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Figure 1-18 Antibodies (and T cell receptors) are encoded by sets of gene segments.
During development of a B (or T) cell, gene segments are joined randomly by DNA recombination (irreversible).
Juxtaposed gene segments encode variable part of the antibody (or T cell receptor).
Different cells join gene segments differently, so receptors are unique.
Each B cell bears many copies of its unique receptor (membrane-bound antibody). Each T cell bears many copies of its unique receptor (T cell receptor; TCR).
The diversity of antigen receptors in both B and T cells is generated by
rearrangements of gene segments
Hypervariable regions fall in loops of V domain structure
Crystal structures show that loops contact antigen
Rename them CDRs (Complementarity Determining Regions)
CDR2 CDR1
CDR3
Figure 3.7
Arrangement of CDRs in Ab combining sites: CDR3s always in center, CDR1 and CDR2 always on sides
CDR1 (H)
CDR2 (H) CDR3 (L)
CDR3 (H) CDR2 (L)
CDR1 (L) Branden and Tooze, Fig. 15.13
Lysozyme
Lysozyme Lysozyme
Antibody variable domains
Hypervariable loops (CDRs)
Hypervariable loops (CDRs)
Hypervariable loops removed
Antibody variable Domains (framework)
Antibody variable Domains
Antibody variable domains bound to an antigen
Janeway et al. (2005) Immunobiology: The Immune system in health and disease Garland Publishing, 6th edition, figure 3-8
Laboratory uses for antibodies: Immunoprecipitation
Microtitre well
coated with p24
Serum added
Anti-human antibody
labeled with enzyme added
Enzyme substrate
added
Enyzme-linked immunosorbent assay (ELISA) to detect anti-HIV-1 p24 antibodies
See Figure 49.15 in Freeman 3/E
Note: False positives can result from cross-reactions.
Lyse (break open) laboratory strain of HIV-1 in detergent
Separate HIV proteins by SDS-PAGE*
Transfer proteins to membrane (blot)
Cut membrane and incubate in blood sample from individual being tested
Detect bound antibodies
Western blot to detect anti-HIV-1 antibodies
-gp160
-p66 -p55
-p24
-pg41
-p32
-p17
-gp120
Days after HIV-1 infection
30 6
SDS is a detergent that binds to and unfolds proteins; PAGE: polyacrylamide gel electrophoresis
Clicker question In SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins migrate towards the positive electrode because:
1) The polypeptide backbone is negatively charged. 2) The sidechains are negatively charged. 3) SDS is negatively charged. 4) Migration is random; ~half of proteins migrate
towards the positive electrode.
Laboratory uses for antibodies: Immunofluorescence microscopy and flow
cytometry
Kuby, Kindt, Goldsby, Osborne Immunology Textbook
Flow cytometry --Fluorescence Activated
Cell Sorting (FACS)
A two-color FACS analysis
Kuby, Kindt, Goldsby, Osborne Immunology Textbook
Single color FACS analysis (e.g., using anti-A antibody) -- note this is a log scale
A- cells
A+ cells
Another way to sort cells using antibodies
Kuby, Kindt, Goldsby, Osborne Immunology Textbook
Having two identical antigen binding sites allows antibodies to bind tightly to surfaces
with repeating epitopes
Note -- Fc regions of antibodies are exposed when Fabs bind to surface antigens.
Fc
Affinity versus avidity Affinity is a measure of the strength of a binding interaction:
A + B <--> AB Equilibrium dissociation constant (KD) = [A][B]/[AB]
Strength of binding between a multivalent protein and an antigen or ligand containing multiple binding sites is the AVIDITY.
High avidity can compensate for low affinity.
Moderate affinity when only one Fab
is bound Higher “apparent” affinity
due to avidity effects
IgM IgG
No avidity effects if antigen is not tethered to a surface
Fab IgG
Fab IgG
No effects of tethering to a
surface if only one Fab is bound
What antibodies do • Don’t directly kill anything
• Can block entry of a virus into a host cell or prevent virus from replicating (neutralizing antibodies)
CD4 CCR5
gp120
gp41
IgG
What antibodies do • Can tag invaders for destruction (first of three ways)
– By complement -- binding of IgM or IgG to repeated epitope on invader surface triggers “classical” pathway (movie in extra material at end of Antibodies Lecture 1)
Electron micrographs of ~100 Å diameter membrane attack complex channels that are one of the end results of complement activation.
