Lecture 16 Antigen-Antibody Interactions Part 1

Primary antigen-antibody interactions

Initial interaction between an antigen and antibody
-rapid
-reversible
-without visible effects (during diagnostic testing)

Secondary antigen-antibody interactions

Interactions between multivalent antigens and antibodies
-slow
-irreversible
-with visible effects (during diagnostic testing)

What serves as the basis for many immune assays?

antigen-antibody reactions

Valence

the combining capacity (# of binding sites) of a molecule (antigen or antibody)
-univalent
-bi- or divalent
-multivalent

univalent binding

a complex between one antibody and one epitope

Divalent binding

a complex between one antibody and two epitopes

Multivalent binding

-A complex between one antibody and three or more epitopes
(Pertaining to a multimeric antibody containing many antigen binding sites) (IgM or IgA - Multivalent antibody)

Unideterminant

single type of antigenic determinant (epitope)
-ex. haptens or many polysaccharides, homopolymers

Multideterminant

Many different types of antigenic determinants (epitopes)
-ex. proteins

Cross-link

a physical or chemical bond that associates antibodies and antigens together in a network of interconnecting components

What is required for precipitation, agglutination, or complement activation to take place?

Various antigen molecules need to be cross-linked by antibodies.

Can precipitation, agglutination, or complement activation occur with a monovalent antigen?

No, it is only possible if the antigen is multivalent.

What kind of antibody is required for cross-linking to occur?

The antibody must be divalent or multivalent.

What kind of antigen is required for cross-linking with antibodies?

The antigen must be multivalent.

Cross-linked complexes

facilitate several goals of the immune system such as neutralizing, destroying, or eliminating pathogens.
-Pathogenic microorganisms are good examples of multideterminant, multivariant antigens since they express an array of epitopes that B and T cells respond to during and immune response.

what types of forces are involved in the antigen-antibody interaction?

No covalent bonds. Van der Waals forces, Electrostatic interactions, Hydrogen bonds, hydrophobic interactions.

-forces are relatively weak

-requires very close proximity among the interacting moieties

-must occur over an area large enough to allow for all available connections

-The fit between antigen and antibody is specific, like a "lock and key"

-Antigen-antibody complexes can be readily dissociated by: low/high pH and high salt concentrations.

Association constant

a measurement of the affinity of the antibody for the antigenic epitope
-The primary interaction between an antigen and antibody is usually in flux.

Intrinsic association constant

when all the associated antibodies are identical

Average association constant

when all the associated antibodies are heterogenous

equilibrium dialysis method

a way to determine the association constant
-ligand= hapten or antigenic epitope
-a semipermeable membrane allows passage of appropriately sized molecules
-Antibodies are too large to pass through the membrane
-Ligand will diffuse across the membrane
-Total amount of ligand will be greater on the side containing Ab
The difference in ligand concentration between the two compartments represents the ligand bound to antibody concentration (AgAb)

The higher the affinity of the antibody,

the more ligand is bound

Affinity

the strength of a single antigen-antibody interaction
-It is the intrinsic association constant that characterizes identical antibodies binding with a univalent epitope or hapten.
-Once an antibody's first antigen binding arm attaches to an antigen on a solid support, the chances of a bivalent interaction are greatly improved.

What does avidity refer to?

the strength of all antigen-antibody interactions combined.

How is avidity related to multivalent interactions?

Multivalent interactions may form large, stable (high avidity) structures.
-Several antibodies may bind the antigen, each recognizing a different epitope.

How does IgM antibody avidity compare to IgG antibody avidity?

IgM antibodies have a higher avidity than IgG antibodies (related to the valency of the antibody).

Agglutination reactions

antibody cross-linked reactions with multivalent particulate (insoluble) antigens

-dependent on the correct proportion of antibody

-high dilutions of serum do not usually cause antigen agglutination

-The highest dilution of serum that can still cause agglutination is termed the titer.

Zeta potential

an electrical potential created between charged particles that prevents them from getting very close to each other.
-Introduces agglutination difficulties between charged particles and antibodies

Coombs test (anti-immunoglobulin test)

-Immunoglobulins of one species are immunogenic when injected into another species
-Anti-immunoglobulins bind with antigenic determinants present on the Fc portion of the antibody, leaving the Fab portion to react with antigen.
-Both direct and indirect tests use heterologous anti-immunoglobulins to detect a reaction between Ig and antigens.

Direct coombs

anti-immunoglobulins are added to the particles suspected of having antibodies bound on their surfaces
-measures Ab already bound to RBCs

Indirect Coombs

Used to detect the presence of antibodies specific to antigens on a particle
-measures antibody in the serum

Direct Agglutination

Agglutination that occurs between antibodies and antigens when the antigen is a natural constituent of a particle

Passive Agglutination

Agglutination that occurs between antibodies and soluble antigen that had been attached to an insoluble particle

Precipitation reaction

-Take place when antibodies and soluble antigens are mixed

-Occurs when divalent antibodies cross-link with multivalent antigen molecules to form a lattice

-At a certain size, the AgAb complex loses solubility and precipitates out of solution

What does the precipitate formed depend on?

it depends on the optimal proportions of the reactant antigens and antibodies

Nephelometry

measurement of the turbidity (light scatter) caused by AgAb complexes in solution

Gel Precipitation reactions

-involves separating a mixture of proteins in an electrical field
-Diffused antibodies are used to detect the proteins

Western Blots (immunoblots)

-Antigen (or a mixture of antigens) is separated in a gel
-Separated material is transferred onto protein-binding sheets (ex. nitrocellulose) using electroblotting method
-Antibody is applied to the sheet to bind to the specific antigen
-Labeled antibody or labeled anti-immunoglobulin is used to localize the antigen-antibody complex

Used widely in research and clinical laboratories to detect and characterize antigens (ex. HIV infection confirmation)

What do immune complexes activate?

the classical complement pathway, assembling a membrane attack complex (MAC)
-MAC adheres to and lyses a pathogen cell membrane

What do immune complexes bring together?

high-density Fc antibody regions interacting with antigens
-Increases likelihood phagocytic cells expressing similar Fc receptors will bind to these Fc antibody regions and initiate phagocytosis