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The basic antibody is
a dimer of dimer (
heavy chain-light
chain pairs)
composed of repeats
of a single structural
unit known as the
“immunoglobulin
domain”
A Brief Review of Antibody Structure A Brief Review of Antibody Structure Primary structure Secondary structure Quaternary structure Tertiary structure Antigens & Antibodies II Definitions A comparison of antigen recognition by B and T cells Factors that influence immunogenicity Quantitating the strength of antibody-antigen interactions Equilibrium constants equilibrium dialysis impact of multivalency Cross-reactivity of antibodies Measuring antibody-antigen binding Polyclonal antibodies vs Monoclonal antibodies
Polyclonal antibodies: antibody preparations from
immunized animals. Consist of complex mixtures of
different antibodies produced by many different B cell
clones
Monoclonal Antibody: homogeneous antibody preparations
produced in the laboratory. Consist of a single type of
antigen binding site, produced by a single B cell clone
(later we’ll talk about how these are made).
Affinity between two macromolecules can measured using a biosensor Technique: Surface Plasmon Resonance Instrument: Biocore
- Resonance units are proportional to the degree of binding of soluble ligand to the immobilized receptor. (or soluble antibody to immobilized antigen, as shown here )
- Determining the amount of binding at equilibrium with different known concentrations of receptor (antibody) and ligand (protein antigen) allows you to calculate equilibrium constants (Ka, Kd). -Rate of dissociation and association (koff, kon) can also be calculated.
Affinity refers to strength of binding of single
epitope to single antigen binding site.
But antibodies have 2 or more identical binding
sites.
Most antigens are multivalent.
What is impact of valence on strength of
binding?
Avidity (strength of binding) is influenced by both
Affinity (Ka of single binding site) x Valence of
interaction (number of interacting binding sites)
Antibody-antigen interactions
are multivalent when both the
antibody and the antigen have
multiple binding sites.
Decavalent IgM Bivalent IgG
low affinity interactions can have high avidity if valence is high. IgM tend to bind tightly, but have less specificity. Avid binding due to high affinity. Binding of IgG tends to be more specific. (more perfect “fit” between antigen binding site and antigen)
Antigens & Antibodies II
Definitions and comparison of B and T cell antigen recognition Quantitating the strength of antibody-antigen interactions: affinity and avidity Equilibrium constants equilibrium dialysis impact of multivalency Cross-reactivity of antibodies Definition of cross-reactivity Example: ABO blood groups Cause of rheumatic fever (streptolysin) Useful for vaccine generation Problems for self-tolerance Measuring antibody-antigen binding
Cross-reactive Antigens Cross-reactive Antigens
Antigens & Antibodies II
Definitions and comparison of B and T cell antigen recognition Quantitating the strength of antibody-antigen interactions: affinity and avidity Equilibrium constants equilibrium dialysis impact of multivalency Cross-reactivity of antibodies Definition of cross-reactivity Example: ABO blood groups Cause of rheumatic fever (streptolysin) Useful for vaccine generation Problems for self-tolerance Measuring antibody-antigen binding
Rheumatic Fever
- Complication arising from infection with
Streptococcus pyogenes
- antibodies to bacterial proteins (M antigen or
streptolysin) cross-react with myocardial and
muscle proteins.
- Immunization with cowpox (vaccinia virus) induces
immunity to smallpox (variola virus). (Jenner)
- Vaccination to one type of influenza virus provides
resistance to other forms of influenza.
Taking advantage of cross-
reactivity in vaccine design
- Our bodies contain many epitopes that resemble the epitopes found on pathogens.
- By avoiding reactivity to those self-antigens, we restrict the ability of our immune systems to recognize certain pathogens.
- Tolerance to polysaccaride antigens on RBC prevents the production of certain antibodies reactive to microbial antigens.
Cross-reactivity and self-tolerance
Antigens & Antibodies II
Definitions and comparison of B and T cell antigen recognition Quantitating the strength of antibody-antigen interactions: affinity and avidity Equilibrium constants equilibrium dialysis Cross-reactivity of antibodies Measuring antibody-antigen binding lattice formation and precipitation reactions secondary antibodies: antibodies reactive with other antibodies medical tests based on antibody-antigen precipitation reactions A variety of different assays have been developed to detect antibody-antigen interactions. Some of these are based on the tendency of antibody- antigen complexes to come out of solution called “precipitation reaction”. Some are based on the ability of antibodies to stick cells together, called an “agglutination reaction”.