What antibodies do • Can tag invaders for destruction (2nd of three ways)
– By macrophages: antibodies opsinize (decorate the surface of) invaders -- Fc receptors on macrophages bind to exposed Fcs to increase phagocytosis
What antibodies do • Can tag invaders for destruction (3rd of three ways)
– By Natural Killer (NK) cells: Fc receptors on NK cells bind to exposed Fcs to activate Antibody-Dependent Cellular Cytotoxicity (ADCC)
• NK cells bear surface Fc receptors (CD16). If Fc regions of IgG are clustered or aggregated by antigen on a target cell, they bind to CD16.
• Binding to CD16 causes contents of granules inside cells to be released --> lysis of target cells.
Protein antigens
Haptens
Figure courtesy of Ian Wilson,
Scripps Institute
Antibodies can bind to different epitopes on the same antigen -- The immune response to any antigen is polyclonal
Note that these three Fabs bind to different regions of the model antigen lysozyme
What if you want a single, chemically-homogeneous antibody against an antigen? Answer: you make a monoclonal antibody -- see next slide and link to Köhler and Milstein’s 1984 Nobel Prize lecture on Bi1 website.
Figure A-14 part 1 of 2 B cell hybridomas secrete monoclonal antibodies
Polyclonal B cells secreting antibodies against antigen A cannot be grown in tissue culture, so can’t produce a clone secreting a single type of antibody.
Fuse B cells with myeloma (malignant tumor) cells. These cells have been immortalized (can be grown in tissue culture).
Use myeloma cells that lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT) enzyme.
Resulting hybrid cells (hybridomas) secrete antibody and can be grown in tissue culture.
Make clones of single hybridomas.
Monoclonal antibodies have many uses in biology, biotechnology, medicine
• Used to detect presence and/or quantity of an antigen; e.g., Western blot, ELISA, immunofluorescence microscopy, immunoelectron microscopy, flow cytometry.
• Used to purify antigens; e.g., immunoprecipitation (e.g., CHiP), immunoaffinity chromatograpy.
• Used for medical applications, especially for the treatment of cancer. 160 different monoclonal antibodies in clinical trials or awaiting FDA approval (August 2006).
Examples: Therapeutic uses of monoclonal antibodies
• Rituximab (Genentech) -- against CD20 antigen on surface of normal and malignant B cells. Used to treat non-Hodgkin’s lymphomas (B cell lymphomas).
• Herceptin (Genentech) -- against HER2 antigen. Given to patients with metastatic breast cancer whose tumors overexpress the HER2 protein (growth factor receptor). (HER2-positive breast cancers are more aggressive than HER2-negative breast cancers.)
Figure 14-17
Different ways monoclonal antibodies are used to eliminate tumors
e.g., ricin or Pseudomonas toxin
Can also link antibody to a chemotherapy drug (e.g., adriamycin)
(ADCC)
Figure A-15
In vitro selection to produce human monoclonal antibodies or increase affinity of existing monoclonal antibody
Generate library of heavy and light chain variable
regions using spleen DNA. Or introduce random mutations
into variable regions genes of a specific
antibody.
Clone into a phage so that each phage expresses one
VH-VL surface fusion protein.
Multiply phage display library in
bacteria, bind phage to surface coated with antigen. Wash away unbound phage.
Repeat procedure (multiply recovered
phage, bind to antigen, wash away unbound phage) for
several cycles. Recover specific
high-affinity antigen binding
VH-VL regions.
Epitope : CDR
1) HV region: Ag
2) Ab : Ig
3) Fab : Fc
4) Ag : Ab
Clicker question
Clicker question After an egg is fertilized, the DNA in the egg is
copied. Copies are passed to daughter cells, copied again, passed to new daughter cells, etc. etc.
With the exception of errors arising during copying (mutations), all somatic* cells end up with same DNA as the fertilized egg.
*somatic = not a germline cell; i.e., not sperm or egg
1) True? 2) False?
Extra slides for reference
V regions encoded by >1 gene segment (light chains)
Kuby Immunology textbook
Important point: Rearrangement for antibody genes is at the DNA level -- different from RNA splicing, which occurs in many genes
V regions encoded by >1 gene segment (heavy chains)
Kuby Immunology textbook
Complement activation - classical pathway
Anaphylatoxin: peptides produced during complement fixation that mediate inflammation. Induce anaphylactic shock when injected into animals.
Don’t need to memorize the details.
Gene segment recombination
Opsinization of invader by antibodies enhances phagocytosis