Chap 10 - precipitation and agglutination reactions

What is precipitation in immunology?

The process of combining soluble antigens with soluble antibodies to produce visible insoluble complexes.

What is agglutination?

The process by which particulate antigens, such as cells, aggregate to form larger complexes in the presence of a specific antibody.

What are the three phases of antibody and antigen reactions?

1. Primary: binding of an antibody's individual site to a single epitope on an antigen. 2. Secondary: includes precipitation, agglutination, and complement fixation. 3. Tertiary: involves inflammation, phagocytosis, deposition of immune complexes, immune adherence, and chemotaxis.

What is affinity in the context of antigen-antibody binding?

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

What does avidity represent?

The sum of all attractive forces between an antigen and antibody, indicating the strength of binding for multivalent antibodies to multivalent antigens.

What is the Law of Mass Action in relation to antigen-antibody binding?

It states that all antigen-antibody binding is reversible and governed by the equilibrium between free reactants and bound reactants.

What is the prozone phenomenon in precipitation reactions?

A condition where antibody excess leads to antigen combining with only one or two antibody molecules, preventing cross-linkages.

What is the zone of equivalence in precipitation reactions?

The state where the number of multivalent sites of antigen and antibody are approximately equal.

What occurs in the postzone of a precipitation reaction?

Antigen excess surrounds small aggregates, preventing the formation of a lattice network.

What is immunoturbidimetry?

A method of measuring precipitation where initial turbidity is followed by precipitation, with detection light aligned with incident light.

What is nephelometry?

A technique that measures light scattered at a specific angle as it passes through a suspension, correlating the amount of light scattered to the complexes formed.

What is endpoint nephelometry?

A method where the reaction is allowed to run to completion, but particles may fall out of solution, decreasing light scatter.

What is kinetic (rate) nephelometry?

A method that measures the rate of increase of light scattering immediately after reagent addition, directly related to the concentration of the analyte.

What is passive immunodiffusion?

A technique where electrical current is used to accelerate the diffusion process, utilizing agar and agarose as the medium.

What is radial immunodiffusion?

A method where antibody is uniformly distributed in a gel, and antigen is applied to a well, leading to diffusion and precipitation.

What does the endpoint method in radial immunodiffusion measure?

The square of the diameter of the precipitate is directly proportional to the concentration of the antigen.

What is the Fahey method in radial immunodiffusion?

A measurement taken before the endpoint is reached, where the diameter is proportional to the logarithm of the concentration.

What is Ouchterlony double diffusion?

A method where both antigen and antibody diffuse in a semisolid medium, allowing comparison of antigens based on patterns formed.

What does fusion of lines in Ouchterlony double diffusion indicate?

Serologic identity between the antigens being compared.

What factors affect the rate of diffusion in passive immunodiffusion?

Size of particles, temperature, gel viscosity, amount of hydration, and interactions between the matrix and reactants.

What is the significance of the zone of equivalence in precipitation reactions?

It is the optimal state for maximum precipitation where antigen and antibody concentrations are balanced.

How does the concentration of antibody affect the rate of kinetic nephelometry?

The rate change in light scattering is directly related to the concentration of the analyte if the antibody concentration is kept constant.

What is the purpose of the central well in passive immunodiffusion?

It allows antigens to be compared with one another.

What does fusion of lines at their junction indicate in serological testing?

Serologic identity between the antigens.

What does a spur in the fusion of two lines indicate?

Partial identity, with the spur pointing to the simpler antigen.

List two sources of error in passive immunodiffusion.

1. Overfilling/underfilling the wells. 2. Incorrect incubation time/temperature.

What is the principle behind electrophoresis?

It separates molecules according to differences in their electric charge.

Describe the process of immunoelectrophoresis.

A double diffusion technique where the antigen is electrophoresed, and an antibody is placed in a trough to diffuse, forming precipitin lines where specific antigen-antibody combinations occur.

What is the purpose of immunofixation electrophoresis?

To trap antigen-antibody complexes in the gel after electrophoresis for easier viewing.

Identify a source of error in electrophoresis related to current application.

Application of current in the wrong direction.

What can happen if the current in electrophoresis is too strong?

It may generate heat that can denature the proteins.

What is agglutination in immunology?

The visible aggregation of particles caused by the combination with antibodies.

What are agglutinins?

Antibodies that produce agglutination reactions.

What are the two steps of the agglutination process?

1. Sensitization - initial binding of antibodies. 2. Lattice Formation - formation of large aggregates.

How does the class of antibody affect sensitization in agglutination?

IgM (valence of 10) is more efficient than IgG (valence of 2) in binding.

What factors enhance lattice formation in agglutination?

