zone of Equivalence in the context of precipitation reactions?

<p>The Zone of Equivalence is the situation where multivalent sites of antigen and antibody are approximately equal, leading to optimal precipitation.</p> Signup and view all the answers

Differentiate between single and double diffusion in immunodiffusion methods.

<p>Single diffusion involves a single layer of gel where only one reactant diffuses, while double diffusion involves two reactants diffusing in separate directions within the gel.</p> Signup and view all the answers

Explain the significance of the sensitization phase in the agglutination process.

<p>The sensitization phase is crucial as it initiates the antigen-antibody binding required for the subsequent lattice formation.</p> Signup and view all the answers

What is turbidity and how is it measured in a fluid medium?

<p>Turbidity is the cloudiness of a solution, measured by the reduction in light intensity caused by scattering and absorption using a detection device.</p> Signup and view all the answers

What factors influence lattice formation during agglutination?

<p>Lattice formation is influenced by environmental conditions and the relative concentrations of antigen and antibody.</p> Signup and view all the answers

What is the method used to count particles in PACIA?

<p>A laser beam in an optical particle counter is used to count particles.</p> Signup and view all the answers

How does agglutination affect the counting of particles in the PACIA method?

<p>Agglutination increases the size of clumps, making larger clumps uncountable.</p> Signup and view all the answers

What size are the latex particles used in the PACIA reaction?

<p>The latex particles have a diameter smaller than 1 μm.</p> Signup and view all the answers

List two causes of false positives in agglutination tests.

<p>Overcentrifugation and contaminated glassware are two causes of false positives.</p> Signup and view all the answers

What effect can a delay in testing have on agglutination results?

<p>A delay in testing can lead to false negatives.</p> Signup and view all the answers

Explain the significance of under centrifugation in agglutination tests.

<p>Under centrifugation can cause false negatives by not adequately separating agglutinated from non-agglutinated particles.</p> Signup and view all the answers

What phenomenon might occur if there is excessive antigen present in a sample?

<p>The Prozone/Post zone phenomenon might occur with excessive antigen.</p> Signup and view all the answers

Identify one reason why adequate washing of cells is important in agglutination tests.

<p>Inadequate washing can result in false negatives.</p> Signup and view all the answers

What is the principle behind nephelometry?

<p>Nephelometry measures the amount of light scattered at specific angles as it passes through a suspension, which reflects the concentration of the solution.</p> Signup and view all the answers

What range of angles do nephelometers typically measure?

<p>Nephelometers measure light scatter at angles ranging from 10 degrees to about 90 degrees.</p> Signup and view all the answers

Which immunoglobulins can be quantified using rate nephelometry?

<p>Rate nephelometry can quantify IgG, IgA, IgM, and IgE, but not IgD.</p> Signup and view all the answers

What is passive immunodiffusion?

<p>Passive immunodiffusion is the process where antigen diffuses through a support medium without electrical current to speed up the reaction.</p> Signup and view all the answers

In Oudin single diffusion, how does precipitation occur?

<p>Precipitation occurs as the antigen moves down into the agarose gel, creating a gradient proportional to the amount of antigen present.</p> Signup and view all the answers

Describe the procedure of radial immunodiffusion.

<p>In radial immunodiffusion, a well is cut into the gel containing uniformly distributed antibody, and the antigen is applied to this well, allowing diffusion.</p> Signup and view all the answers

What factors affect the rate of diffusion in passive immunodiffusion?

<p>The rate of diffusion is affected by particle size, temperature, gel viscosity, and hydration levels.</p> Signup and view all the answers

What happens at the zone of equivalence in radial immunodiffusion?

<p>At the zone of equivalence, antigen-antibody combinations occur in changing proportions, resulting in a stable lattice network in the gel.</p> Signup and view all the answers

What is the significance of protein A in Staphylococcus aureus?

<p>Protein A binds the FC portion of IgG antibodies, enhancing the bacterium's ability to evade the immune response.</p> Signup and view all the answers

Explain the principle behind agglutination inhibition reactions.

<p>Agglutination inhibition reactions are based on the competition between particulate and soluble antigens for antibody sites, where lack of agglutination indicates a positive result.</p> Signup and view all the answers

What role do red blood cells (RBCs) play in hemagglutination inhibition tests?

<p>RBCs serve as the indicator particles that show agglutination when viral particles link them together, which can be inhibited by the presence of patient antibodies.</p> Signup and view all the answers

Differentiate between the direct and indirect antiglobulin tests.

<p>The direct test detects in vivo RBC sensitization, while the indirect test assesses in vitro sensitization and is used for crossmatching and antibody detection.</p> Signup and view all the answers

What are the applications of the Coomb's test?

<p>The Coomb's test is used to investigate hemolytic disease of the newborn, transfusion reactions, autoimmune hemolytic anemia, and drug-induced hemolytic anemia.</p> Signup and view all the answers

Describe the source of polyclonal antibodies in AHG preparation.

<p>Polyclonal antibodies are prepared from various animal sources, such as rabbits and goats, using conventional technology.</p> Signup and view all the answers

What is the classic example of an agglutination inhibition reaction?

<p>The human chorionic gonadotropin (hCG) test is a classic example of agglutination inhibition.</p> Signup and view all the answers

How do viruses trigger spontaneous agglutination of RBCs?

<p>Viruses link RBCs together through their receptors, causing spontaneous agglutination when they are present.</p> Signup and view all the answers

Study Notes

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.

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Precipitation and Agglutination PDF

Description

Explore the fundamentals of precipitation reactions, where soluble antigens combine with antibodies to form visible complexes. Understand key concepts like affinity, avidity, and the law of mass action, alongside the significance of the precipitation curve in interpreting reaction results.

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