Trends in Biotechnology

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Trends in Biotechnology

• Week 13 Immunology and some uses

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• V. Immune System Disorders
• A. Hypersensitivity
• 1. Allergies
• 2. Autoimmune Disorders
• Immunodeficiencies
• a) HIV and AIDS

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• VI. Monoclonal Antibodies
• A. Biotech Revolution Future of Monoclonal
  Antibody Production
• VII. Tools of Immunology
• A. Western Blotting
• B. Fluorescent Antibody Technique
• C. Enzyme-Linked Immunosorbent Assay

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• Name some ways vaccines are created. How do
  vaccines prepare the immune system for an
  infection?
• Know some of the disorders of the immune system.
• 8. Know how polyclonal and monoclonal antibodies
  are made, and why monoclonal antibodies are so
  important in biotechnology.

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• 9. Western blotting, fluorescent antibody
  technique, and the enzyme-linked immunosorbent
  assay are three techniques in biotechnology that
  use antibodies. Know how each of these techniques
  is performed. Compare the techniques, and say
  when one technique is better than the others.

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• II. Acquired or Adaptive Immunity (Figure
  4.8).
• A. Recognizes foreign invaders and responds
  to the invader, called an antigen (Figure 4.9).
• B. Can also recognize the body as self and
  the tissues of others as non-self.
• C. Antigens can be protein, glycoprotein,
  polysaccharides, and nucleic acids, and small
  parts of the antigen can trigger a response
  (called the antigenic determinant).
• D. Based on the complex interactions of
  different types of cells and other components
  (Figure 4.10).

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B Cells
Class II MHC and processed antigen are displayed
Antigen-specific B cell receptor
Antigen
Antibodies
B cell
Plasma cell
Lymphokines
Antigen-presenting bacteria
Activated helper T cell
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Fig. 4.10 Acquired immunity, both cell mediated
and antibody mediated, requires the interaction
of many different types of cells by complex
signaling.
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• There are two types of Acquired Immunity
• 1. Cell-mediated this is controlled by
  cells called T lymphocytes (T cells).
• 2. Antibody-mediated this is controlled by
  cells called B lymphocytes (B cells).

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• E. Lymphocytes are circulated in the blood
  (by blood vessels) and lymphatic system (by
  lymphatic vessels) and in organs such as the
  spleen, tonsils, and thymus gland.

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Markers of Self Major Histocompatibility Complex
Antigenic peptide
Antigenic peptide
Antigenic peptide
Viral infection
MHC Class II
MHC Class I
MHC Class I
Antigen-presenting cell uses MHC Class I or II
Infected cell
Cell membrane
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Fig. 4.11 T cells react to antigens on the
surface of modified body cells.
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Fig. 4.12 An antibody molecular binds to an
antigenic determinant (epitope) of a cell that
has different antigens on its surface.
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• Antigenic Determinants (Epitopes)
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter32/animation_quiz_5.html

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Activation of T Cells Helper
Antigen is processed
Processed antigen and Class II MHC are displayed
Antigen
Macrophage
Helper T cell receptor recognizes processed
antigen plus Class II MHC
Class II MHC
Monokines
Antigen-presenting cell
Resting helper T cell
Lymphokines
MHC Class II
Activated helper T cell
Antigenic peptide
T cell receptor
CD4 protein
Helper T cell
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Activation of T Cells Cytotoxic
Processed antigen and Class II MHC are displayed
Antigen is processed
Antigen
Macrophage
Resting helper T cell receptor recognizes
processed antigen plus Class II MHC
Class II MHC
Monokines
Resting helper T cell
Lymphokines
Activated helper T cell
Class I MHC
Processed antigen and Class I MHC
Cytotoxic T cell becomes activated
Infected cell
Antigen (virus)
MHC Class I
CD8 protein
Activated cytotoxic T cell
Cytotoxic T cell
Infected cell
Processed antigen (viral protein)
Cell dies
Cytotoxic T cell
Antigenic peptide
T cell receptor
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Fig. 4.15 (b) T cells respond to changes in
histocompatibility antigens as in this
virus-infected cell.
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• Cytotoxic T-Cell Activity against Target Cells
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter32/animation_quiz_1.html

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Activation of B Cells to Make Antibody
Circulating antibody
Antigen
Antigen-presenting cell
Class II MHC and processed antigen are displayed
Antigen-specific B cell receptor
Antigen is processed
Antigen
Class II MHC
Lymphokines
Antibodies
B cell
Activated helper T cell
Antigen-presenting cell
Plasma cell
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• T-Cell Dependent Antigens
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter32/animation_quiz_6.html