Precipitation Reactions:
Antibody and Antigen
interactions in solution can lead to the
formation of a lattice and precipitation of
immune complexes.
Antibody and antigen must be multivalent.
Occurs most efficiently when antigen and
antibody are at similar concentration.
Can be generated by repeated immunization of animal (rabbit) with antigen (with adjuvant). polyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen.
Polyclonal antisera
Polyclonal antibodies vs Monoclonal antibodies
Polyclonal antibodies: antibody preparations from
immunized animals. Consist of complex mixtures of
different antibodies produced by many different B cell
clones
Monoclonal Antibody: homogeneous antibody preparations
produced in the laboratory. Consist of a single type of
antigen binding site, produced by a single B cell clone
(later we’ll talk about how these are made).
Polyclonal antibodies can form
lattices with homogeneous,
monomeric protein antigens because
each antibody can interact with a
different epitope on the antigen.
Monoclonal antibodies do not form
lattices with homogeneous,
monomeric proteins, because only
they can bind to only one epitope on
the antigen.
When do antibody-antigen lattices form?
Polyclonal antibody and antigen with multiple Distinct epitopes monoclonal antibody and antigen with repeating pattern of identical epitopes
When do antibody-antigen lattices form?
Not with monoclonal antibody and antigen with multiple distinct epitopes
Haptens are not
immunogenic
unless they are
coupled to a
carrier protein.
Agglutination-based pregnancy test Antigens & Antibodies II Definitions and comparison of B and T cell antigen recognition Quantitating the strength of antibody-antigen interactions: affinity and avidity Equilibrium constants equilibrium dialysis Cross-reactivity of antibodies Measuring antibody-antigen binding lattice formation and precipitation reactions secondary antibodies: antibodies reactive with other antibodies medical tests based on antibody-antigen precipitation reactions Antibodies that bind to other antibodies (secondary antibodies) Immunize animal (rabbit) with purified antibodies from another species (human). The human Ig is the antigen, and the antibodies raised in the rabbit bind specifically to human Ig. These anti-human Ig (secondary antibodies) can be used to detect presence of human Ig. Use of anti-Ig antibodies increases degree of cross-linking and can increase lattice formation. human Ig Rabbit anti-human Ig antibody
Some secondary antibodies recognize portions of the
constant regions that are characteristic of a particular
antibody isotype. (These are the most common types of
secondary antibodies.)
Some secondary antibodies recognize portions of the the
antibody that are variable between different individuals
(different allotypes) in the species.
Some secondary antibodies recognize unique portions of
the variable domain of the antibody: the antigen binding
sites (idiotypes). Anti-idiotypic antibodies are rare.
Agglutination as a
clinical assay-- Testing
for Rh incompatibility
Disease : Erythroblastosis fetalis Cause : Mother produces IgG that bind to an antigen (Rh) on RBC of fetus Detection : Expose RBC to anti-human Ab and look for agglutination Treatment of mothers with antibodies to Rh (RhoGam) at time of 1st
delivery can prevent her from developing anti-Rh antibodies. Immunological Techniques
Monoclonal Antibodies
Radioimmune Assay (RIA)
Enyzme Linked Immune Sorbant Assay (ELISA)
Western blot
Immunoprecipitation
Flow cytometry
Expression cloning
P olyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen. Each antisera preparation differs in specificity, averge affinity, cross- reactive specificies, etc. Supply is limited.
Limitations
of polyclonal
antisera
Monoclonal antibodies
Kohler and Milstein, 1975
a technique to generate inexhaustible supply of
homogeneous antibody with useful specificities.
Basic strategy: fuse 2 cell types to generate a “hybridoma”.
Takes advantage of the properties of myeloma cell
(unlimited growth capacity and cellular machinery to
produce antibodies) and the antigen specificity of primary
B cells.
Advantages of Monoclonal Abs
• Consistent
• Limitless supply of specific reagent
• More easily tested for cross-reactivity
