Fundamentals of Acquired Immunity

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Fundamentals of Acquired Immunity
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• I. Definitions

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• Immunology the study of all aspects of host
  defense against infection and of adverse
  consequences of immune response
• Immunity the state of protection fro infectious
  diseases utilizing both innate and acquired
  mechanisms

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• Immune response - a specific and complex series
  of events throughout the animals body that helps
  it defend against disease-causing organisms or
  substances
• Antigen particular foreign molecules that
  stimulate and immune response
• Often immunogenic on its own, otherwise requires
  a carrier molecule)
• Non-self substances to which lymphocytes can
  respond

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• Innate Immunity non-specific protective
  mechanisms conferring basic resistance to disease

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• general, physical, chemical, and biological
  barriers against disease
• discussed in previous unit
• also referred to as innate or natural immunity

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II. Different Types of Acquired Immunity
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• Acquired Immunity specific protective
  mechanisms displaying specificity, diversity,
  memory and self/non-self discrimination
• Often dependent on innate immunity for full
  activation
• Can be acquired actively or passively

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• Naturally acquired active immunity - An
  individual comes in contact with an antigen (Ag)
  and produces sensitized lymphocytes and/or
  antibodies that inactivate the antigen
• Ag is encountered naturally
• Immune responses are activated
• Antibodies made
• Lymphocytes sensitized
• Result Ag inactivated or destroyed
• Memory generated (long-lasting immunity)

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• Naturally acquired passive immunity - transfer of
  antibodies (Abs) from one individual to another
• Abs transferred from a donor to a recipient
  (adoptive transfer)
• Maternal transfer of IgG Abs to fetus across the
  placenta
• Maternal transfer of IgA Abs to newborn in
  colostrom and breast milk
• Newborn has immunity to infections encountered by
  mother previously
• Short-lived (weeks to months)

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• Artificially acquired active immunity -
  deliberate exposure of an individual to a vaccine
  with subsequent development of an immune response
• Vaccine preparation of attenuated or killed
  microbes or inactivated toxins (toxoids)

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• Artificially acquired passive immunity -
  deliberate introduction of antibodies into an
  individual
• Abs made deliberately in another animal or
  synthesized using in vitro methods
• Antiserum
• Antitoxins
• Antivenom
• Monoclonal antibodies (in vitro)

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• III. Origin, Maturation and Function of
  Lymphocytes

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B Lymphocytes

• Birds Bursa of fabricius
• Part of the chicken cloaca where B cells mature
• Absent in mammals
• Humans
• Fetal liver
• Bone marrow stem cells
• Two types
• T-dependent B cells (require T cell help)
• T-independent B cells (do not require T cell help)

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T Lymphocytes

• Originate in the thymus
• DiGeorge Syndrome athymic no T cells
• Specialized anatomicl locations responsible for
  educating T cells
• Cortex
• Medulla
• Education involves two main processes
• Positive selection
• Negative selection

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• Three types
• TH1 Inflammatory T cells
• Help macrophages (and B cells)
• TH2 Helper T cells
• Help B cells
• Tcyt Cytotoxic T cells (killer T cells)
• Kill target cells using perforin and granzyme

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Natural Killer Cells (NK cells)

• Probably derived from prethymic lymphocytes
• Do not have the characteristics of either B-cells
  or T-cells
• AKA Large Granular Lymphocytes (LGL)
• AKA Null cells
• Not equivalent to killer T cells but kill their
  target cells using same mechanism (perforin and
  granzyme)
• Participate in ADCC

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• Humoral Immunity

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B Cells and Their Role in Humoral Immunity

• Involvement of Ab in immune responses
• T-dependent
• T-independent
• Protects against pathogens that exist in fluid
  spaces
• Bacteria and their toxins
• Viruses
• Protozoans
• Helminths
• When B cells begin to produce antibody upon
  activation by antigen, they differentiate into
  plasma cells

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Cell-Mediated Immunity
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T Cells and Their Role in Cell-Mediated Immunity

• Involvement of T cells subsets
• TH1
• TH2
• Tcyt
• Requirement for direct interaction with infected
  cell or foreign cells
• Involves complex of T cell receptor (TCR), Major
  Histocompatibility Complex molecule (MHC) and
  Antigenic peptide fragment

