Antibodies (Chapter 4)

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Antibodies(Chapter 4)

• Proteins produced by B-lymphocytes in response to
  infection
• Major components of blood plasma and lymph
• Their function is to bind pathogens and their
  toxins
• Antibodies are highly specific each antibody
  can bind only one type of antigen
• Antibody repertoire The total number of
  different specific antibodies that can be made by
  an individual (1016)

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Antibodies are the secreted form of
immunoglobulins Plasma cells release antibody of
the same antigen specificity as the membrane
bound immunoglobulin expressed by their B-cell
precursor.
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Antibodies are glycoproteins built from two
identical heavy chains (H-chains) and two
identical light chains (L- chains). Carbohydrate
is attached to the H-chains. Antibodies have
characteristic Y-shaped structure. There are 5
different classes of antibodies (Ig) IgA, IgD,
IgE, IgG, and IgM. IgG the most abundant Ig. Mw
of IgG is 150 kD (50 kD for H-chain and 25 kD for
L-chain).
Antibody structure
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Antibodies are composed of polypeptides with
variable and constant regions
The N-terminal regions (called variable regions
V-regions) vary greatly in amino-acid sequences,
and are responsible for the antibody specificity,
and binding to the antigen. The remaining parts
have more conserved amino-acid sequences, and are
called constant (C-regions). They are responsible
for binding to other immune cells.
VH VL
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The Y-shaped Ig molecule can be cleaved by
proteases
Fragment antigen binding Fragment crystallizable

• Flexible hinge region in the middle of the heavy
  chain
• Can be cleaved by different proteases

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Antibody structure
Arms of Ig are called Fab Stem of Ig is called Fc
region
Hinge region is the region at which the arms of
the antibody molecule forms a Y. It is called the
hinge region because there is some flexibility in
the molecule at this point.
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Differences in the heavy C-regions define five
main isotypes (classes) of Ig IgA, IgD, IgE, IgG
and IgM

• The isotypes differ in
• Length of the heavy C-regions
• Location of disulfide bonds linking the chains
• Hinge region (present in IgG, IgD and IgA, and
  absent in IgM and IgE)
• Distribution of carbohydrate groups

• In the membrane-bound form, all Igs are
  monomers.
• In the secreted form ( antibodies), IgD, IgE
  and IgG are monomers IgM is a pentamer and IgA
  can exist as a monomer or a dimer.

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Two isotypes of the light chain

• Kappa (k) 2/3 of antibody molecules in humans
• Lambda (l) 1/3 of antibody molecules in humans
• No functional difference
• Each antibody contains either k or l

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Immunoglobulin chains are folded into compact
and stable protein domains
Heavy and light chains consist of motifs of
100-110 amino acids that are folded into compact
domains, called immunoglobulin domains. The light
chain has one variable domain (VL) and one
constant domain (CL). The heavy chain has one
variable domain (VH) and three constant domains
(CH1, CH2, and CH3). VH and VL domains together
form the antigen-binding site.
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Hypervariable regions

• The differences in amino acid sequence in the V
  domains of heavy and light chains are
  concentrated in hypervariable regions (HV), also
  called complementarity-determining regions (CDR).
  
• HVs (CDRs) are flanked by much less variable
  framework regions (FR).
• Each V domain has three HVs and four FRs.

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Antigen-binding sites are formed from the
hypervariable regions of VL and VH
The pairing of a heavy and a light chain brings
together the HV loops (CDRs), which form the
antigen-binding site.
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Antigenic determinant (epitope)

• Individual antigens are usually composed of a
  cluster of AA or a part of polysaccharide.
• Epitope is the specific part of the antigen to
  which the antibody binds
• Antigen that contains more than one epitope
  multivalent antigen.
• There can be two forms of multivalent antigens
  1) with different epitopes and 2) with multiple
  copies of the same repeated epitope.

Two kinds of multivalent antigen
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Chemical nature of antigens (epitopes)

• Antibodies can be made to any chemical structure
• Most often, antigens are carbohydrates or
  proteins
• In allergic reactions or autoimmune diseases,
  antigens can be drugs (penicillin), environmental
  substances (pollen), metals (allergy to
  jewelry), DNA (lupus), or antibodies (arthritis).

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Recognition of antigens (epitopes) by B-cell
receptors (BCR) is highly specific
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Antibody binds to antigen by non-covalent forces
Antigen-binding sites of antibodies are usually
rich in aromatic AA (Phe, Tyr, Trp), which can
form strong hydrophobic interactions
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Main characteristics of antibodies

• IgM The first antibody produced in response to
  pathogen. By isotype switching, synthesis of IgM
  gives a way to synthesis of IgG.
• IgG The most abundant antibody. IgG is smaller
  and more flexible than IgM. During pregnancy, it
  can be transferred across placenta to provide the
  fetus with protective antibodies from the mother.
• IgA The main antibody in body fluids - tears,
  sweat, breast milk and saliva.
• IgE Highly specialized induces activation of
  the mast cells involved in parasitic infections.
  Responsible for allergies when produced against
  harmless antigen.
• IgD Present in serum in low amounts its
  function is not fully understood.

