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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

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Page 1: Figure 1-18

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

Page 2: Figure 1-18

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

Page 3: Figure 1-18

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

Page 4: Figure 1-18

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

Page 5: Figure 1-18
Page 6: Figure 1-18

Janeway et al. (2005) Immunobiology: The Immune system in health and disease Garland Publishing, 6th edition, figure 3-8

Page 7: Figure 1-18

Laboratory uses for antibodies: Immunoprecipitation

Page 8: Figure 1-18

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.

Page 9: Figure 1-18

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

Page 10: Figure 1-18

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.

Page 11: Figure 1-18

Laboratory uses for antibodies: Immunofluorescence microscopy and flow

cytometry

Kuby, Kindt, Goldsby, Osborne Immunology Textbook

Page 12: Figure 1-18

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

Page 13: Figure 1-18

Another way to sort cells using antibodies

Kuby, Kindt, Goldsby, Osborne Immunology Textbook

Page 14: Figure 1-18

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

Page 15: Figure 1-18

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

Page 16: Figure 1-18

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

Page 17: Figure 1-18

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.

Page 18: Figure 1-18

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

Page 19: Figure 1-18

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.

Page 20: Figure 1-18

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.

Page 21: Figure 1-18

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.

Page 22: Figure 1-18

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).

Page 23: Figure 1-18

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.)

Page 24: Figure 1-18

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)

Page 25: Figure 1-18

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.

Page 26: Figure 1-18

Epitope : CDR

1) HV region: Ag

2) Ab : Ig

3) Fab : Fc

4) Ag : Ab

Clicker question

Page 27: Figure 1-18

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?

Page 28: Figure 1-18

Extra slides for reference

Page 29: Figure 1-18

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

Page 30: Figure 1-18

V regions encoded by >1 gene segment (heavy chains)

Kuby Immunology textbook

Page 31: Figure 1-18

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.

Page 32: Figure 1-18

Gene segment recombination

Page 33: Figure 1-18

Opsinization of invader by antibodies enhances phagocytosis


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