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19 Cards in this Set

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combined strength of non-covalent interactions between a single Ag-binding site on an Ab with a single epitope
takes into consideration the sum of all interactions between the combining sites of the Ab (ex. two for IgG) and all the epitopes of the Ag
-not a simple “sum” since multivalent binding of the Ag to Ab confers an enhancing effect
-Ex. IgG Ab binding to Ag on virus
-Hypothetical numbers:
affinity = 1; avidity = 100 (because of enhancing effect)
for IgM: affinity = 0.5 (less than IgG) but with more combining sites, it has a greater enhancing effect, avidity = 2000.
low affinity Ab may have the problem of _______
cross reactivity
_________ Ab have less cross reactivity than __________
polyclonal Abs
have a mixture of multiple epitope specificities
ABO blood groups
-glycoproteins expressed on RBCs
-subtle differences in the terminal sugar residues determine “type”
-Humans have Abs in our blood that are specific for a microbial Ag
-anti-microbial Abs bind (cross-react) to sugars on RBCs
Steps in generation of monoclonal antibodies (mAb)
1. Immunize
2. Isolate cells (spleen)
3. Fuse cells with myeloma
4. Select for Ab producers
5. Mass produce
Describe precipitation
-Occurs when Ab binds to soluble Ags (eg. free protein or carbohydrate)

Ag-Ab forms a large lattice that leads to increased turbidity (can be measured by spectrophotometry) and eventually, preciptiation
Use of precipitation in clinical situations
Check for neutralization ability

Radial immunodiffusion (RID)
Radial immunodiffusion (RID)
-Precipitation reaction in a gel
-Check for presence of specific Abs to an Ag
-Compare relative concentration of Ag or Ab
Two examples of Radial immunodiffusion (RID)
Mancini method – testing one variable

Ouchterlony method (double immunodiffusion) – can compare reactivity of an Ab with various Ags
Describe Agglutination
-Used for cell bound Ags (insoluble)
-Ab binding to Ag will cause clumping of cells that can be visualized
Describe Hemagglutination
routinely used for testing blood types
-Ab binding to carbohydrates on RBCs will cause clumping and cells will NOT settle to a small “button”
-Can be used to check relative concentration of an Ab (as long as the Ab binds to RBCs)
Immunoelectrophoresis (IEF)
Two step procedure
1) Electrophoretic separation of the Ags in agarose gel

2)Diffusion of Abs from a trough cut in the gel parallel with the direction of migration of the Ag
Immunoblotting/Western blotting
-Electrophoresis of the protein (Ag) of interest
-Transfer to nitrocellulose
-Incubate nitrocellulose with Ab (primary antibody) specific for Ag
-Add enzyme-linked Ab (secondary Ab) that will bind to primary Ab
-Add substrate to view the bands
Western blot for HIV detection
-Lyse HIV with detergent (break up the virus into individual proteins)
-Run on gel and transfer to nitrocellulose
-Add patient samples (potential anti-HIV antibodies) to nitrocellulose
-Detect patient antibodies by adding enzyme-linked anti-human Ig
Enzyme-linked immunosorbent assay
useful when Ag is in low concentration
Detection of serum proteins (ex. cytokines): Quantitative
-Attach a fluorochrome to Ab
-Ab added to cells with Ag
Visualize cells or tissues via MICROSCOPY or FACS (fluorescence activated cell sorter)
-Flow cytometer
-Can be used to quantify number of Ag positive cells
-Can isolate/purify cells of interest (research
Other Ab-based immunological assays
Radioimmunoassay (RIA)


Immunoelectron microscopy

Antibody microarrays