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• Superantigens
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter32/animation_quiz_4.html

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Fig. 4.17 B cell activation.
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Fig. 4.18 Steps in antibody-mediated immunity.
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Antibody
Heavy chain
Light chain
Antigen-binding region
Constant region
Assembled antibody molecule
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Fig. 4.20 Structure of an antibody.
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Immunoglobulins
IgG, IgD, IgE, and IgA
IgA
IgM
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Fig. 4.21 Five classes of antibodies.
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Antibody Genes
V V V D D J J J J C
Heavy chain
Light chain
V D J C
Antigen-binding region
Constant region
Assembled antibody molecule
Rearranged gene components encoding a heavy chain
Gene components scattered through one chromosome
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Fig. 4.22 The generation of a functional antibody
L-chain by recombination of DNA regions.
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• Antibody Diversity
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter32/animation_quiz_2.html

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Fig. 4.23 Steps in cell-mediated immunity.
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• Hypersensitivity
• Sometimes the immune system responds the wrong
  way to the presence of antigen. These responses
  are hypersensitivities.
• There are four different types of
  hypersensitivities. These types are classified
  as
• Type I Immediate Hypersensitivity
• Type II Cytotoxic Hypersensitivity
• Type III Immune Complex Hypersensitivity
• Type IV Delayed Hypersensitivity

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• Type I, antibody-mediated allergy, immediate
  hypersensitivity
• ?? ? ????? ?? (bee venom) ? ?? ?????? (allergen)
  ? ?? ?????? ????? ????. ? ??? ?? ????? ????. ???
  ??? ????? ?? ?? ??? (immediate hypersensitivity)
  ?? ??? ??

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• Initial introduction of antigen produces an
  antibody response, the synthesis of IgE antibody
  in particular.
• Immunoglobulin IgE binds very specifically to
  receptors on the surface of mast cells, which
  remain circulating.

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• Reintroduced antigen interacts with IgE on mast
  cells causing the cells to degranulate and
  release large amounts of histamine, lipid
  mediators and chemotactic factors that cause
  smooth muscle contraction, vasodilation,
  increased vascular permeability,
  broncoconstriction and edema. These reactions
  occur very suddenly, causing death.

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• Examples of Type I hypersensitivities include
  allergies to penicillin, insect bites, molds,
  etc. People who are hypersensitive to such
  allergens must avoid contact with large amounts
  to prevent anaphylactic shock.

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• IgE Mediated (Type 1) Hypersensitivity
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter33/animation_quiz_2.html

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• Type II, Cytotoxic hypersensitivity
• ??? ? ?? ????? ????? ??? ??? ??? ? ????
  (????????? ??? ????).?? ? ?? ??????? ???? ????
  ??? ?? ?????? ??

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• Type II or Cytotoxic Hypersensitivity involves
  antibody-mediated reactions. The immunoglobulin
  class (isotype) is generally IgG (or IgM). This
  process involves K-cells. K-cells are involved in
  antibody-dependent cell-mediated cytotoxicity
  (ADCC).

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• Type II hypersensitivity may also involve
  complement that binds to cell-bound antibody. The
  antibodies are specific for (or able to
  cross-react with) "self" antigens. When these
  circulating antibodies react with a host cell
  surface, tissue damage may result.

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• Cytotoxic (Type II Hypersensitivity)
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter33/animation_quiz_5.html

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• Type III, Immune-complex hypersensitivity
• ??? ??? ????? ??? ??? ???? ??? ???? ????? ????. ?
  ???? ? ?? ???? ???? ????

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• Type III or Immune Complex hypersensitivity
  involves circulating antibody that reacts with
  free antigen.
• These circulating complexes can then become
  deposited on tissues.
• Tissue deposition may lead to reaction with
  complement, causing tissue damage.

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• This type of hypersensitivity develops as a
  result of systematic exposure to an antigen and
  is dependent on
• the type of antigen and antibody and
• the size of the resulting complex.
• Complexes that are too small remain in
  circulation complexes too large are removed by
  the glomerulus intermediate complexes may become
  lodged in the glomerulus leading to kidney
  damage.

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• Immune Complex Type 3 Hypersensitivity
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter33/animation_quiz_3.html

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• Type IV, Cell-mediated hypersensitivity
• ???? ? ?? ?? ??? ???? ??? ????? ??? ??? ???? .T
  ??? ????? ???? ?? ?? ????. ?? ???? ???? ????? ?
  ?? ??? ??? ??? ??? ???? ???. ? ?? ??? ????? ?? ??
  ??? (delayed hypersensitivity) ?? ??? ??