1. Decreasing ionic strength of the buffer. 2. Use of albumin. 3. Increasing viscosity. 4. Agitation and centrifugation. 5. Temperature.

What is the effect of temperature on IgM and IgG during agglutination?

IgM reacts best at 4° to 27°C, while IgG reacts best at 30° to 37°C.

What is direct agglutination?

A type of agglutination reaction where antigens are directly agglutinated by antibodies.

What is the significance of the ionic strength of the buffer in agglutination?

Lower ionic strength enhances the ability of antibodies to bridge the gap between particles.

What role does albumin play in agglutination reactions?

It neutralizes surface charge, facilitating the association of particles.

How does increasing viscosity affect agglutination?

It enhances the likelihood of particle association.

What is one consequence of incorrect concentrations of antigen or antibody in electrophoresis?

It can lead to incomplete separation of the components.

What happens if the electrophoresis time is incorrect?

It can result in inadequate separation of molecules.

What is the role of agitation in enhancing agglutination?

It increases cell-to-cell contact, promoting the formation of aggregates.

What is the main application of immunoelectrophoresis?

It is used as a screening tool for differentiating more than 30 serum proteins, including immunoglobulins.

What is bacterial serotyping?

A method that involves mixing a suspension of bacteria with standardized antiserum to identify bacterial strains.

What is hemagglutination and its best example?

A process used to determine the presence of A or B antigens on red blood cells, best exemplified by blood typing using IgM antibodies.

What is passive agglutination?

A technique that employs particles coated with antigens not normally found on their surface, such as red cells or latex, to detect various antibodies.

What treatments are used to prepare cells for passive agglutination tests?

Cells are treated with formalin or glutaraldehyde to prevent lysis, and proteins can be more easily attached after treatment with tannic acid.

What conditions can passive agglutination tests detect?

Rheumatoid factor, antinuclear antibodies, antibodies to group A streptococcus, and viruses like CMV, rubella, and varicella-zoster.

What is reverse passive agglutination?

A technique where antibodies are attached to carrier particles to detect microbial antigens, with active sites facing outward.

What microbial antigens can be detected using reverse passive agglutination?

Group B streptococcus, Staphylococcus aureus, Neisseria meningitidis, Group A streptococcus, and Candida albicans.

What is agglutination inhibition?

A method based on competition between particulate and soluble antigens for limited antibody combining sites, where lack of agglutination indicates a positive reaction.

What is the Coomb's test?

Also known as antiglobilin mediated agglutination, it detects non-anti-body by mixing with a second antibody, using anti-human globulin as a key component.

What does the direct antiglobulin test indicate?

It demonstrates the in vivo attachment of antibody or complement, serving as an indicator of autoimmune hemolytic anemia, hemolytic disease of the newborn, or transfusion reactions.

What is the process of the indirect antiglobulin test?

A two-step process where red blood cells and antibody combine at 37°C, then antihuman globulin is added to detect bound antibodies.

What is the purpose of turbidimetry and nephelometry in immunological testing?

These are techniques used to measure the concentration of particles in a solution, often used in immunoassays.

What is the principle behind the Particle-enhanced turbidimetric inhibition immunoassay (PETINIA)?

In this assay, latex beads coated with the analyte of interest are incubated with a reagent antibody; high analyte levels prevent turbidity, while low levels cause aggregate formation.

What is cross-reactivity in immunological tests?

It occurs when antibodies react with similar antigenic determinants, potentially leading to false results.

How can cross-reactivity be minimized?

Using monoclonal antibodies can help avoid most cross-reactivity.

What is a heterophile antibody?

An antibody that can agglutinate red cells by reacting with an antigen on the cell other than the one being tested.

What is the significance of rheumatoid factor in reverse passive agglutination?

Rheumatoid factor can react with any IgG present, complicating the interpretation of results.

What is the role of tannic acid in passive agglutination tests?

Tannic acid treatment enhances the attachment of proteins to cells, improving the efficacy of the tests.

What is the importance of quality control in immunological testing?

Quality control ensures the accuracy and reliability of test results, minimizing false positives and negatives.

What is the expected outcome in agglutination inhibition assays?

A lack of agglutination indicates a positive reaction, confirming the presence of the target antigen.

What is the role of indicator particles in agglutination inhibition?

Indicator particles contain the same antigen being measured and compete with the patient's soluble antigens for antibody binding.

What is the significance of the strength of reaction in the Coomb's test?

The strength of the reaction correlates with the amount of antibody coating the red blood cells, indicating the level of immune response.

What types of antibodies can be detected using the indirect antiglobulin test?

It can detect unexpected antibodies during compatibility testing and determine blood group antigens.