micro 20.2

In vitro assay

A laboratory test performed outside the body, such as in a test tube or on a laboratory surface.

Antigen-antibody complex formation in solution

They can form large antigen-antibody lattices that become visible as a precipitate.

Precipitin

A visible antigen-antibody complex formed when soluble antigens and antibodies create a large lattice.

Precipitin reaction

An in vitro assay that produces a visible precipitin through antigen-antibody lattice formation.

Polyclonal antibodies

They recognize multiple epitopes on an antigen, making extensive lattice formation more likely.

Monoclonal antibodies

They recognize only one epitope, so they are less likely to link many antigen molecules into a large lattice.

Lattice in antigen-antibody reaction

A large network formed when antibodies bind multiple antigen molecules together.

Antibody affinity and precipitation

Higher-affinity antibodies generally enhance precipitation because they bind antigens more strongly.

Antigen-antibody interaction stability

Antigen-antibody binding relies on relatively weak noncovalent bonds, which can break and reform.

Optimal antigen-antibody ratio

An optimal ratio must be present; this occurs in the zone of equivalence.

Zone of antibody excess

The condition in which there is too much antibody relative to antigen, preventing effective lattice formation and visible precipitation.

Zone of equivalence

The range where the antigen-to-antibody ratio is optimal for lattice formation and maximum precipitation occurs.

Zone of antigen excess

The condition in which there is too much antigen relative to antibody, causing precipitation to decrease.

Antigen addition in zone of antibody excess

Initially, little or no visible precipitate forms.

Precipitation at zone of equivalence

Maximum precipitation occurs because optimal lattice formation takes place.

Antigen concentration beyond zone of equivalence

Precipitation decreases because antigen becomes excessive.

Precipitin ring test

The relative amount of antigen-specific antibody in a serum sample.

Setting up the precipitin ring test

Antigen solution is placed at the bottom of test tubes, glycerol is added, and serially diluted antiserum is carefully layered above it.

Purpose of glycerol in precipitin ring test

It prevents the antigen and antibody solutions from mixing so they interact only at their interface.

Location of precipitin ring in test

At the interface between the antigen and antibody solutions when their ratio falls within the zone of equivalence.

Antibody titer

The reciprocal of the highest dilution of serum that still produces a positive reaction.

Antibody titer calculation example

If the highest dilution producing a visible precipitin ring is 1/16, the antibody titer is 16.

Significance of antibody titer

It provides a measure of the biological activity of antibodies directed against a particular antigen.

Utility of antibody titer vs mass measurement

Titer indicates how much functional antibody activity is directed against the antigen of interest rather than simply how much total antibody is present.

Ouchterlony assay

A qualitative antigen-antibody precipitation test performed in an agar gel using wells containing antigen and antiserum.

Another name for Ouchterlony assay

Double immunodiffusion.

Mechanism of Ouchterlony assay

Antigens and antibodies diffuse through agar toward one another; when they meet at the zone of equivalence, they form visible precipitin arcs.

Visibility of precipitin arcs in agar

The antigen-antibody lattice is too large to diffuse through the gel, so it becomes locked in place.

Important use of Ouchterlony assay

Detecting cross-reactivity by comparing an antiserum's reactions with closely related antigens.

Radial immunodiffusion vs Ouchterlony assay

Radial immunodiffusion quantitatively measures antigen concentration, whereas Ouchterlony is primarily a qualitative comparison of antigen-antibody reactions.