Chapter 6, Lecture 1
Richard L. Myers, Ph.D.
Department of Biology
Southwest Missouri State
Temple Hall 227
Telephone: 417-836-5307
Email: [email protected]
• TopClass:
Antigen-Antibody interactions
• Similar to enzyme-substrate reactions
• Involves noncovalent interactions
– between epitope and CDRs of antibody
– the reaction is very specific
• used in diagnosing disease
• monitoring level of the humoral response
• identifying certain molecules
• Ag-Ab reactions differ and speed and
Strength of Ag-Ab reactions
• Involves noncovalent interactions
hydrogen bonds
ionic bonds
hydrophobic interactions
van der Waals interactions
• Requires many of these interactions
– operating over a small distance (1 angstrom)
• Therefore, depends upon a very close fit
Antibody affinity
• Defined as the strength of the sum total of
noncovalent interactions
• Low-affinity antibodies bind antigen weakly
• High-affinity bind more tightly
• Ag-Ab association can be determined by
equilibrium dialysis
Antibody avidity
• Defined as the strength of multiple
interactions between multivalent antibody
and antigen
• IgM has a low affinity compared to IgG, but
has a high avidity
• Ag-Ab reactions are highly specific, but
– Ab may cross-react with an unrelated Ag
– affinity is usually less
• An example of a cross-reacting Ab is the
ABO blood-group antigens
– originally elicited by microbial antigens
– will cross-react with similar antigens on RBCs
Precipitin reactions
• Results when antigen and antibody interact
• Forms a lattice that becomes visible
– antibody must be bivalent
– called precipitins
• Reaction may take place in fluids
– dilute the antibody, hold antigen constant
– used as a rapid test for presence of Ag or Ab
• the interfacial or ring test is very useful
Precipitin reactions (cont)
• Reactions may occur in gels
– produces a visible line of precipitation
– called immunodiffusion
• used to determine relative concentration of Ag or Ab
• used to compare antigens
• or determine relative purity of an antigen
• Mancini method (radial diffusion)
– antigen placed in a well
– diluted antibody placed into agar
Precipitin reactions (cont)
• Ouchterlony method
double diffusion in agar
antigen and antibody diffuse toward each other
form visible precipitation line where they meet
show relationships between different systems
• identity
• nonidentity
• partial identity
Precipitin reactions (cont)
• Immunoelectrophoresis
seperation of proteins with electrophoresis
followed by double diffusion in gel
can detect concentrations of 3 - 20 mg/ml
rocket electrophoresis is similar but can detect
level of protein as low as 0.2 mg/ml
• 2-D immunoelectrophoresis
– electrophoresis followed by overlay with gel
with antiserum
• Read Chapter 6,
• Review question 6 (pg
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