immunologic procedures - precipitation and diffusion assays

What is specificity?

The ability of a particular antibody to combine with one antigen over another

What site on the antibody is responsible for specificity?

Fab region, or variable region

How do antigens bind to antibodies?

With weak, reversible, non-covalent bonds

What influences immune complex formation?

The goodness of fit, strong forces, and high affinity

What is affinity?

The bond that can form between a single antigenic determinant and a paratope (variable region on Ab)

What is avidity?

The overall bond strength between a multivalent antibody and the antigen

What is cross reactivity?

When a second structurally similar antigen can bind to an antibody

What are factors that influence antigen:antibody binding (or reaction)?

The pH, temperature, Ig type, and concentration of Ab or Ag

Which Ig is the most efficient for forming immune complexes?

IgM

What is sensitivity?

The lowest detectable amount of an Ag or Ab required to elicit an immune response

What is precipitation?

The combination of soluble Ag to soluble Ab to produce insoluble complexes, or the aggregation of soluble antigens

What is the prozone?

The zone where antibody concentration exceeds antibody concentration

Ratio is NOT 1:1

No lattice formation

What is the zone of equivalence?

The zone of precipitation where there are almost equal concentrations of antigens and antibodies

Ratio is 1:1

Lattice formation occurs and precipitation is seen

What is the postzone?

The zone of precipitation where the concentration of antigens exceeds antibodies

Ratio is NOT 1:1

No lattice formation occurs

What is the result if a precipitation test shows no reaction?

A false negative

What causes false negatives in precipitation reactions?

When there is either too much antibody (prozone false negative) or too much antigen (postzone false negative)

How do we correct for a false negative?

Serially dilute the specimen

How do we report out the result of a precipitation test?

The titer, or the highest dilution of serum that shows a positive reaction (clumping)

What is immunodiffusion?

Where antigens and antibodies precipitate through passive diffusion, no electrical current

What is the Oudin single diffusion test? What does it detect?

Single diffusion test that occurs in a tube

It detects antigens or antibodies

What is the radial diffusion test? What does it detect?

Single diffusion that measures the diameter of the precipitation ring after 18 hours

Detects blood proteins, or antigens in general

What is the Ouchterlony double diffusion test? What does it detect?

Double diffusion of both antigen and antibody in agar that are opposite of each other. When antigen and antibody cross paths, they form a line

Detects fungal antigens and autoantigens

What does the identity pattern look like on an Ouchterlony test? What is the interpretation?

When two antigens with the same epitope come into contact with the antibody, an arc line forms in the gel

Interpretation: identity of both epitopes, common determinant

What does the non-identity pattern look like on an Ouchterlony test? What is the interpretation?

When two different epitopes interact with an antibody, the precipitation lines cross

Interpretation: Non-identity, no common determinant

What does the partial identity pattern look like on an Ouchterlony test? What is the interpretation?

When two antigens with a shared common determinant and one non-shared determinant interact with the antibody, an arc line is formed with the more complex antigen creating a spur line pointing towards the simpler antigen

Interpretation: partial identity and partial common determinant

How can we measure precipitation in a fluid model?

Through turbidity and nephelometry

For turbidity, how do we measure the results?

Through light absorbed, reflected, or scattered

What is nephelometry?

Light scattering from a liquid sample at a particular angle

For nephelometry, what do we measure?

The relative light scatter of macromolecular complexes, which is proportional to the immunoglobulin concentration