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• Functions
• Lysis of host cells infected by viruses
• Elimination of cancer cells
• Productin of cytokines to assist other cells in
  eradicating foreign Ags
• Cytokines Molecules secreted by one cell and
  affecting a different cell type ? specific
  regulatory interactions
• Trasplantation rejection
• Due to differences in MHC class I and class II
  molecules (alloantigens)

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NK Cells and Their Role in Cell-Mediated Immunity

• Nonspecifically kill tumor cells, virus-infected
  cells, and other parasite-infected cells
• Play a role in regulating the immune response
• Exhibit antibody dependent cell-mediated
  cytotoxicity (ADCC)

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Figure 31.20
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• IV. The Nature of Antigens

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SELF VERSUS FOREIGN
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Self

• Prior to and after birth, the immune system
  develops and is able to differentiate between
  self proteins and foreign proteins
• T cells that are responsive to self Ags are
  eliminated early in their developmental pathway
• Negative selection
• Apoptosis occurs in the thymus
• Never enter periphery

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• B cells interacting with self Ags either apoptose
  or anergize
• Apoptosis programmed cell death
• Anergy induction of non-responsive state

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Self-Tolerance

• The process of elimination of self-reactive
  lymphocytes is called self tolerance
• Leads to removal of what could initiate harmful
  autoreactive response Autoimmunity
• Is very efficient, but not complete

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FOREIGN
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Immunogenicity

• The term immunogen is often used synonymously
  with the term antigen
• Careful!!!!
• All immunogens are antigens, but not all antigens
  are immunogens
• An immunogen is any substance that can mediate an
  immune response

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• Types of Antigens (immunogens)
• Antigens recognized by B cells
• Proteins, peptides, glycoproteins,
  nucleoproteins, polysaccharides, lipids,
  glycolipids, and small chemical groups (haptens)
• B cell receptor Antibody (Ab)
• Antigens recognized by T cells
• Peptide in association with MHC molecule
• T cell receptor TCR

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Epitopes

• Epitopes (antigenic determinant sites) are areas
  of an antigen that can stimulate production of
  specific antibodies and that can combine with
  them
• Some antigens have more than one site capable of
  interacting with Abs

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• Valence - the number of epitopes on an antigen
  determines number of antibody molecules an
  antigen can combine with at one time
• Generally the high the valency, the more
  immunogenic the substance
• Epitopes (determinants) that are more easily
  accessible stimulate better immune response
  (projections or NH2/COOH ends)

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Figure 32.3 The number of antigenic determinant
sites (epitopes) on an antigen is its valence.
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• Hapten - a small organic molecule that is not
  itself antigenic but that may become antigenic
  when bound to a larger carrier molecule
• Examples
• Dinitrophenol (DNP)
• Penicillin

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Figure 32.4 Effect of carrier on immunogenicity
of hapten.
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• V. Structure and Function of Antibodies
  (Immunoglobulins)

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• What is an antibody?

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Antibodies

• A group of glycoproteins in the blood, serum, and
  tissue fluids of mammals
• Produced in response to an antigen and can
  combine specifically with that antigen
• In the serum, four major classes can be measured
  electrophoretically, but there are actually five
  classes

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Figure 32.6 Electrophoresis of human serum
showing distribution of serum proteins and four
major classes of immunoglobulins.
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• Immunoglobulin structure
• Multiple antigen-combining sites (usually two
  some can form multimeric antibodies with up to
  ten combining sites)
• Composed of four polypeptide chains (two heavy or
  long, and two light or short) that form a
  flexible Y with a hinge region
• Heavy chain 440 aa, 50-70kDal (GAMED)
• Light chain 220 aa, 25kDal (kappa or lambda)

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• The stalk of the Y (called the Fc) is constant in
  amino acid sequence (i.e., the amino acid
  sequences of antibodies of the same subclass do
  not vary significantly)
• Constant regions are relatively invariable
  between Igs belonging to the same class (
  isotype, IgG, IgA, IgM, IgE, IgD)
• Constant regions confer different biological
  properties upon the Ig classes (in vivo half
  life, anatomical location, interactions with
  other molecules)

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• B cells can switch from production of one type of
  Ig to another type (isotype) by a mechanism known
  as ISOTYPE SWITCHING
• Isotype switching is mediated by signals received
  during ongoing immune responses (e.g. T cell
  cytokines)