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• Type IV hypersensitivity is often called delayed
  type hypersensitivity as the reaction takes two
  to three days to develop. Unlike the other types,
  it is not antibody mediated but rather is a type
  of cell-mediated response.

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• CD8 cytotoxic T cells and CD4 helper T cells
  recognize antigen in a complex with either type 1
  or 2 major histocompatibility complex. The
  antigen-presenting cells in this case are
  macrophages which secrete IL-12, which stimulates
  the proliferation of further CD4 T cells.

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• CD4 T cells secrete IL-2 and interferon gamma,
  further inducing the release of other Type 1
  cytokines, thus mediating the immune response.
  Activated CD8 T cells destroy target cells on
  contact while activated macrophages produce
  hydrolytic enzymes and, on presentation with
  certain intracellular pathogens, transform into
  multinucleated giant cells.

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• Delayed (Type IV) Hypersensitivity
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter33/animation_quiz_4.html

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Comparison of Different Types of hypersensitivity Comparison of Different Types of hypersensitivity Comparison of Different Types of hypersensitivity Comparison of Different Types of hypersensitivity Comparison of Different Types of hypersensitivity
characteristics type-I(anaphylactic) type-II(cytotoxic) type-III(immune complex) type-IV(delayed type)
antibody IgE IgG, IgM IgG, IgM None
antigen exogenous cell surface soluble tissues organs
response time 15-30 minutes minutes-hours 3-8 hours 48-72 hours
appearance Skin swelling and redness lysis and tissue death (necrosis) redness and swelling, necrosis redness and hardening
histology basophils and eosinophil antibody and complement complement and neutrophils monocytes and lymphocytes
transferred with antibody antibody antibody T-cells
examples allergic asthma, hay fever erythroblastosis fetalis, Goodpasture's nephritis SLE, farmer's lung disease   tuberculin test, poison ivy, granuloma
 
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Fig. 4.24 Structure of HIV.
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Fig. 4.25 Replication of HIV.
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• Watch animation of HIV replication
• http//highered.mcgraw-hill.com/sites/0072495855/s
  tudent_view0/chapter24/animation__how_the_hiv_infe
  ction_cycle_works.html

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• Monoclonal Antibody Production http//highered.mcg
  raw-hill.com/sites/0072556781/student_view0/chapte
  r32/animation_quiz_3.html

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• IF on rat trigeminal using Mouse monoclonal
  antibody to rat p75NTR (green), Rabbit antibody
  to internal part of cFos (c-fos) whole serum and
  DAPI counterstained appearing in blue.

http//www.osenses.com/mouse-monoclonal-antibody-p
75ntr-affinity-receptor-mc192-p-576.html
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• Monoclonal bodies have a wide variety of
  academic, medical and commercial uses.
• Antibodies are used in several diagnostic tests
  to detect small amounts of drugs, toxins or
  hormones.

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• Antibodies are used in the radioimmunodetection
  and radioimmunotherapy of cancer, and some new
  methods can even target only the cell membranes
  of cancerous cells.
• Monoclonal antibodies can be used to treat viral
  diseases, traditionally considered "untreatable".

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• Monoclonal antibodies can be used to classify
  strains of a single pathogen.
• Researchers use monoclonal antibodies to identify
  and to trace specific cells or molecules in an
  organism.
• Some antibodies to the T3 antigen of T cells, is
  used to alleviate the problem of organ rejection
  in patients who have had organ transplants.

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Carter P (November 2001). "Improving the efficacy
of antibody-based cancer therapies" Nature
Reviews Cancer 1 (2)118-29
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• Antigen-Antibody Interactions In Vitro
• Many of the antigen-antibody interactions that
  occur in vivo also occur in vitro and are
  frequently the basis of diagnostic procedures
  serology is the branch of immunology concerned
  with these in vitro reactions.

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• Agglutination-visible clumps or aggregates of
  cells or of coated latex microspheres
• Widal Test-direct agglutination test for
  diagnosing typhoid fever
• Latex agglutination tests are used in pregnancy
  test to diagnose mycotic, helminthic, and
  bacterial infections and in drug testing
• Viral agglutination inhibition tests are used to
  diagnose influenza and other viral infections
• Agglutination tests can be used to measure
  antibody titer (the reciprocal of the greatest
  dilution showing agglutination reaction)

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• Complement fixation
• used to detect the presence of serum antibodies
  to a pathogen currently used to diagnose certain
  viral, fungal, rickettsial, chlamydial and
  protozoan diseases

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• Complement Fixation Test
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter31/animation_quiz_4.html