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Polyclonal vs. monoclonal antibodies
Polyclonals Monoclonals
Immunize animals (rabbits) with the appropriate antigen Prepare antisera from their blood IN VIVO The specificity of the antibody is highly dependent on the purity of the antigen ? Can be made only if antigen is available in highly purified form. Fuse B-cells producing antibodies with tumor plasma cells to form hybridomas Test the cells which produce the desired antibody, and clone them IN VITRO Does not require to have purified antigen. The antibodies produced by hybridomas are all identical (produced from the same clone).
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POLYCLONAL ANTIBODIES

• Obtained by bleeding animal following response to
  antigen.  Usually 4-6 weeks after initial
  injection (or immunization). Takes about 3 weeks
  for initial immune response required to develop a
  population of B-cells making high affinity IgGs. 
  
• In response to antigen, B-lymphocytes will
  differentiate and produce clones of cells that
  make various IgGs that recognize the antigen. 
  Hence the term  POLYCLONAL ANTIBODIES.  Each
  antibody has the potential to recognize different
  sites on the molecule or the same site in
  different ways. IgGs are secreted in blood. 
• Following bleeding of the animal, the serum (or
  ANTISERUM) can be used directly for many
  immunological assays.  Antiserum will contain
  many blood proteins and many IgGs in addition to
  the ones elicited specifically against the
  antigen of interest.  (In fact, only 1-5 of the
  IgG fraction will be against your antigen).
                 
• ADVANTAGES AND DISADVANTAGES OF POLYCLONAL
  ANTIBODIES
•         1) Easiest and cheapest way to prepare
  antibodies.  Animal does all the work.
•         2) Obtain many types of IgGs that
  recognize the same protein. 
•         3) Antiserum only as good as the antigen
  preparation you injected. If the antigen is
  contaminated with other substances, antibodies to
  those contaminants also maybe produced. 
•        

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Production of monoclonal antibodies
Nobel Prize in Medicine in 1984 was awarded to
Milstein, Kohler, and Jerne for the discovery of
the technology to produce monoclonal antibodies
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Production of monoclonal antibodies
Polyethylene glycol-mediated fusion of
antibody-producing cells with myeloma cells
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Uses for MAbs Diagnosis

• Identification of tumors and classification of
  leukemia
• Screening for prostate cancer (PSA)

• Identification of pathogens
• HIV testing

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Uses for MAbs Treatment

• To suppress the immune system
• Remicade Monoclonal antibody neutralizing TNF
  used in rheumatoid arthritis

• Cancer immunotherapies
• Herceptin Binds protein (HER2) expressed on some
  tumor cells (breast cancers, lymphomas) inhibits
  cancer cell proliferation

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Immunological methods based on antibodies

• ELISA
• Western blotting
• Immunofluorescence
• Immunoprecipitation
• Based on an antigen-antibody interaction used
  in medicine and molecular biology

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Enzyme-Linked Immunosorbent Assay (ELISA)

• Can be used to detect antigens or antibodies in a
  sample

The higher the concentration of the antigen in
the sample, the higher the concentration of the
color product, and absorbance
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Introduction to Western Blotting

• The term blotting refers to the transfer of
  biological samples from a gel to a membrane, and
  their subsequent detection on the surface of the
  membrane.
• Western blotting (also called immunoblotting
  because an antibody is used to specifically
  detect its antigen) was introduced by Towbin, et
  al. in 1979, and is now a routine technique for
  protein analysis.
• The specificity of the antibody-antigen
  interaction enables a single protein to be
  identified in the midst of a complex protein
  mixture.
• Western blotting is commonly used to positively
  identify a specific protein in a complex mixture
  and to obtain qualitative and semiquantitative
  data about that protein.

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Immunoblotting (Western Blot)

• Procedure
• proteins separated by electrophoresis on a
  protein SDS-gel
• proteins transferred to (nitrocellulose or PVDF)
  membrane sheets
• protein bands visualized with enzyme-tagged
  antibodies
• Applications
• Diagnosis (diseases)
• Research (analysis of protein expression)

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Western Blotting

• In Western blotting, proteins are first separated
  by SDS electrophoresis (SDS-PAGE).
• As the proteins migrate through the gel they are
  separated based upon size and charge.
• Characteristically, smaller proteins migrate
  through the gel faster than larger proteins.

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ELISA
Western Blotting
Time (h) 0 0.5 3 9 18
IL-8 Actin
Intracellular levels of IL-8 in human neutrophils
analyzed by western blotting
IL-8 release from prostate cancer cells measured
by ELISA
Confocal immunofluorescence
Intracellular localization of IL-8 in a single
human neutrophil analyzed by confocal
immunofluorescence microscopy
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Antibodies Summary

• Antibodies are made of four polypeptide chains
  two identical heavy chains and two identical
  light chains.
• Each antibody has a V region that contains the
  antigen-binding site and determines specificity
  of the antibody, and a C region, which determines
  the antibody isotype and its effector functions.
• In the V-domain, the sequence variability is
  concentrated into three hyper-variable regions.
• The type of antigen bound by an antibody depends
  on the shape of the antigen-binding site each
  antibody molecule has two antigen binding sites.
• Monoclonal antibodies are antibodies of a single
  specificity that originate from one clone of
  identical antibody-producing cells. They are
  produced by fusion of mouse plasma cells with
  cancer cells, and are used in diagnostic tests
  and as therapeutic agents.