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• The arms of the Y have variable regions and
  constitute the antigen-binding domains (Fab)
• Each Ab has 2 Ag-combining sites (bivalent) made
  up of H and L chain regions encoded by the
  variable portion of the Ig (VLVH)
• Some Abs exist as dimers (IgA) or pentamers (IgM)
  and so have 4 or 10 Ag-binding sites, respectively

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• Intrachain disulfide bonds create loops on both
  heavy and light chain
• Each loop contains 25 aa making up a single
  domain
• Interchain disulfide bonds hold the heavy and
  light chains together
• H to H
• H to L

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• Within the Fab segment are hypervariable (or
  complementarity determining) regions
• these regions are responsible for the diversity
  of antibodies

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Figure 32.8 Constant and Variable Domains in
Heavy and Light Chains Dark blue
hypervariable regions in the variable
domains Hypervariable regions are also known as
complementarity determining regions (CDRs).
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Pockets Grooves Extended Surfaces Peptide Ag
Red HIV peptide Orange HEL F(ab) frag. 5
CDRs used Red Ab contact sites Blue
Backbone Yellow Ag
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• There are five types of heavy chains that
  determine the five classes (isotypes) of
  immunoglobulins (IgG, IgA, IgM, IgD, and IgE)
• In IgG there are four subclasses, and in IgA
  there are two subclasses

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• Categories of immunoglobulin types
• Isotypes - variations in the constant regions of
  heavy chains that are associated with different
  classes and subclasses
• Allotypes - genetically controlled allelic forms
  of the immunoglobulin molecule
• Idiotypes - individual-specific immunoglobulin
  molecules that differ in the hypervariable
  regions of the Fab segments

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• Review of Immunoglobulin Function
• Fab region binds to antigen
• Fc region mediates binding to host tissue, to
  various cells of the immune system, to some
  phagocytic cells, or to the first component of
  the complement system

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• Binding of antibody to an antigen does not
  destroy the antigen, but marks (targets) the
  antigen for immunological attack or attack by the
  nonspecific defense mechanisms that do destroy it
• Opsonization - coating a bacterium with
  antibodies to stimulate phagocytosis

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Figure 31.15
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• Immunoglobulin classes (Isotypes)
• IgG - monomeric protein, 70 to 75 of Ig pool
• Antibacterial and antiviral
• Enhances opsonization
• Neutralizes toxins
• Only IgG is able to cross placenta (naturally
  acquired passive immunity for newborn)

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• Activates the complement system by the classical
  pathway
• Four subclasses with some differences in function
• IgG1
• IgG2
• IgG3
• IgG4

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• IgM - pentameric protein, 10 of Ig pool
• First antibody made during B-cell maturation
• First antibody secreted into serum during primary
  antibody response
• Never leaves the bloodstream
• Activates complement by classical pathway
• Enhances phagocytosis of target cells

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• Agglutinates bacteria and foreign red blood cells
• Up to 5 may be hexameric which is better able to
  activate the complement system than pentameric IgM

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Figure 32.11
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• IgA - dimeric protein, 15 of Ig pool
• Associated with secretory mucosal surfaces
• Protects gastrointestinal tract, respiratory
  tract, and genitourinary tract
• Also found in saliva, tears, and breast milk
  (protects nursing newborns)
• Secretory form (sIgA) helps rid the body of
  antibody-antigen complexes by excretion into the
  gut lumen and subsequent excretion from the body
• Adherence Inhibition

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Figure 32.12
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• IgD - monomeric protein, trace amounts in serum
• Abundant on surface of B cells
• May play a role in B-cell recognition of antigens
• May play a role in B cell anergy
• Does not activate the complement system
• Cannot cross the placenta

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Figure 32.13
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• IgE - monomeric protein, less than 1 of Ig pool
• Skin-sensitizing and anaphylactic antibodies
• When an antigen cross-links two molecules of IgE
  on the surface of a mast cell or basophil, it
  triggers release of histamine, and it increases
  intestinal motility, which helps to eliminate
  helminthic parasites

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Figure 32.14
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• Diversity of Antibodies

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• A number of mechanisms contribute to the
  generation of antibody diversity
• Somatic recombination (gene shuffling)
• Combinatorial joining (unique H and L chains)
• Somatic Hypermutation
• Isotype switching