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• Immunoblotting (Western Blot)-proteins are
  separated by electrophoresis, blotted to
  nitrocellulose sheets, then treated with solution
  containing enzyme-tagged antibodies

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Fig. 4.26 Western blotting for antibodies to HIV
proteins.
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• Immunofluorescence-dyes coupled to antibody
  molecules will fluoresce (emit visible light)
  when irradiated with ultraviolet light
• Direct-used to detect antigen-bearing organisms
  fixed on a microscope slide
• Indirect-used to detect the presence of serum
  antibodies

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Fig. 4.27 Direct and indirect fluorescent
antibody methods. (a) The direct antibody assay
where tagged antibody interacts directly whit
antigens on a cell surface.
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Fig. 4.27 (b) The microorganism Legionella
pneumophila fluoresces after its cell surface
antigens are bound with fluorescent-tagged
antibody using the direct binding methods.
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Fig. 4.27 (c) In the direct method, untagged
antibodies are sandwiched between the known
antigen and a fluorescent-tagged antibody.
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• Flow cytometry and fluorescence
• Detects single or multiple microorganisms on the
  basis of a cytometric parameter or by means of
  fluorochromes
• Flow cytometer forces cells through a laser beam
  and measures light scatter or fluorescence as the
  cells pass through the beam cells can be tagged
  with fluorescent antibody directed against
  specific surface antigen

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• Video of a HIV test being carried out.

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• Enzyme-linked immunosorbent assay
  (ELISA)-involves linking labeled enzymes to an
  antibody
• Double antibody sandwich assay-detects antigens
  in a sample
• Wells of a microtiter plate are coated with
  antibody specific to the antigen of interest
• Test sample is placed in well if it contains the
  antigen of interest, the antigen will be retained
  in the well after washing
• Second antibody is added it is conjugated to an
  enzyme and is specific to the antigen the second
  antibody will be retained in the well after
  washing if the antigen was retained in the
  previous step
• Substrate of enzyme is added reaction only
  occurs if conjugated enzyme (and therefore
  antigen) is present in the well produces a
  colored product that can be detected

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• Indirect immunosorbent assay-detects serum
  antibody
• Well of a micro titer plate is coated with
  antigen specific to the antibody of interest
• Test serum is added if antibodies are present,
  they will bind antigen and will be retained after
  washing
• An antibody against the test immunoglobulin is
  added the second antibody is conjugated to an
  enzyme and will only be retained in the well
  after washing if the test antibody is present in
  the well
• Substrate of the enzyme is added reaction only
  occurs if conjugated antibody (and therefore test
  antibody) are present in the well the colored
  product of the reaction can be detected
  spectrophotometrically

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Fig. 4.28 The ELISA method. (a) The sandwich of
substrate-bound antibody, specific antigen, and a
second antibody whit a bound enzyme, with the
subsequent conversion of a substrate to a colored
product.
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Fig. 4.28 (b) The steps in conducting an ELISA
assay.
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Fig. 4.28 (b)-1 The steps in conducting an ELISA
assay.
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• The ELISA Method
• positive
• negative
• http//www.biology.arizona.edu/immunology/activiti
  es/elisa/technique.html

http//www.biology.arizona.edu
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• ELISA Enzyme-Linked Immunosorbent Assay
• http//highered.mcgraw-hill.com/sites/0072556781/s
  tudent_view0/chapter33/animation_quiz_1.html

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• Video of HIV antibody test

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• Immunodiffusion-involves the precipitation of
  immune complexes in an agar gel
• Single radial immunodiffusion (RID) assay is
  quantitative
• Double diffusion assay-lines of precipitation
  form where antibodies and antigens have diffused
  and met determines whether antigens share
  identical determinants
• Immunoelectrophoresis-antigens are first
  separated by electrophoresis according to charge,
  and are then visualized by the precipitation
  reaction greater resolution than diffusion assay
  
• Immunoprecipitation-soluble antigens form
  insoluble immune complexes that can be detected

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• Liposomes-artificially created microscopic lipid
  vesicles that contain a colored dye in its
  aqueous compartment and specific antibodies (or
  antigens) on its surface will bind to
  complementary antigens (or antibodies) in a test
  sample and this is detected by presence of color
  
• Neutralization-an antibody that is mixed with a
  toxin or a virus will neutralize the effects of
  the toxin or the infectivity of the virus this
  is determined by subsequent assay in lab animals
  or tissue culture
• Radioimmunoassay (RIA)-purified antigen labeled
  with a radioisotope competes with unlabeled
  sample for antibody binding
• Serotyping-antigen-antibody specificity is used
  to differentiate among various strains (serovars)
  of an organism