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• Somatic Recombination
• Ig genes contain multiple segments of variable
  encoding regions of heavy and light chains (VDJ
  for heavy VJ for light)
• During differentiation of B cells, these genes
  are rearranged on the chromosome to form various
  combinations
• The number of different antibodies possible is
  the product of the number of light chains
  possible and the number of heavy chains possible

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• Imprecise joining - during combinations, the same
  segments can be joined at different nucleotides,
  thus increasing the number of codons and the
  possible diversity (N and P nucleotides)
• Somatic mutations - the V regions of germ-line
  DNA are susceptible to a high rate of somatic
  mutation following B cell activation
• The total diversity produces more than 2x108
  different antibody molecules

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Fig. 32.15
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Figure 32.16
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Figure 32.17
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• Specificity of Antibodies

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• The Clonal Selection Theory

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• Because of somatic recombination, combinatorial
  joining and somatic mutation, there are a small
  number of B cells capable of responding to any
  given antigen

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• Each group of cells is derived asexually from a
  parent cell and is referred to as a clone
• There is a large, diverse population of B-cell
  clones that collectively are capable of
  responding to many possible antigen
• The surface receptor molecules of the B cells
  bind to the appropriate antigen

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• The cell is then stimulated to divide and
  differentiate into two populations of cells
• Plasma cells
• Memory cells
• Plasma cells are protein factories that produce
  about 2,000 antibodies per second for their brief
  life span (5-7 days)

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Figure 32.18
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• Memory cells, like the original B cells, can
  differentiate into plasma cells if they are
  stimulated by being bound to the antigen
• because there are more memory cells than original
  B cells, the secondary (anamnestic) response can
  be (and usually is) faster and larger than the
  primary response
• they have long life spans (years or decades)

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• Sources of Antibodies (pure homogeneous
  preparations)

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• Immunization

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• Procedure
• Inject animal with antigen to stimulate primary
  immune response
• Initial lag phase of several days
• Log phase antibody titer rises logarithmically
• Plateau phase antibody titer stabilizes
• Decline phase antibody titer decreases because
  the antibodies are metabolized or cleared from
  the circulation
• Mostly IgM low-affinity antibodies

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Fig. 32.19
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• After a period of time, give animal a series of
  booster injections with same antigen to stimulate
  secondary immune response, or anamnestic response
• Shorter lag phase, higher antibody titer
• Mostly IgG high-affinity antibodies (affinity
  maturation)
• Withdraw blood from animal allow it to clot and
  remove fluid (serum), which is referred to as
  antiserum since it is from a specifically
  immunized host

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Fig. 32.19
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• Limitations
• This method results in polyclonal antibodies
  which have different epitope specificities thus
  sensitivity is lower, and the antibodies often
  cross-react with closely related antigens
• Repeated injections with antiserum from one
  species into another can cause serious allergic
  reactions
• Antiserum contains a mixture of antibodies, not
  all of which are of interest

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• Hybridomas - overcome some of the limitations of
  antisera
• Inject animals with antigen
• Separate spleen cells (which contain plasma
  cells)
• Fuse spleen cells with myeloma cells (tumor cells
  of the immune system that produce large
  quantities of antibodies and that are easy to
  culture)

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• Culture fused cells (hybridomas) so that each
  grows into a separate colony
• Some plasma cells that fused with a myeloma cell
  will produce the desired antibody
• Screen colonies for those producing desired
  antibody

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• Can grow many desired colonies to obtain large
  amounts of antibody
• Antibodies produced by this method are monoclonal
  (react with only one epitope) since they come
  from the fusion of a single plasma cell with the
  tumor cell

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Figure 32.20
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• Have a variety of uses in which high specificity
  is required
• Tissue typing for transplants
• Identification and epidemiological study of
  infectious microorganisms
• Identification of tumor and other surface antigens

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• Classification of leukemias and T-cell
  populations
• Sensitive diagnostic procedures
• Immunotoxins - used in the targeted delivery of a
  toxic substance to a particular cell type
  chemotherapeutic agents

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Box 32.1
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• Catalytic Antibodies

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• They are monoclonal antibodies made by subjecting
  an animal to an enzyme-substrate transition-state
  analogue and then producing a hybridoma
• The binding pocket on the antibody lowers the
  energy of activation of a reaction by ensuring
  the proper orientation of the reactant(s)

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• Currently, catalytic antibodies have been
  produced that can transform relatively simple
  compounds, but the potential is great if
  antibodies that act on proteins and nucleic acids
  can be produced